> top > docs > PMC:1942070 > annotations

PMC:1942070 JSONTXT

Annnotations TAB JSON ListView MergeView

2_test

Id Subject Object Predicate Lexical cue
17349631-10859332-30580518 1348-1349 10859332 denotes 3
17349631-12506120-30580518 1348-1349 12506120 denotes 3
17349631-12506120-30580519 1662-1663 12506120 denotes 6
17349631-9765302-30580519 1662-1663 9765302 denotes 6
17349631-11062248-30580519 1662-1663 11062248 denotes 6
17349631-11410586-30580519 1662-1663 11410586 denotes 6
17349631-15590638-30580520 1807-1808 15590638 denotes 7
17349631-10471840-30580521 1884-1886 10471840 denotes 12
17349631-10856238-30580521 1884-1886 10856238 denotes 12
17349631-11410587-30580521 1884-1886 11410587 denotes 12
17349631-12646243-30580521 1884-1886 12646243 denotes 12
17349631-12676944-30580521 1884-1886 12676944 denotes 12
17349631-11912133-30580522 1994-1996 11912133 denotes 16
17349631-11514571-30580522 1994-1996 11514571 denotes 16
17349631-16899224-30580522 1994-1996 16899224 denotes 16
17349631-12505989-30580523 2026-2028 12505989 denotes 18
17349631-14654790-30580523 2026-2028 14654790 denotes 18
17349631-14563314-30580524 2057-2059 14563314 denotes 19
17349631-16678913-30580525 2218-2220 16678913 denotes 20
17349631-12505989-30580526 2382-2384 12505989 denotes 23
17349631-15604256-30580526 2382-2384 15604256 denotes 23
17349631-15226414-30580526 2382-2384 15226414 denotes 23
17349631-15728188-30580527 2438-2440 15728188 denotes 24
17349631-16875491-30580528 2527-2529 16875491 denotes 25
17349631-16449666-30580529 2708-2710 16449666 denotes 28
17349631-15738054-30580529 2708-2710 15738054 denotes 28
17349631-15623513-30580529 2708-2710 15623513 denotes 28
17349631-15367659-30580529 2708-2710 15367659 denotes 28
17349631-16449666-30580530 2855-2856 16449666 denotes 1
17349631-16449666-30580531 3155-3156 16449666 denotes 1
17349631-16449666-30580532 3922-3923 16449666 denotes 1
17349631-16449666-30580533 4035-4036 16449666 denotes 1
17349631-7730364-30580534 4113-4115 7730364 denotes 29
17349631-16449666-30580535 4822-4823 16449666 denotes 1
17349631-16449666-30580536 5518-5519 16449666 denotes 1
17349631-15728188-30580537 5605-5607 15728188 denotes 24
17349631-16678913-30580538 7023-7025 16678913 denotes 20
17349631-12505989-30580539 8270-8272 12505989 denotes 32
17349631-16166634-30580539 8270-8272 16166634 denotes 32
17349631-16421204-30580539 8270-8272 16421204 denotes 32
17349631-15145937-30580539 8270-8272 15145937 denotes 32
17349631-16006559-30580540 10394-10396 16006559 denotes 35
17349631-14963034-30580540 10394-10396 14963034 denotes 35
17349631-8995387-30580541 10937-10939 8995387 denotes 36
17349631-12505989-30580542 12280-12282 12505989 denotes 37
17349631-15604256-30580542 12280-12282 15604256 denotes 37
17349631-16166634-30580542 12280-12282 16166634 denotes 37
17349631-15024053-30580542 12280-12282 15024053 denotes 37
17349631-15743830-30580543 12377-12379 15743830 denotes 38
17349631-16356855-30580544 12445-12447 16356855 denotes 40
17349631-16301747-30580544 12445-12447 16301747 denotes 40
17349631-1332886-30580545 14289-14291 1332886 denotes 42
17349631-16678913-30580546 14723-14725 16678913 denotes 23

MyTest

Id Subject Object Predicate Lexical cue
17349631-10859332-30580518 1348-1349 10859332 denotes 3
17349631-12506120-30580518 1348-1349 12506120 denotes 3
17349631-12506120-30580519 1662-1663 12506120 denotes 6
17349631-9765302-30580519 1662-1663 9765302 denotes 6
17349631-11062248-30580519 1662-1663 11062248 denotes 6
17349631-11410586-30580519 1662-1663 11410586 denotes 6
17349631-15590638-30580520 1807-1808 15590638 denotes 7
17349631-10471840-30580521 1884-1886 10471840 denotes 12
17349631-10856238-30580521 1884-1886 10856238 denotes 12
17349631-11410587-30580521 1884-1886 11410587 denotes 12
17349631-12646243-30580521 1884-1886 12646243 denotes 12
17349631-12676944-30580521 1884-1886 12676944 denotes 12
17349631-11912133-30580522 1994-1996 11912133 denotes 16
17349631-11514571-30580522 1994-1996 11514571 denotes 16
17349631-16899224-30580522 1994-1996 16899224 denotes 16
17349631-12505989-30580523 2026-2028 12505989 denotes 18
17349631-14654790-30580523 2026-2028 14654790 denotes 18
17349631-14563314-30580524 2057-2059 14563314 denotes 19
17349631-16678913-30580525 2218-2220 16678913 denotes 20
17349631-12505989-30580526 2382-2384 12505989 denotes 23
17349631-15604256-30580526 2382-2384 15604256 denotes 23
17349631-15226414-30580526 2382-2384 15226414 denotes 23
17349631-15728188-30580527 2438-2440 15728188 denotes 24
17349631-16875491-30580528 2527-2529 16875491 denotes 25
17349631-16449666-30580529 2708-2710 16449666 denotes 28
17349631-15738054-30580529 2708-2710 15738054 denotes 28
17349631-15623513-30580529 2708-2710 15623513 denotes 28
17349631-15367659-30580529 2708-2710 15367659 denotes 28
17349631-16449666-30580530 2855-2856 16449666 denotes 1
17349631-16449666-30580531 3155-3156 16449666 denotes 1
17349631-16449666-30580532 3922-3923 16449666 denotes 1
17349631-16449666-30580533 4035-4036 16449666 denotes 1
17349631-7730364-30580534 4113-4115 7730364 denotes 29
17349631-16449666-30580535 4822-4823 16449666 denotes 1
17349631-16449666-30580536 5518-5519 16449666 denotes 1
17349631-15728188-30580537 5605-5607 15728188 denotes 24
17349631-16678913-30580538 7023-7025 16678913 denotes 20
17349631-12505989-30580539 8270-8272 12505989 denotes 32
17349631-16166634-30580539 8270-8272 16166634 denotes 32
17349631-16421204-30580539 8270-8272 16421204 denotes 32
17349631-15145937-30580539 8270-8272 15145937 denotes 32
17349631-16006559-30580540 10394-10396 16006559 denotes 35
17349631-14963034-30580540 10394-10396 14963034 denotes 35
17349631-8995387-30580541 10937-10939 8995387 denotes 36
17349631-12505989-30580542 12280-12282 12505989 denotes 37
17349631-15604256-30580542 12280-12282 15604256 denotes 37
17349631-16166634-30580542 12280-12282 16166634 denotes 37
17349631-15024053-30580542 12280-12282 15024053 denotes 37
17349631-15743830-30580543 12377-12379 15743830 denotes 38
17349631-16356855-30580544 12445-12447 16356855 denotes 40
17349631-16301747-30580544 12445-12447 16301747 denotes 40
17349631-1332886-30580545 14289-14291 1332886 denotes 42
17349631-16678913-30580546 14723-14725 16678913 denotes 23

pmc-enju-pas

Id Subject Object Predicate Lexical cue
T49 180-187 NN denotes protein
T50 188-194 NN denotes kinase
T51 195-196 NN denotes D
T52 197-198 -LRB- denotes (
T53 198-201 NN denotes PKD
T54 201-202 -RRB- denotes )
T55 203-210 NN denotes enzymes
T56 211-213 PRP denotes we
T57 214-223 VB denotes generated
T58 224-225 DT denotes a
T59 226-234 JJ denotes PKD-null
T60 235-239 NN denotes DT40
T61 240-252 NN denotes B-lymphocyte
T62 253-257 NN denotes cell
T63 258-262 NN denotes line
T64 264-274 RB denotes Previously
T65 275-277 PRP denotes we
T66 278-282 VB denotes have
T67 283-288 VB denotes shown
T68 289-293 IN denotes that
T69 294-298 NN denotes PKDs
T70 299-303 VB denotes have
T71 304-306 DT denotes an
T72 307-316 JJ denotes essential
T73 317-321 NN denotes role
T74 322-324 IN denotes in
T75 325-335 VB denotes regulating
T76 336-341 NN denotes class
T77 342-344 CD denotes II
T78 345-352 NN denotes histone
T79 353-365 NN denotes deacetylases
T80 366-368 IN denotes in
T81 369-373 NN denotes DT40
T82 374-381 NN denotes B-cells
T83 382-383 -LRB- denotes [
T84 383-391 NNP denotes Matthews
T85 391-392 -COMMA- denotes ,
T86 393-397 NNP denotes S.A.
T87 397-398 -COMMA- denotes ,
T88 399-402 NNP denotes Liu
T89 402-403 -COMMA- denotes ,
T90 404-406 NNP denotes P.
T91 406-407 -COMMA- denotes ,
T92 408-416 NNP denotes Spitaler
T93 416-417 -COMMA- denotes ,
T94 418-420 NNP denotes M.
T95 420-421 -COMMA- denotes ,
T96 422-427 NNP denotes Olson
T97 427-428 -COMMA- denotes ,
T98 429-433 NNP denotes E.N.
T99 433-434 -COMMA- denotes ,
T100 435-443 NNP denotes McKinsey
T101 443-444 -COMMA- denotes ,
T102 445-449 NNP denotes T.A.
T103 449-450 -COMMA- denotes ,
T104 451-459 NNP denotes Cantrell
T105 459-460 -COMMA- denotes ,
T106 461-465 NNP denotes D.A.
T107 466-469 CC denotes and
T108 470-481 NNP denotes Scharenberg
T109 481-482 -COMMA- denotes ,
T110 483-487 NNP denotes A.M.
T111 488-489 -LRB- denotes (
T112 489-493 CD denotes 2006
T113 493-494 -RRB- denotes )
T114 495-504 JJ denotes Essential
T115 505-509 NN denotes role
T116 510-513 IN denotes for
T117 514-521 NN denotes protein
T118 522-528 NN denotes kinase
T119 529-530 NN denotes D
T120 531-537 NN denotes family
T121 538-545 NN denotes kinases
T122 546-548 IN denotes in
T123 549-552 DT denotes the
T124 553-563 NN denotes regulation
T125 564-566 IN denotes of
T126 567-572 NN denotes class
T127 573-575 CD denotes II
T128 576-583 NN denotes histone
T129 584-596 NN denotes deacetylases
T130 597-599 IN denotes in
T131 600-601 NN denotes B
T132 602-613 NN denotes lymphocytes
T133 615-619 NNP denotes Mol.
T134 620-624 NNP denotes Cell
T135 625-630 NNP denotes Biol.
T136 631-633 CD denotes 26
T137 633-634 -COMMA- denotes ,
T138 635-644 CD denotes 1569–1577
T139 644-645 -RRB- denotes ]
T140 647-649 PRP denotes We
T141 650-653 RB denotes now
T142 654-658 VB denotes show
T143 659-663 IN denotes that
T144 664-668 NN denotes PKDs
T145 669-672 VB denotes are
T146 673-677 RB denotes also
T147 678-686 VB denotes required
T148 687-689 TO denotes to
T149 690-698 VB denotes regulate
T150 699-704 NN denotes HSP27
T151 705-720 NN denotes phosphorylation
T152 721-723 IN denotes in
T153 724-728 NN denotes DT40
T154 729-736 NN denotes B-cells
T155 738-745 RB denotes However
T156 745-746 -COMMA- denotes ,
T157 747-749 IN denotes in
T158 750-758 NN denotes contrast
T159 759-761 TO denotes to
T160 762-770 JJ denotes previous
T161 771-783 NN denotes observations
T162 784-786 IN denotes in
T163 787-792 JJ denotes other
T164 793-797 NN denotes cell
T165 798-803 NN denotes types
T166 803-804 -COMMA- denotes ,
T167 805-808 NN denotes PKD
T168 809-816 NN denotes enzymes
T169 817-819 VB denotes do
T170 820-823 RB denotes not
T171 824-832 VB denotes regulate
T172 833-838 JJ denotes basic
T173 839-847 JJ denotes cellular
T174 848-857 NN denotes processes
T175 858-862 JJ denotes such
T176 863-865 IN denotes as
T177 866-879 NN denotes proliferation
T178 880-882 CC denotes or
T179 883-891 NN denotes survival
T180 892-901 NN denotes responses
T181 901-902 -COMMA- denotes ,
T182 903-906 CC denotes nor
T183 907-911 NN denotes NFκB
T184 912-927 JJ denotes transcriptional
T185 928-936 NN denotes activity
T186 937-947 RB denotes downstream
T187 948-950 IN denotes of
T188 951-954 DT denotes the
T189 955-956 NN denotes B
T190 957-961 NN denotes cell
T191 962-969 NN denotes antigen
T192 970-978 NN denotes receptor
T193 980-984 RB denotes Thus
T194 984-985 -COMMA- denotes ,
T195 986-990 NN denotes PKDs
T196 991-995 VB denotes have
T197 996-997 DT denotes a
T198 998-1007 JJ denotes selective
T199 1008-1012 NN denotes role
T200 1013-1015 IN denotes in
T201 1016-1020 NN denotes DT40
T202 1021-1027 NN denotes B-cell
T203 1028-1035 NN denotes biology
T532 635-1057 NN denotes 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The
T533 1058-1065 NN denotes protein
T534 1066-1072 NN denotes kinase
T535 1073-1074 NN denotes D
T536 1075-1076 -LRB- denotes (
T537 1076-1079 NN denotes PKD
T538 1079-1080 -RRB- denotes )
T539 1081-1097 NN denotes serine/threonine
T540 1098-1104 NN denotes kinase
T541 1105-1111 NN denotes family
T542 1112-1115 VB denotes has
T543 1116-1121 CD denotes three
T544 1122-1129 NN denotes members
T545 1129-1130 -COLON- denotes :
T546 1131-1135 NN denotes PKD1
T547 1135-1136 -COMMA- denotes ,
T548 1137-1141 NN denotes PKD2
T549 1142-1145 CC denotes and
T550 1146-1150 NN denotes PKD3
T551 1152-1156 JJ denotes Most
T552 1157-1161 NN denotes cell
T553 1162-1167 NN denotes types
T554 1168-1175 VB denotes express
T555 1176-1178 IN denotes at
T556 1179-1184 JJ denotes least
T557 1185-1188 CD denotes two
T558 1189-1192 NN denotes PKD
T559 1193-1201 NN denotes isoforms
T560 1202-1205 CC denotes but
T561 1206-1209 NN denotes PKD
T562 1210-1217 NN denotes enzymes
T563 1218-1221 VB denotes are
T564 1222-1232 RB denotes especially
T565 1233-1239 RB denotes highly
T566 1240-1249 VB denotes expressed
T567 1250-1252 IN denotes in
T568 1253-1267 JJ denotes haematopoietic
T569 1268-1273 NN denotes cells
T570 1273-1274 -COMMA- denotes ,
T571 1275-1280 WRB denotes where
T572 1281-1285 PRP denotes they
T573 1286-1289 VB denotes are
T574 1290-1299 VB denotes activated
T575 1300-1302 IN denotes in
T576 1303-1311 NN denotes response
T577 1312-1314 TO denotes to
T578 1315-1322 NN denotes antigen
T579 1323-1332 NN denotes receptors
T580 1333-1344 NN denotes stimulation
T581 1345-1346 -LRB- denotes [
T582 1346-1349 CD denotes 2,3
T583 1349-1350 -RRB- denotes ]
T584 1352-1353 DT denotes A
T585 1354-1363 VB denotes conserved
T586 1364-1374 NN denotes signalling
T587 1375-1382 NN denotes pathway
T588 1383-1390 VB denotes linking
T589 1391-1398 NN denotes antigen
T590 1399-1408 NN denotes receptors
T591 1409-1411 TO denotes to
T592 1412-1416 NN denotes PKDs
T593 1417-1425 VB denotes involves
T594 1426-1429 DT denotes the
T595 1430-1440 NN denotes activation
T596 1441-1443 IN denotes of
T597 1444-1448 NN denotes PLCγ
T598 1449-1452 CC denotes and
T599 1453-1456 DT denotes the
T600 1457-1467 JJ denotes subsequent
T601 1468-1478 NN denotes production
T602 1479-1481 IN denotes of
T603 1482-1496 NN denotes diacylglycerol
T604 1497-1498 -LRB- denotes (
T605 1498-1501 NN denotes DAG
T606 1501-1502 -RRB- denotes )
T607 1503-1508 WDT denotes which
T608 1509-1519 VB denotes stimulates
T609 1520-1529 JJ denotes classical
T610 1530-1536 CC denotes and/or
T611 1537-1542 JJ denotes novel
T612 1543-1550 NN denotes protein
T613 1551-1557 NN denotes kinase
T614 1558-1560 NN denotes Cs
T615 1561-1562 -LRB- denotes (
T616 1562-1565 NN denotes PKC
T617 1565-1566 -RRB- denotes )
T618 1567-1571 WDT denotes that
T619 1572-1585 VB denotes phosphorylate
T620 1586-1589 CD denotes two
T621 1590-1593 JJ denotes key
T622 1594-1604 JJ denotes regulatory
T623 1605-1611 NN denotes serine
T624 1612-1620 NN denotes residues
T625 1621-1623 IN denotes in
T626 1624-1627 DT denotes the
T627 1628-1638 NN denotes activation
T628 1639-1643 NN denotes loop
T629 1644-1646 IN denotes of
T630 1647-1650 NN denotes PKD
T631 1651-1658 NN denotes kinases
T632 1659-1660 -LRB- denotes [
T633 1660-1663 CD denotes 3–6
T634 1663-1664 -RRB- denotes ]
T635 1666-1669 DT denotes The
T636 1670-1680 JJ denotes N-terminal
T637 1681-1691 JJ denotes regulatory
T638 1692-1698 NN denotes region
T639 1699-1701 IN denotes of
T640 1702-1705 NN denotes PKD
T641 1706-1713 NN denotes enzymes
T642 1714-1722 VB denotes contains
T643 1723-1724 DT denotes a
T644 1725-1728 NN denotes DAG
T645 1729-1736 NN denotes binding
T646 1737-1743 NN denotes domain
T647 1744-1747 CC denotes and
T648 1748-1754 JJ denotes direct
T649 1755-1762 NN denotes binding
T650 1763-1765 IN denotes of
T651 1766-1769 NN denotes DAG
T652 1770-1774 RB denotes also
T653 1775-1786 VB denotes contributes
T654 1787-1789 TO denotes to
T655 1790-1794 NN denotes PKD1
T656 1795-1805 NN denotes activation
T657 1806-1807 -LRB- denotes [
T658 1807-1808 CD denotes 7
T659 1808-1809 -RRB- denotes ]
T660 1810-1812 RB denotes as
T661 1813-1817 RB denotes well
T662 1818-1820 IN denotes as
T663 1821-1831 VB denotes regulating
T664 1832-1835 DT denotes the
T665 1836-1843 JJ denotes spatial
T666 1844-1852 NN denotes location
T667 1853-1855 IN denotes of
T668 1856-1859 NN denotes PKD
T669 1860-1867 NN denotes enzymes
T670 1868-1874 IN denotes within
T671 1875-1880 NN denotes cells
T672 1881-1882 -LRB- denotes [
T673 1882-1886 CD denotes 8–12
T674 1886-1887 -RRB- denotes ]
T675 1889-1892 NN denotes PKD
T676 1893-1900 NN denotes enzymes
T677 1901-1905 VB denotes have
T678 1906-1910 VB denotes been
T679 1911-1919 VB denotes proposed
T680 1920-1922 TO denotes to
T681 1923-1931 VB denotes regulate
T682 1932-1940 JJ denotes numerous
T683 1941-1949 JJ denotes cellular
T684 1950-1959 NN denotes functions
T685 1959-1960 -COMMA- denotes ,
T686 1961-1970 VB denotes including
T687 1971-1975 NN denotes cell
T688 1976-1989 NN denotes proliferation
T689 1990-1991 -LRB- denotes [
T690 1991-1996 CD denotes 13–16
T691 1996-1997 -RRB- denotes ]
T692 1997-1998 -COMMA- denotes ,
T693 1999-2013 JJ denotes anti-apoptotic
T694 2014-2021 NN denotes signals
T695 2022-2023 -LRB- denotes [
T696 2023-2028 CD denotes 17,18
T697 2028-2029 -RRB- denotes ]
T698 2030-2033 CC denotes and
T699 2034-2043 NN denotes thymocyte
T700 2044-2055 NN denotes development
T701 2056-2057 -LRB- denotes [
T702 2057-2059 CD denotes 19
T703 2059-2060 -RRB- denotes ]
T704 2062-2072 NN denotes Expression
T705 2073-2075 IN denotes of
T706 2076-2082 JJ denotes mutant
T707 2083-2096 RB denotes catalytically
T708 2097-2105 JJ denotes inactive
T709 2106-2109 CC denotes and
T710 2110-2124 RB denotes constitutively
T711 2125-2134 VB denotes activated
T712 2135-2139 NN denotes PKDs
T713 2140-2143 MD denotes can
T714 2144-2148 RB denotes also
T715 2149-2155 VB denotes modify
T716 2156-2161 NNP denotes Golgi
T717 2162-2170 NN denotes function
T718 2170-2171 -COMMA- denotes ,
T719 2172-2176 NN denotes cell
T720 2177-2185 NN denotes adhesion
T721 2186-2189 CC denotes and
T722 2190-2194 NN denotes cell
T723 2195-2203 NN denotes motility
T724 2204-2205 -LRB- denotes (
T725 2205-2213 VB denotes reviewed
T726 2214-2216 IN denotes in
T727 2217-2218 -LRB- denotes [
T728 2218-2220 CD denotes 20
T729 2220-2221 -RRB- denotes ]
T730 2221-2222 -RRB- denotes )
T731 2224-2226 IN denotes In
T732 2227-2237 JJ denotes particular
T733 2237-2238 -COMMA- denotes ,
T734 2239-2243 NN denotes PKDs
T735 2244-2248 VB denotes have
T736 2249-2253 VB denotes been
T737 2254-2260 RB denotes widely
T738 2261-2267 VB denotes linked
T739 2268-2270 TO denotes to
T740 2271-2274 DT denotes the
T741 2275-2285 NN denotes activation
T742 2286-2288 IN denotes of
T743 2289-2292 DT denotes the
T744 2293-2297 NN denotes NFκB
T745 2298-2311 NN denotes transcription
T746 2312-2318 NN denotes factor
T747 2319-2322 CC denotes and
T748 2323-2325 IN denotes in
T749 2326-2336 VB denotes regulating
T750 2337-2341 NN denotes cell
T751 2342-2350 NN denotes survival
T752 2351-2357 IN denotes during
T753 2358-2367 JJ denotes oxidative
T754 2368-2374 NN denotes stress
T755 2375-2376 -LRB- denotes [
T756 2376-2384 CD denotes 17,21–23
T757 2384-2385 -RRB- denotes ]
T758 2387-2394 DT denotes Another
T759 2395-2403 RB denotes recently
T760 2404-2412 VB denotes proposed
T761 2413-2417 NN denotes PKD1
T762 2418-2427 NN denotes substrate
T763 2428-2430 VB denotes is
T764 2431-2436 NN denotes HSP27
T765 2437-2438 -LRB- denotes [
T766 2438-2440 CD denotes 24
T767 2440-2441 -RRB- denotes ]
T768 2441-2442 -COMMA- denotes ,
T769 2443-2444 DT denotes a
T770 2445-2450 JJ denotes small
T771 2451-2455 NN denotes heat
T772 2456-2461 NN denotes shock
T773 2462-2469 NN denotes protein
T774 2470-2478 VB denotes involved
T775 2479-2481 IN denotes in
T776 2482-2492 VB denotes regulating
T777 2493-2497 NN denotes cell
T778 2498-2507 NN denotes migration
T779 2508-2511 CC denotes and
T780 2512-2516 NN denotes cell
T781 2517-2525 NN denotes survival
T782 2526-2527 -LRB- denotes [
T783 2527-2529 CD denotes 25
T784 2529-2530 -RRB- denotes ]
T785 2532-2534 DT denotes An
T786 2535-2544 JJ denotes essential
T787 2545-2549 NN denotes role
T788 2550-2553 IN denotes for
T789 2554-2557 NN denotes PKD
T790 2558-2565 NN denotes enzymes
T791 2566-2568 IN denotes in
T792 2569-2579 VB denotes regulating
T793 2580-2585 NN denotes class
T794 2586-2588 CD denotes II
T795 2589-2596 NN denotes histone
T796 2597-2609 NN denotes deacetylases
T797 2610-2611 -LRB- denotes (
T798 2611-2616 NN denotes HDACs
T799 2616-2617 -RRB- denotes )
T800 2617-2618 -COMMA- denotes ,
T801 2619-2626 NN denotes enzymes
T802 2627-2631 WDT denotes that
T803 2632-2639 VB denotes repress
T804 2640-2654 JJ denotes MEF2-dependent
T805 2655-2659 NN denotes gene
T806 2660-2673 NN denotes transcription
T807 2673-2674 -COMMA- denotes ,
T808 2675-2678 VB denotes has
T809 2679-2683 RB denotes also
T810 2684-2688 VB denotes been
T811 2689-2701 VB denotes demonstrated
T812 2702-2703 -LRB- denotes [
T813 2703-2710 CD denotes 1,26–28
T814 2710-2711 -RRB- denotes ]
T815 2713-2715 TO denotes To
T816 2716-2727 VB denotes investigate
T817 2728-2731 DT denotes the
T818 2732-2742 JJ denotes biological
T819 2743-2747 NN denotes role
T820 2748-2750 IN denotes of
T821 2751-2755 NN denotes PKDs
T822 2756-2758 PRP denotes we
T823 2759-2763 VB denotes have
T824 2764-2773 VB denotes generated
T825 2774-2778 NN denotes DT40
T826 2779-2780 NN denotes B
T827 2781-2785 NN denotes cell
T828 2786-2791 NN denotes lines
T829 2792-2796 WDT denotes that
T830 2797-2801 VB denotes lack
T831 2802-2812 NN denotes expression
T832 2813-2815 IN denotes of
T833 2816-2819 CD denotes one
T834 2820-2822 CC denotes or
T835 2823-2827 JJ denotes more
T836 2828-2835 NN denotes members
T837 2836-2838 IN denotes of
T838 2839-2842 DT denotes the
T839 2843-2846 NN denotes PKD
T840 2847-2853 NN denotes family
T841 2854-2855 -LRB- denotes [
T842 2855-2856 CD denotes 1
T843 2856-2857 -RRB- denotes ]
T844 2857-2858 -COMMA- denotes ,
T845 2859-2867 VB denotes allowing
T846 2868-2870 PRP denotes us
T847 2871-2873 TO denotes to
T848 2874-2885 VB denotes investigate
T849 2886-2889 DT denotes the
T850 2890-2898 NN denotes function
T851 2898-2899 -LRB- denotes (
T852 2899-2900 NN denotes s
T853 2900-2901 -RRB- denotes )
T854 2902-2904 IN denotes of
T855 2905-2908 NN denotes PKD
T856 2909-2917 NN denotes isoforms
T857 2918-2927 VB denotes following
T858 2928-2929 NN denotes B
T859 2930-2934 NN denotes cell
T860 2935-2942 NN denotes antigen
T861 2943-2951 NN denotes receptor
T862 2952-2953 -LRB- denotes (
T863 2953-2956 NN denotes BCR
T864 2956-2957 -RRB- denotes )
T865 2958-2969 NN denotes stimulation
T866 2969-2970 -COMMA- denotes ,
T867 2971-2973 RB denotes as
T868 2974-2978 RB denotes well
T869 2979-2989 VB denotes addressing
T870 2990-2993 DT denotes the
T871 2994-2999 NN denotes issue
T872 3000-3002 IN denotes of
T873 3003-3013 JJ denotes functional
T874 3014-3024 NN denotes redundancy
T875 3025-3032 IN denotes between
T876 3033-3036 DT denotes the
T877 3037-3046 JJ denotes different
T878 3047-3050 NN denotes PKD
T879 3051-3057 NN denotes family
T880 3058-3065 NN denotes members
T881 3067-3075 JJ denotes Previous
T882 3076-3083 NN denotes studies
T883 3084-3088 VB denotes have
T884 3089-3094 VB denotes shown
T885 3095-3099 IN denotes that
T886 3100-3104 NN denotes PKDs
T887 3105-3108 VB denotes are
T888 3109-3122 JJ denotes indispensable
T889 3123-3126 IN denotes for
T890 3127-3131 NN denotes HDAC
T891 3132-3142 NN denotes regulation
T892 3143-3145 IN denotes in
T893 3146-3147 NN denotes B
T894 3148-3153 NN denotes cells
T895 3154-3155 -LRB- denotes [
T896 3155-3156 CD denotes 1
T897 3156-3157 -RRB- denotes ]
T898 3159-3165 RB denotes Herein
T899 3166-3168 PRP denotes we
T900 3169-3173 VB denotes show
T901 3174-3178 IN denotes that
T902 3179-3183 NN denotes PKDs
T903 3184-3187 VB denotes are
T904 3188-3192 RB denotes also
T905 3193-3206 JJ denotes indispensable
T906 3207-3210 IN denotes for
T907 3211-3216 NN denotes HSP27
T908 3217-3232 NN denotes phosphorylation
T909 3233-3235 IN denotes in
T910 3236-3237 NN denotes B
T911 3238-3243 NN denotes cells
T912 3245-3252 RB denotes However
T913 3252-3253 -COMMA- denotes ,
T914 3254-3262 JJ denotes PKD-null
T915 3263-3267 NN denotes DT40
T916 3268-3269 NN denotes B
T917 3270-3275 NN denotes cells
T918 3276-3279 VB denotes are
T919 3280-3286 JJ denotes viable
T920 3287-3290 CC denotes and
T921 3291-3302 VB denotes proliferate
T922 3303-3311 RB denotes normally
T923 3313-3321 RB denotes Moreover
T924 3321-3322 -COMMA- denotes ,
T925 3323-3327 NN denotes loss
T926 3328-3330 IN denotes of
T927 3331-3334 DT denotes the
T928 3335-3341 JJ denotes entire
T929 3342-3350 JJ denotes cellular
T930 3351-3355 NN denotes pool
T931 3356-3358 IN denotes of
T932 3359-3362 NN denotes PKD
T933 3363-3367 VB denotes does
T934 3368-3371 RB denotes not
T935 3372-3382 RB denotes critically
T936 3383-3389 VB denotes affect
T937 3390-3399 JJ denotes oxidative
T938 3400-3406 NN denotes stress
T939 3407-3416 NN denotes responses
T940 3417-3419 IN denotes in
T941 3420-3421 NN denotes B
T942 3422-3427 NN denotes cells
T943 3428-3431 CC denotes nor
T944 3432-3434 VB denotes do
T945 3435-3438 NN denotes PKD
T946 3439-3446 NN denotes kinases
T947 3447-3451 VB denotes play
T948 3452-3454 DT denotes an
T949 3455-3464 JJ denotes essential
T950 3465-3469 NN denotes role
T951 3470-3472 IN denotes in
T952 3473-3483 VB denotes regulating
T953 3484-3488 NN denotes NFκB
T954 3489-3504 JJ denotes transcriptional
T955 3505-3513 NN denotes activity
T956 3515-3523 RB denotes Together
T957 3523-3524 -COMMA- denotes ,
T958 3525-3530 DT denotes these
T959 3531-3539 NN denotes findings
T960 3540-3546 VB denotes reveal
T961 3547-3551 IN denotes that
T962 3552-3554 IN denotes in
T963 3555-3556 NN denotes B
T964 3557-3568 NN denotes lymphocytes
T965 3568-3569 -COMMA- denotes ,
T966 3570-3573 NN denotes PKD
T967 3574-3581 NN denotes kinases
T968 3582-3585 VB denotes are
T969 3586-3589 RB denotes not
T970 3590-3598 JJ denotes critical
T971 3599-3609 NN denotes regulators
T972 3610-3612 IN denotes of
T973 3613-3617 JJ denotes many
T974 3618-3620 IN denotes of
T975 3621-3624 DT denotes the
T976 3625-3633 JJ denotes cellular
T977 3634-3643 NN denotes processes
T978 3644-3654 RB denotes previously
T979 3655-3663 VB denotes ascribed
T980 3664-3666 TO denotes to
T981 3667-3671 PRP denotes them
T982 3672-3674 IN denotes in
T983 3675-3680 JJ denotes other
T984 3681-3689 JJ denotes cellular
T985 3690-3697 NN denotes systems
T1674 0-3735 NN denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell
T1675 3736-3743 NN denotes culture
T1676 3743-3744 -COMMA- denotes ,
T1677 3745-3754 JJ denotes transient
T1678 3755-3768 NN denotes transfections
T1679 3769-3772 CC denotes and
T1680 3773-3777 NN denotes cell
T1681 3778-3789 NN denotes stimulation
T1682 3790-3793 DT denotes The
T1683 3794-3804 NN denotes generation
T1684 3804-3805 -COMMA- denotes ,
T1685 3806-3813 NN denotes culture
T1686 3814-3817 CC denotes and
T1687 3818-3828 NN denotes activation
T1688 3829-3831 IN denotes of
T1689 3832-3839 NN denotes PKD1−/−
T1690 3839-3840 -COMMA- denotes ,
T1691 3841-3848 NN denotes PKD3−/−
T1692 3849-3852 CC denotes and
T1693 3853-3862 NN denotes PKD1/3−/−
T1694 3863-3871 NN denotes knockout
T1695 3872-3876 NN denotes DT40
T1696 3877-3878 NN denotes B
T1697 3879-3883 NN denotes cell
T1698 3884-3889 NN denotes lines
T1699 3890-3894 VB denotes have
T1700 3895-3899 VB denotes been
T1701 3900-3909 VB denotes described
T1702 3910-3920 RB denotes previously
T1703 3921-3922 -LRB- denotes [
T1704 3922-3923 CD denotes 1
T1705 3923-3924 -RRB- denotes ]
T1706 3926-3931 NN denotes Cells
T1707 3932-3936 VB denotes were
T1708 3937-3942 VB denotes lysed
T1709 3943-3946 CC denotes and
T1710 3947-3954 NN denotes protein
T1711 3955-3963 NN denotes extracts
T1712 3964-3968 VB denotes were
T1713 3969-3977 VB denotes analysed
T1714 3978-3980 IN denotes in
T1715 3981-3988 JJ denotes Western
T1716 3989-3997 NN denotes blotting
T1717 3998-4009 NN denotes experiments
T1718 4010-4012 IN denotes as
T1719 4013-4023 RB denotes previously
T1720 4024-4033 VB denotes described
T1721 4034-4035 -LRB- denotes [
T1873 4151-4152 -LRB- denotes (
T1874 4152-4159 CD denotes 2 × 106
T1875 4160-4165 NN denotes cells
T1876 4166-4169 IN denotes per
T1877 4170-4175 NN denotes point
T1878 4175-4176 -RRB- denotes )
T1879 4177-4181 VB denotes were
T1880 4182-4193 VB denotes resuspended
T1881 4194-4196 IN denotes in
T1882 4197-4203 NN denotes 200 μl
T1883 4204-4210 NN denotes buffer
T1884 4211-4212 -LRB- denotes (
T1885 4212-4216 NN denotes RPMI
T1886 4217-4221 CD denotes 1640
T1887 4222-4227 NN denotes media
T1888 4227-4228 -COMMA- denotes ,
T1889 4229-4230 CD denotes 1
T1890 4230-4231 NN denotes %
T1891 4232-4238 JJ denotes foetal
T1892 4239-4243 NN denotes calf
T1893 4244-4249 NN denotes serum
T1894 4249-4250 -RRB- denotes )
T1895 4251-4261 VB denotes containing
T1896 4262-4274 JJ denotes anti-chicken
T1897 4275-4277 NN denotes M1
T1898 4278-4288 JJ denotes monoclonal
T1899 4289-4297 NN denotes antibody
T1900 4298-4308 VB denotes conjugated
T1901 4309-4311 TO denotes to
T1902 4312-4316 NN denotes FITC
T1903 4317-4320 IN denotes for
T1904 4321-4327 NN denotes 20 min
T1905 4328-4330 IN denotes on
T1906 4331-4334 NN denotes ice
T1907 4336-4339 DT denotes The
T1908 4340-4345 NN denotes cells
T1909 4346-4350 VB denotes were
T1910 4351-4357 VB denotes washed
T1911 4358-4363 RB denotes twice
T1912 4364-4367 CC denotes and
T1913 4368-4379 JJ denotes fluorescent
T1914 4380-4389 NN denotes intensity
T1915 4390-4393 VB denotes was
T1916 4394-4402 VB denotes analysed
T1917 4403-4405 IN denotes by
T1918 4406-4410 NN denotes flow
T1919 4411-4420 NN denotes cytometry
T1920 4422-4425 DT denotes All
T1921 4426-4433 NN denotes results
T1922 4434-4439 VB denotes shown
T1923 4440-4443 VB denotes are
T1924 4444-4458 JJ denotes representative
T1925 4459-4461 IN denotes of
T1926 4462-4464 IN denotes at
T1927 4465-4468 CD denotes two
T1928 4469-4471 TO denotes to
T1929 4472-4476 CD denotes four
T1930 4477-4488 JJ denotes independent
T1931 4489-4500 NN denotes experiments
T1932 4501-4507 IN denotes unless
T1933 4508-4517 RB denotes otherwise
T1934 4518-4527 VB denotes indicated
T2288 0-4551 NN denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss
T2289 4552-4554 IN denotes of
T2290 4555-4560 NN denotes HSP27
T2291 4561-4576 NN denotes phosphorylation
T2292 4577-4579 IN denotes in
T2293 4580-4584 NN denotes DT40
T2294 4585-4586 NN denotes B
T2295 4587-4592 NN denotes cells
T2296 4593-4600 VB denotes lacking
T2297 4601-4611 NN denotes expression
T2298 4612-4614 IN denotes of
T2299 4615-4618 NN denotes PKD
T2300 4619-4625 NN denotes family
T2301 4626-4633 NN denotes kinases
T2302 4634-4638 NN denotes DT40
T2303 4639-4640 NN denotes B
T2304 4641-4646 NN denotes cells
T2305 4647-4654 VB denotes express
T2306 4655-4658 CD denotes two
T2307 4659-4662 NN denotes PKD
T2308 4663-4671 NN denotes isoforms
T2309 4671-4672 -COMMA- denotes ,
T2310 4673-4677 NN denotes PKD1
T2311 4678-4681 CC denotes and
T2312 4682-4686 NN denotes PKD3
T2313 4686-4687 -COMMA- denotes ,
T2314 4688-4691 CC denotes and
T2315 4692-4694 IN denotes as
T2316 4695-4705 RB denotes previously
T2317 4706-4715 VB denotes described
T2318 4716-4718 PRP denotes we
T2319 4719-4723 VB denotes have
T2320 4724-4732 RB denotes recently
T2321 4733-4742 VB denotes generated
T2322 4743-4747 NN denotes DT40
T2323 4748-4749 NN denotes B
T2324 4750-4754 NN denotes cell
T2325 4755-4760 NN denotes lines
T2326 4761-4765 WDT denotes that
T2327 4766-4770 VB denotes lack
T2328 4771-4781 NN denotes expression
T2329 4782-4784 IN denotes of
T2330 4785-4791 CC denotes either
T2331 4792-4796 NN denotes PKD1
T2332 4797-4799 CC denotes or
T2333 4800-4804 NN denotes PKD3
T2334 4805-4807 CC denotes or
T2335 4808-4812 DT denotes both
T2336 4813-4820 NN denotes enzymes
T2337 4821-4822 -LRB- denotes [
T2338 4822-4823 CD denotes 1
T2339 4823-4824 -RRB- denotes ]
T2340 4826-4828 IN denotes In
T2341 4829-4839 VB denotes generating
T2342 4840-4843 DT denotes the
T2343 4844-4850 JJ denotes double
T2344 4851-4859 NN denotes knockout
T2345 4860-4864 NN denotes cell
T2346 4865-4870 NN denotes lines
T2347 4871-4873 PRP denotes we
T2348 4874-4882 VB denotes targeted
T2349 4883-4886 DT denotes the
T2350 4887-4891 NN denotes PKD1
T2351 4892-4896 NN denotes loci
T2352 4897-4899 IN denotes in
T2353 4900-4901 DT denotes a
T2354 4902-4909 NN denotes PKD3−/−
T2355 4910-4914 NN denotes cell
T2356 4915-4919 NN denotes line
T2357 4920-4924 WDT denotes that
T2358 4925-4934 VB denotes expressed
T2359 4935-4936 DT denotes a
T2360 4937-4946 NN denotes Flag-PKD3
T2361 4947-4956 NN denotes transgene
T2362 4957-4962 IN denotes under
T2363 4963-4966 DT denotes the
T2364 4967-4974 NN denotes control
T2365 4975-4977 IN denotes of
T2366 4978-4979 DT denotes a
T2367 4980-5001 JJ denotes doxycycline-inducible
T2368 5002-5010 NN denotes promoter
T2369 5012-5017 RB denotes Hence
T2370 5017-5018 -COMMA- denotes ,
T2371 5019-5021 IN denotes in
T2372 5022-5025 DT denotes the
T2373 5026-5034 NN denotes presence
T2374 5035-5037 IN denotes of
T2375 5038-5049 NN denotes doxycycline
T2376 5049-5050 -COMMA- denotes ,
T2377 5051-5060 NN denotes Flag-PKD3
T2378 5061-5071 NN denotes expression
T2379 5072-5074 IN denotes in
T2380 5075-5081 NN denotes PKD1/3
T2381 5082-5088 JJ denotes double
T2382 5089-5097 JJ denotes knockout
T2383 5098-5103 NN denotes cells
T2384 5104-5106 VB denotes is
T2385 5107-5117 JJ denotes comparable
T2386 5118-5120 TO denotes to
T2387 5121-5131 JJ denotes endogenous
T2388 5132-5136 NN denotes PKD3
T2389 5137-5144 JJ denotes present
T2390 5145-5147 IN denotes in
T2391 5148-5157 JJ denotes wild-type
T2392 5158-5162 NN denotes DT40
T2393 5163-5168 NN denotes cells
T2394 5169-5172 CC denotes and
T2395 5173-5180 NN denotes removal
T2396 5181-5183 IN denotes of
T2397 5184-5195 NN denotes doxycycline
T2398 5196-5200 IN denotes from
T2399 5201-5204 DT denotes the
T2400 5205-5212 NN denotes culture
T2401 5213-5218 NN denotes media
T2402 5219-5222 IN denotes for
T2403 5223-5224 CD denotes 5
T2404 5225-5229 NN denotes days
T2405 5230-5237 VB denotes results
T2406 5238-5240 IN denotes in
T2407 5241-5242 DT denotes a
T2408 5243-5253 RB denotes completely
T2409 5254-5258 JJ denotes null
T2410 5259-5262 NN denotes PKD
T2411 5263-5272 NN denotes phenotype
T2412 5273-5274 -LRB- denotes (
T2413 5274-5278 NN denotes Fig.
T2414 5279-5281 NN denotes 1A
T2415 5281-5282 -RRB- denotes )
T2416 5284-5294 RB denotes Previously
T2417 5294-5295 -COMMA- denotes ,
T2418 5296-5298 PRP denotes we
T2419 5299-5303 VB denotes have
T2420 5304-5316 VB denotes demonstrated
T2421 5317-5321 IN denotes that
T2422 5322-5337 NN denotes phosphorylation
T2423 5338-5341 CC denotes and
T2424 5342-5349 JJ denotes nuclear
T2425 5350-5359 NN denotes exclusion
T2426 5360-5362 IN denotes of
T2427 5363-5368 NN denotes class
T2428 5369-5371 CD denotes II
T2429 5372-5379 NN denotes histone
T2430 5380-5392 NN denotes deacetylases
T2431 5393-5394 -LRB- denotes (
T2432 5394-5399 NN denotes HDACs
T2433 5399-5400 -RRB- denotes )
T2434 5401-5407 IN denotes during
T2435 5408-5411 NN denotes BCR
T2436 5412-5422 NN denotes engagement
T2437 5423-5425 VB denotes is
T2438 5426-5435 JJ denotes defective
T2439 5436-5438 IN denotes in
T2440 5439-5448 NN denotes PKD1/3−/−
T2441 5449-5450 NN denotes B
T2442 5451-5456 NN denotes cells
T2443 5457-5460 CC denotes and
T2444 5461-5464 MD denotes can
T2445 5465-5473 VB denotes restored
T2446 5474-5478 IN denotes upon
T2447 5479-5492 NN denotes re-expression
T2448 5493-5495 IN denotes of
T2449 5496-5497 DT denotes a
T2450 5498-5504 JJ denotes single
T2451 5505-5508 NN denotes PKD
T2452 5509-5516 NN denotes isoform
T2453 5517-5518 -LRB- denotes [
T2454 5518-5519 CD denotes 1
T2455 5519-5520 -RRB- denotes ]
T2456 5522-5525 DT denotes The
T2457 5526-5531 JJ denotes small
T2458 5532-5536 NN denotes heat
T2459 5537-5542 NN denotes shock
T2460 5543-5550 NN denotes protein
T2461 5551-5556 NN denotes HSP27
T2462 5557-5560 VB denotes has
T2463 5561-5569 RB denotes recently
T2464 5570-5574 VB denotes been
T2465 5575-5583 VB denotes proposed
T2466 5584-5586 IN denotes as
T2467 5587-5588 DT denotes a
T2468 5589-5593 NN denotes PKD1
T2469 5594-5603 NN denotes substrate
T2470 5604-5605 -LRB- denotes [
T2471 5605-5607 CD denotes 24
T2472 5607-5608 -RRB- denotes ]
T2473 5609-5612 CC denotes and
T2474 5613-5615 PRP denotes we
T2475 5616-5627 RB denotes accordingly
T2476 5628-5636 VB denotes assessed
T2477 5637-5644 IN denotes whether
T2478 5645-5653 JJ denotes PKD-null
T2479 5654-5658 NN denotes DT40
T2480 5659-5664 NN denotes cells
T2481 5665-5669 VB denotes have
T2482 5670-5679 JJ denotes defective
T2483 5680-5695 NN denotes phosphorylation
T2484 5696-5698 IN denotes of
T2485 5699-5704 NN denotes HSP27
T2486 5705-5707 IN denotes on
T2487 5708-5714 NN denotes serine
T2488 5715-5717 CD denotes 82
T2489 5717-5718 -COMMA- denotes ,
T2490 5719-5722 DT denotes the
T2491 5723-5731 VB denotes proposed
T2492 5732-5736 NN denotes PKD1
T2493 5737-5746 NN denotes substrate
T2494 5747-5755 NN denotes sequence
T2495 5757-5759 PRP denotes We
T2496 5760-5769 RB denotes initially
T2497 5770-5782 VB denotes investigated
T2498 5783-5786 DT denotes the
T2499 5787-5797 NN denotes regulation
T2500 5798-5800 IN denotes of
T2501 5801-5806 NN denotes HSP27
T2502 5807-5822 NN denotes phosphorylation
T2503 5823-5825 IN denotes in
T2504 5826-5832 JJ denotes single
T2505 5833-5841 NN denotes knockout
T2506 5842-5846 NN denotes DT40
T2507 5847-5848 NN denotes B
T2508 5849-5854 NN denotes cells
T2509 5855-5862 VB denotes lacking
T2510 5863-5869 CC denotes either
T2511 5870-5874 NN denotes PKD1
T2512 5875-5877 CC denotes or
T2513 5878-5882 NN denotes PKD3
T2514 5884-5886 IN denotes As
T2515 5887-5892 VB denotes shown
T2516 5893-5895 IN denotes in
T2517 5896-5900 NNP denotes Fig.
T2518 5901-5903 NN denotes 1B
T2519 5903-5904 -COMMA- denotes ,
T2520 5905-5915 NN denotes activation
T2521 5916-5918 IN denotes of
T2522 5919-5922 DT denotes the
T2523 5923-5926 NN denotes BCR
T2524 5927-5929 CC denotes or
T2525 5930-5939 NN denotes treatment
T2526 5940-5944 IN denotes with
T2527 5945-5948 DT denotes the
T2528 5949-5960 JJ denotes DAG-mimetic
T2529 5961-5965 NN denotes PdBu
T2530 5966-5975 VB denotes increased
T2531 5976-5979 DT denotes the
T2532 5980-5986 NN denotes levels
T2533 5987-5989 IN denotes of
T2534 5990-5995 NN denotes HSP27
T2535 5996-6011 NN denotes phosphorylation
T2536 6012-6014 IN denotes at
T2537 6015-6018 NN denotes S82
T2538 6019-6021 IN denotes in
T2539 6022-6031 JJ denotes wild-type
T2540 6032-6036 NN denotes DT40
T2541 6037-6038 NN denotes B
T2542 6039-6044 NN denotes cells
T2543 6046-6049 NN denotes BCR
T2544 6050-6053 CC denotes and
T2545 6054-6061 NN denotes phorbol
T2546 6062-6067 NN denotes ester
T2547 6068-6075 NN denotes signals
T2548 6076-6080 VB denotes were
T2549 6081-6085 RB denotes also
T2550 6086-6090 JJ denotes able
T2551 6091-6093 TO denotes to
T2552 6094-6102 VB denotes increase
T2553 6103-6108 NN denotes HSP27
T2554 6109-6124 NN denotes phosphorylation
T2555 6125-6127 IN denotes in
T2556 6128-6132 NN denotes PKD1
T2557 6133-6135 CC denotes or
T2558 6136-6140 NN denotes PKD3
T2559 6141-6147 JJ denotes single
T2560 6148-6156 NN denotes knockout
T2561 6157-6161 NN denotes DT40
T2562 6162-6163 NN denotes B
T2563 6164-6169 NN denotes cells
T2564 6170-6171 -LRB- denotes (
T2565 6171-6175 NN denotes Fig.
T2566 6176-6178 NN denotes 1B
T2567 6178-6179 -RRB- denotes )
T2568 6181-6188 RB denotes However
T2569 6188-6189 -COMMA- denotes ,
T2570 6190-6194 NN denotes BCR-
T2571 6195-6198 CC denotes and
T2572 6199-6206 NN denotes phorbol
T2573 6207-6220 JJ denotes ester-induced
T2574 6221-6236 NN denotes phosphorylation
T2575 6237-6239 IN denotes of
T2576 6240-6245 NN denotes HSP27
T2577 6246-6248 IN denotes on
T2578 6249-6252 NN denotes S82
T2579 6253-6256 VB denotes was
T2580 6257-6266 VB denotes abolished
T2581 6267-6269 IN denotes in
T2582 6270-6271 NN denotes B
T2583 6272-6277 NN denotes cells
T2584 6278-6282 WDT denotes that
T2585 6283-6289 VB denotes lacked
T2586 6290-6294 CC denotes both
T2587 6295-6299 NN denotes PKD1
T2588 6300-6303 CC denotes and
T2589 6304-6308 NN denotes PKD3
T2590 6309-6310 -LRB- denotes (
T2591 6310-6314 NN denotes Fig.
T2592 6315-6317 NN denotes 1C
T2593 6317-6318 -RRB- denotes )
T2594 6320-6333 RB denotes Significantly
T2595 6333-6334 -COMMA- denotes ,
T2596 6335-6354 JJ denotes doxycycline-induced
T2597 6355-6365 NN denotes expression
T2598 6366-6368 IN denotes of
T2599 6369-6372 DT denotes the
T2600 6373-6382 NN denotes Flag-PKD3
T2601 6383-6392 NN denotes transgene
T2602 6393-6395 IN denotes in
T2603 6396-6399 DT denotes the
T2604 6400-6406 JJ denotes double
T2605 6407-6415 NN denotes knockout
T2606 6416-6421 NN denotes cells
T2607 6422-6425 VB denotes was
T2608 6426-6436 JJ denotes sufficient
T2609 6437-6439 TO denotes to
T2610 6440-6447 VB denotes restore
T2611 6448-6454 JJ denotes normal
T2612 6455-6465 NN denotes regulation
T2613 6466-6468 IN denotes of
T2614 6469-6474 NN denotes HSP27
T2615 6475-6490 NN denotes phosphorylation
T2616 6491-6492 -LRB- denotes (
T2617 6492-6496 NN denotes Fig.
T2618 6497-6499 NN denotes 1C
T2619 6499-6500 -RRB- denotes )
T2620 6502-6504 IN denotes In
T2621 6505-6513 NN denotes contrast
T2622 6513-6514 -COMMA- denotes ,
T2623 6515-6525 NN denotes expression
T2624 6526-6528 IN denotes of
T2625 6529-6530 DT denotes a
T2626 6531-6547 JJ denotes kinase-deficient
T2627 6548-6552 NN denotes PKD3
T2628 6553-6559 JJ denotes mutant
T2629 6560-6567 NN denotes protein
T2630 6568-6570 IN denotes in
T2631 6571-6574 DT denotes the
T2632 6575-6581 JJ denotes double
T2633 6582-6590 NN denotes knockout
T2634 6591-6596 NN denotes cells
T2635 6597-6600 VB denotes was
T2636 6601-6604 RB denotes not
T2637 6605-6609 JJ denotes able
T2638 6610-6612 TO denotes to
T2639 6613-6620 VB denotes restore
T2640 6621-6625 NN denotes BCR-
T2641 6626-6628 CC denotes or
T2642 6629-6636 NN denotes phorbol
T2643 6637-6650 JJ denotes ester-induced
T2644 6651-6656 NN denotes HSP27
T2645 6657-6672 NN denotes phosphorylation
T2646 6673-6674 -LRB- denotes (
T2647 6674-6678 NN denotes Fig.
T2648 6679-6681 NN denotes 1D
T2649 6681-6682 -RRB- denotes )
T2650 6684-6689 RB denotes Hence
T2651 6689-6690 -COMMA- denotes ,
T2652 6691-6695 NN denotes PKD3
T2653 6696-6698 RB denotes as
T2654 6699-6703 RB denotes well
T2655 6704-6706 IN denotes as
T2656 6707-6711 NN denotes PKD1
T2657 6712-6715 MD denotes can
T2658 6716-6724 VB denotes regulate
T2659 6725-6730 NN denotes HSP27
T2660 6731-6746 NN denotes phosphorylation
T2661 6747-6750 CC denotes and
T2662 6751-6753 IN denotes in
T2663 6754-6758 NN denotes DT40
T2664 6759-6760 NN denotes B
T2665 6761-6766 NN denotes cells
T2666 6767-6771 PRP denotes they
T2667 6772-6775 VB denotes are
T2668 6776-6788 RB denotes functionally
T2669 6789-6798 JJ denotes redundant
T2670 6799-6801 IN denotes as
T2671 6802-6807 NN denotes HSP27
T2672 6808-6815 NN denotes kinases
T3836 0-6831 JJ denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular
T3837 6832-6845 NN denotes proliferation
T3838 6846-6849 CC denotes and
T3839 6850-6858 NN denotes survival
T3840 6859-6861 IN denotes in
T3841 6862-6866 NN denotes DT40
T3842 6867-6868 NN denotes B
T3843 6869-6874 NN denotes cells
T3844 6875-6882 VB denotes lacking
T3845 6883-6893 NN denotes expression
T3846 6894-6896 IN denotes of
T3847 6897-6900 NN denotes PKD
T3848 6901-6907 NN denotes family
T3849 6908-6915 NN denotes kinases
T3850 6916-6919 NN denotes PKD
T3851 6920-6927 NN denotes enzymes
T3852 6928-6932 VB denotes have
T3853 6933-6943 RB denotes previously
T3854 6944-6948 VB denotes been
T3855 6949-6955 VB denotes linked
T3856 6956-6958 TO denotes to
T3857 6959-6962 DT denotes the
T3858 6963-6973 NN denotes regulation
T3859 6974-6976 IN denotes of
T3860 6977-6981 NN denotes cell
T3861 6982-6995 NN denotes proliferation
T3862 6996-6999 CC denotes and
T3863 7000-7008 NN denotes survival
T3864 7009-7010 -LRB- denotes (
T3865 7010-7018 VB denotes reviewed
T3866 7019-7021 IN denotes in
T3867 7022-7023 -LRB- denotes [
T3868 7023-7025 CD denotes 20
T3869 7025-7026 -RRB- denotes ]
T3870 7026-7027 -RRB- denotes )
T3871 7029-7031 TO denotes To
T3872 7032-7043 VB denotes investigate
T3873 7044-7047 DT denotes the
T3874 7048-7054 NN denotes effect
T3875 7055-7059 IN denotes that
T3876 7060-7064 NN denotes loss
T3877 7065-7067 IN denotes of
T3878 7068-7071 NN denotes PKD
T3879 7072-7079 NN denotes kinases
T3880 7080-7083 VB denotes had
T3881 7084-7086 IN denotes on
T3882 7087-7088 NN denotes B
T3883 7089-7093 NN denotes cell
T3884 7094-7102 NN denotes survival
T3885 7103-7109 CC denotes and/or
T3886 7110-7123 NN denotes proliferation
T3887 7124-7126 PRP denotes we
T3888 7127-7135 VB denotes cultured
T3889 7136-7145 JJ denotes wild-type
T3890 7146-7149 CC denotes and
T3891 7150-7158 JJ denotes PKD-null
T3892 7159-7164 NN denotes cells
T3893 7165-7167 IN denotes in
T3894 7168-7171 DT denotes the
T3895 7172-7180 NN denotes presence
T3896 7181-7182 -LRB- denotes (
T3897 7182-7191 NN denotes PKD1/3−/−
T3898 7191-7192 -COLON- denotes :
T3899 7193-7205 NN denotes Flag-PKD3+ve
T3900 7205-7206 -RRB- denotes )
T3901 7207-7209 CC denotes or
T3902 7210-7217 NN denotes absence
T3903 7218-7219 -LRB- denotes (
T3904 7219-7228 NN denotes PKD1/3−/−
T3905 7228-7229 -RRB- denotes )
T3906 7230-7232 IN denotes of
T3907 7233-7244 NN denotes doxycycline
T3908 7245-7248 CC denotes and
T3909 7249-7258 VB denotes monitored
T3910 7259-7270 JJ denotes exponential
T3911 7271-7277 NN denotes growth
T3912 7279-7281 IN denotes As
T3913 7282-7287 VB denotes shown
T3914 7288-7290 IN denotes in
T3915 7291-7295 NNP denotes Fig.
T3916 7296-7298 NN denotes 2A
T3917 7298-7299 -COMMA- denotes ,
T3918 7300-7309 NN denotes PKD1/3−/−
T3919 7310-7315 NN denotes cells
T3920 7316-7328 VB denotes proliferated
T3921 7329-7342 RB denotes exponentially
T3922 7343-7346 CC denotes and
T3923 7347-7360 NN denotes re-expression
T3924 7361-7363 IN denotes of
T3925 7364-7373 NN denotes Flag-PKD3
T3926 7374-7376 IN denotes in
T3927 7377-7382 DT denotes these
T3928 7383-7388 NN denotes cells
T3929 7389-7392 VB denotes had
T3930 7393-7395 DT denotes no
T3931 7396-7402 NN denotes impact
T3932 7403-7405 IN denotes on
T3933 7406-7409 DT denotes the
T3934 7410-7414 NN denotes rate
T3935 7415-7417 IN denotes of
T3936 7418-7431 NN denotes proliferation
T3937 7433-7444 RB denotes Furthermore
T3938 7444-7445 -COMMA- denotes ,
T3939 7446-7449 DT denotes the
T3940 7450-7459 NN denotes viability
T3941 7460-7462 IN denotes of
T3942 7463-7472 NN denotes PKD1/3−/−
T3943 7473-7474 NN denotes B
T3944 7475-7480 NN denotes cells
T3945 7481-7487 IN denotes during
T3946 7488-7495 JJ denotes routine
T3947 7496-7505 NN denotes culturing
T3948 7506-7509 VB denotes was
T3949 7510-7513 RB denotes not
T3950 7514-7527 RB denotes significantly
T3951 7528-7537 JJ denotes different
T3952 7538-7542 IN denotes from
T3953 7543-7547 DT denotes that
T3954 7548-7550 IN denotes of
T3955 7551-7560 JJ denotes wild-type
T3956 7561-7562 NN denotes B
T3957 7563-7568 NN denotes cells
T3958 7569-7570 -LRB- denotes (
T3959 7570-7574 NN denotes data
T3960 7575-7578 RB denotes not
T3961 7579-7584 VB denotes shown
T3962 7584-7585 -RRB- denotes )
T3963 7587-7589 PRP denotes It
T3964 7590-7593 VB denotes was
T3965 7594-7599 VB denotes noted
T3966 7600-7604 IN denotes that
T3967 7605-7608 DT denotes the
T3968 7609-7619 NN denotes population
T3969 7620-7628 NN denotes doubling
T3970 7629-7633 NN denotes time
T3971 7634-7636 IN denotes of
T3972 7637-7646 NN denotes PKD1/3−/−
T3973 7647-7652 NN denotes cells
T3974 7653-7656 VB denotes was
T3975 7657-7665 RB denotes slightly
T3976 7666-7672 JJ denotes slower
T3977 7673-7677 IN denotes than
T3978 7678-7682 DT denotes that
T3979 7683-7685 IN denotes of
T3980 7686-7690 JJ denotes wild
T3981 7691-7695 NN denotes type
T3982 7696-7700 NN denotes DT40
T3983 7701-7706 NN denotes cells
T3984 7707-7708 -LRB- denotes (
T3985 7708-7720 CD denotes 12.7 ± 2.8 h
T3986 7721-7727 IN denotes versus
T3987 7728-7740 CD denotes 10.2 ± 0.4 h
T3988 7740-7741 -RRB- denotes )
T3989 7742-7745 CC denotes but
T3990 7746-7749 DT denotes the
T3991 7750-7757 NN denotes failure
T3992 7758-7760 IN denotes of
T3993 7761-7765 NN denotes PKD3
T3994 7766-7779 NN denotes re-expression
T3995 7780-7782 TO denotes to
T3996 7783-7789 VB denotes modify
T3997 7790-7793 DT denotes the
T3998 7794-7807 NN denotes proliferation
T3999 7808-7812 NN denotes rate
T4000 7813-7815 IN denotes of
T4001 7816-7825 NN denotes PKD1/3−/−
T4002 7826-7831 NN denotes cells
T4003 7832-7840 VB denotes suggests
T4004 7841-7845 IN denotes that
T4005 7846-7851 DT denotes these
T4006 7852-7857 JJ denotes small
T4007 7858-7869 NN denotes differences
T4008 7870-7874 VB denotes were
T4009 7875-7879 RB denotes most
T4010 7880-7886 RB denotes likely
T4011 7887-7890 DT denotes the
T4012 7891-7897 NN denotes result
T4013 7898-7900 IN denotes of
T4014 7901-7907 JJ denotes clonal
T4015 7908-7917 NN denotes variation
T4016 7918-7921 CC denotes and
T4017 7922-7926 VB denotes were
T4018 7927-7930 RB denotes not
T4019 7931-7937 VB denotes caused
T4020 7938-7950 RB denotes specifically
T4021 7951-7953 IN denotes by
T4022 7954-7958 NN denotes loss
T4023 7959-7961 IN denotes of
T4024 7962-7965 NN denotes PKD
T4025 7966-7973 NN denotes enzymes
T4026 7975-7979 RB denotes Thus
T4027 7979-7980 -COMMA- denotes ,
T4028 7981-7984 NN denotes PKD
T4029 7985-7991 NN denotes family
T4030 7992-7999 NN denotes enzymes
T4031 8000-8003 VB denotes are
T4032 8004-8007 RB denotes not
T4033 8008-8017 JJ denotes essential
T4034 8018-8021 IN denotes for
T4035 8022-8032 VB denotes regulating
T4036 8033-8038 JJ denotes basal
T4037 8039-8047 NN denotes survival
T4038 8048-8051 CC denotes and
T4039 8052-8065 NN denotes proliferation
T4040 8066-8068 IN denotes of
T4041 8069-8073 NN denotes DT40
T4042 8074-8075 NN denotes B
T4043 8076-8081 NN denotes cells
T4044 8083-8086 NN denotes PKD
T4045 8087-8094 NN denotes enzymes
T4046 8094-8095 -COMMA- denotes ,
T4047 8096-8108 RB denotes specifically
T4048 8109-8113 NN denotes PKD1
T4049 8114-8117 CC denotes and
T4050 8118-8122 NN denotes PKD2
T4051 8122-8123 -COMMA- denotes ,
T4052 8124-8128 VB denotes have
T4053 8129-8139 RB denotes previously
T4054 8140-8144 VB denotes been
T4055 8145-8151 VB denotes linked
T4056 8152-8154 TO denotes to
T4057 8155-8156 DT denotes a
T4058 8157-8167 JJ denotes protective
T4059 8168-8172 NN denotes role
T4060 8173-8180 IN denotes against
T4061 8181-8190 JJ denotes oxidative
T4062 8191-8205 JJ denotes stress-induced
T4063 8206-8212 NN denotes injury
T4064 8213-8215 IN denotes in
T4065 8216-8219 NN denotes 3T3
T4066 8220-8230 NN denotes fibroblast
T4067 8230-8231 -COMMA- denotes ,
T4068 8232-8236 NN denotes HeLa
T4069 8237-8240 CC denotes and
T4070 8241-8251 JJ denotes epithelial
T4071 8252-8256 NN denotes cell
T4072 8257-8262 NN denotes lines
T4073 8263-8264 -LRB- denotes [
T4074 8264-8272 CD denotes 17,30–32
T4075 8272-8273 -RRB- denotes ]
T4076 8275-8277 PRP denotes We
T4077 8278-8287 RB denotes therefore
T4078 8288-8297 VB denotes addressed
T4079 8298-8301 DT denotes the
T4080 8302-8306 NN denotes role
T4081 8307-8309 IN denotes of
T4082 8310-8313 NN denotes PKD
T4083 8314-8320 NN denotes family
T4084 8321-8328 NN denotes kinases
T4085 8329-8331 IN denotes in
T4086 8332-8342 VB denotes regulating
T4087 8343-8344 NN denotes B
T4088 8345-8349 NN denotes cell
T4089 8350-8358 NN denotes survival
T4090 8359-8361 IN denotes in
T4091 8362-8370 NN denotes response
T4092 8371-8373 TO denotes to
T4093 8374-8383 JJ denotes oxidative
T4094 8384-8390 NN denotes stress
T4095 8391-8394 CC denotes and
T4096 8395-8400 JJ denotes other
T4097 8401-8407 NN denotes stress
T4098 8408-8415 NN denotes stimuli
T4099 8417-8419 IN denotes As
T4100 8420-8425 VB denotes shown
T4101 8426-8428 IN denotes in
T4102 8429-8433 NNP denotes Fig.
T4103 8434-8436 NN denotes 2B
T4104 8436-8437 -COMMA- denotes ,
T4105 8438-8442 NN denotes loss
T4106 8443-8445 IN denotes of
T4107 8446-8452 NN denotes PKD1/3
T4108 8453-8463 NN denotes expression
T4109 8464-8467 VB denotes had
T4110 8468-8470 DT denotes no
T4111 8471-8482 JJ denotes significant
T4112 8483-8489 NN denotes impact
T4113 8490-8492 IN denotes on
T4114 8493-8496 DT denotes the
T4115 8497-8505 NN denotes survival
T4116 8506-8508 IN denotes of
T4117 8509-8513 NN denotes DT40
T4118 8514-8515 NN denotes B
T4119 8516-8521 NN denotes cells
T4120 8522-8524 IN denotes in
T4121 8525-8533 NN denotes response
T4122 8534-8536 TO denotes to
T4123 8537-8550 JJ denotes mitochondrial
T4124 8551-8557 NN denotes stress
T4125 8558-8565 NN denotes stimuli
T4126 8566-8567 -LRB- denotes (
T4127 8567-8571 NN denotes H2O2
T4128 8572-8574 CC denotes or
T4129 8575-8580 NN denotes serum
T4130 8581-8592 NN denotes deprivation
T4131 8592-8593 -RRB- denotes )
T4132 8593-8594 -COLON- denotes ;
T4133 8595-8598 NN denotes DNA
T4134 8599-8607 NN denotes damaging
T4135 8608-8614 NN denotes agents
T4136 8615-8616 -LRB- denotes (
T4137 8616-8625 NN denotes etoposide
T4138 8626-8628 CC denotes or
T4139 8629-8640 NN denotes doxorubicin
T4140 8640-8641 -RRB- denotes )
T4141 8641-8642 -COLON- denotes ;
T4142 8643-8645 NN denotes ER
T4143 8646-8653 NN denotes pathway
T4144 8654-8660 NN denotes stress
T4145 8661-8664 IN denotes due
T4146 8665-8667 TO denotes to
T4147 8668-8675 NN denotes calcium
T4148 8676-8684 NN denotes overload
T4149 8685-8686 -LRB- denotes (
T4150 8686-8698 NN denotes thapsigargin
T4151 8698-8699 -RRB- denotes )
T4152 8700-8702 CC denotes or
T4153 8703-8712 VB denotes following
T4154 8713-8722 VB denotes prolonged
T4155 8723-8732 NN denotes treatment
T4156 8733-8737 IN denotes with
T4157 8738-8745 NN denotes phorbol
T4158 8746-8752 NN denotes esters
T4159 8753-8755 CC denotes or
T4160 8756-8768 NN denotes Trichostatin
T4161 8769-8770 NN denotes A
T4162 8770-8771 -COMMA- denotes ,
T4163 8772-8774 DT denotes an
T4164 8775-8784 NN denotes inhibitor
T4165 8785-8787 IN denotes of
T4166 8788-8793 NN denotes class
T4167 8794-8798 NN denotes I/II
T4168 8799-8804 NN denotes HDACs
T4169 8806-8810 RB denotes Thus
T4170 8810-8811 -COMMA- denotes ,
T4171 8812-8815 NN denotes PKD
T4172 8816-8823 NN denotes kinases
T4173 8824-8826 VB denotes do
T4174 8827-8830 RB denotes not
T4175 8831-8835 VB denotes play
T4176 8836-8838 DT denotes an
T4177 8839-8848 JJ denotes essential
T4178 8849-8853 NN denotes role
T4179 8854-8856 IN denotes in
T4180 8857-8867 VB denotes regulating
T4181 8868-8869 NN denotes B
T4182 8870-8874 NN denotes cell
T4183 8875-8883 NN denotes survival
T4184 8884-8886 IN denotes in
T4185 8887-8895 NN denotes response
T4186 8896-8898 TO denotes to
T4187 8899-8900 DT denotes a
T4188 8901-8906 NN denotes range
T4189 8907-8909 IN denotes of
T4190 8910-8919 JJ denotes different
T4191 8920-8926 NN denotes stress
T4192 8927-8934 NN denotes stimuli
T5031 0-8949 NN denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen
T5032 8950-8958 NN denotes receptor
T5033 8959-8968 VB denotes regulated
T5034 8969-8979 NN denotes signalling
T5035 8980-8988 NN denotes pathways
T5036 8989-8991 IN denotes in
T5037 8992-9000 JJ denotes PKD-null
T5038 9001-9005 NN denotes DT40
T5039 9006-9007 NN denotes B
T5040 9008-9013 NN denotes cells
T5041 9014-9016 TO denotes To
T5042 9017-9024 RB denotes further
T5043 9025-9032 VB denotes explore
T5044 9033-9036 DT denotes the
T5045 9037-9049 NN denotes contribution
T5046 9050-9052 IN denotes of
T5047 9053-9056 NN denotes PKD
T5048 9057-9064 NN denotes kinases
T5049 9065-9067 TO denotes to
T5050 9068-9072 NN denotes DT40
T5051 9073-9074 NN denotes B
T5052 9075-9079 NN denotes cell
T5053 9080-9087 NN denotes biology
T5054 9088-9090 PRP denotes we
T5055 9091-9103 VB denotes investigated
T5056 9104-9111 IN denotes whether
T5057 9112-9120 JJ denotes specific
T5058 9121-9134 JJ denotes BCR-regulated
T5059 9135-9145 NN denotes signalling
T5060 9146-9152 NN denotes events
T5061 9153-9157 VB denotes were
T5062 9158-9167 JJ denotes defective
T5063 9168-9170 IN denotes in
T5064 9171-9174 DT denotes the
T5065 9175-9183 JJ denotes PKD-null
T5066 9184-9185 NN denotes B
T5067 9186-9191 NN denotes cells
T5068 9193-9200 JJ denotes Initial
T5069 9201-9212 NN denotes experiments
T5070 9213-9221 VB denotes revealed
T5071 9222-9226 IN denotes that
T5072 9227-9234 NN denotes surface
T5073 9235-9245 NN denotes expression
T5074 9246-9248 IN denotes of
T5075 9249-9252 DT denotes the
T5076 9253-9256 NN denotes BCR
T5077 9257-9260 VB denotes was
T5078 9261-9268 VB denotes reduced
T5079 9269-9271 IN denotes in
T5080 9272-9281 NN denotes PKD1/3−/−
T5081 9282-9283 -LRB- denotes (
T5082 9283-9286 CC denotes and
T5083 9287-9289 IN denotes in
T5084 9290-9299 NN denotes PKD1/3−/−
T5085 9299-9300 -COLON- denotes :
T5086 9300-9312 NN denotes Flag-PKD3+ve
T5087 9312-9313 -RRB- denotes )
T5088 9314-9319 NN denotes cells
T5089 9320-9328 VB denotes compared
T5090 9329-9331 TO denotes to
T5091 9332-9341 JJ denotes wild-type
T5092 9342-9346 NN denotes DT40
T5093 9347-9348 NN denotes B
T5094 9349-9354 NN denotes cells
T5095 9355-9356 -LRB- denotes (
T5096 9356-9360 NN denotes Fig.
T5097 9361-9363 NN denotes 3A
T5098 9364-9367 CC denotes and
T5099 9368-9372 NN denotes data
T5100 9373-9376 RB denotes not
T5101 9377-9382 VB denotes shown
T5102 9382-9383 -RRB- denotes )
T5103 9385-9397 RB denotes Nevertheless
T5104 9397-9398 -COMMA- denotes ,
T5105 9399-9415 NN denotes BCR-crosslinking
T5106 9416-9418 IN denotes of
T5107 9419-9428 NN denotes PKD1/3−/−
T5108 9429-9434 NN denotes cells
T5109 9435-9438 VB denotes was
T5110 9439-9449 JJ denotes sufficient
T5111 9450-9452 TO denotes to
T5112 9453-9459 VB denotes induce
T5113 9460-9463 DT denotes the
T5114 9464-9474 NN denotes activation
T5115 9475-9477 IN denotes of
T5116 9478-9479 DT denotes a
T5117 9480-9486 NN denotes number
T5118 9487-9489 IN denotes of
T5119 9490-9500 NN denotes signalling
T5120 9501-9509 NN denotes cascades
T5121 9509-9510 -COMMA- denotes ,
T5122 9511-9518 JJ denotes similar
T5123 9519-9521 TO denotes to
T5124 9522-9526 DT denotes that
T5125 9527-9535 VB denotes observed
T5126 9536-9538 IN denotes in
T5127 9539-9548 JJ denotes wild-type
T5128 9549-9554 NN denotes cells
T5129 9555-9556 -LRB- denotes (
T5130 9556-9560 NN denotes Fig.
T5131 9561-9563 NN denotes 3B
T5132 9563-9564 -RRB- denotes )
T5133 9566-9571 RB denotes Hence
T5134 9571-9572 -COMMA- denotes ,
T5135 9573-9584 JJ denotes BCR-induced
T5136 9585-9595 NN denotes activation
T5137 9596-9598 IN denotes of
T5138 9599-9602 DT denotes the
T5139 9603-9606 NN denotes Akt
T5140 9606-9607 -COMMA- denotes ,
T5141 9608-9616 NN denotes mTOR/p70
T5142 9617-9619 NN denotes S6
T5143 9620-9626 NN denotes kinase
T5144 9627-9628 -LRB- denotes (
T5145 9628-9630 IN denotes as
T5146 9631-9636 VB denotes shown
T5147 9637-9639 IN denotes by
T5148 9640-9642 NN denotes S6
T5149 9643-9652 JJ denotes ribosomal
T5150 9653-9660 NN denotes protein
T5151 9661-9676 NN denotes phosphorylation
T5152 9676-9677 -RRB- denotes )
T5153 9678-9681 CC denotes and
T5154 9682-9686 NN denotes MAPK
T5155 9687-9697 NN denotes signalling
T5156 9698-9706 NN denotes pathways
T5157 9707-9710 VB denotes was
T5158 9711-9718 RB denotes clearly
T5159 9719-9729 JJ denotes detectable
T5160 9730-9732 IN denotes in
T5161 9733-9744 JJ denotes PKD1/3-null
T5162 9745-9746 NN denotes B
T5163 9747-9752 NN denotes cells
T5164 9753-9754 -LRB- denotes (
T5165 9754-9758 NN denotes Fig.
T5166 9759-9761 NN denotes 3B
T5167 9761-9762 -RRB- denotes )
T5168 9764-9775 RB denotes Furthermore
T5169 9775-9776 -COMMA- denotes ,
T5170 9777-9785 VB denotes enhanced
T5171 9786-9794 NN denotes tyrosine
T5172 9795-9810 NN denotes phosphorylation
T5173 9811-9813 IN denotes of
T5174 9814-9822 JJ denotes multiple
T5175 9823-9831 JJ denotes cellular
T5176 9832-9840 NN denotes proteins
T5177 9841-9843 RB denotes as
T5178 9844-9848 RB denotes well
T5179 9849-9851 IN denotes as
T5180 9852-9854 DT denotes an
T5181 9855-9863 NN denotes increase
T5182 9864-9866 IN denotes in
T5183 9867-9880 JJ denotes intracellular
T5184 9881-9888 NN denotes calcium
T5185 9889-9895 NN denotes levels
T5186 9896-9899 VB denotes was
T5187 9900-9904 RB denotes also
T5188 9905-9913 VB denotes observed
T5189 9914-9923 VB denotes following
T5190 9924-9927 NN denotes BCR
T5191 9928-9939 NN denotes stimulation
T5192 9940-9942 IN denotes of
T5193 9943-9954 JJ denotes PKD1/3-null
T5194 9955-9956 NN denotes B
T5195 9957-9962 NN denotes cells
T5196 9963-9964 -LRB- denotes (
T5197 9964-9968 NN denotes data
T5198 9969-9972 RB denotes not
T5199 9973-9978 VB denotes shown
T5200 9978-9979 -RRB- denotes )
T5201 9981-9983 PRP denotes We
T5202 9984-9987 VB denotes did
T5203 9988-9995 VB denotes observe
T5204 9996-10000 IN denotes that
T5205 10001-10004 DT denotes the
T5206 10005-10013 NN denotes strength
T5207 10014-10016 IN denotes of
T5208 10017-10020 NN denotes BCR
T5209 10021-10022 -LRB- denotes (
T5210 10022-10025 CC denotes but
T5211 10026-10029 RB denotes not
T5212 10030-10037 NN denotes phorbol
T5213 10038-10043 NN denotes ester
T5214 10043-10044 -RRB- denotes )
T5215 10044-10052 JJ denotes -induced
T5216 10053-10063 NN denotes regulation
T5217 10064-10066 IN denotes of
T5218 10067-10070 DT denotes the
T5219 10071-10080 NN denotes Erk1-RSK1
T5220 10081-10091 NN denotes signalling
T5221 10092-10099 NN denotes pathway
T5222 10100-10103 VB denotes was
T5223 10104-10111 VB denotes reduced
T5224 10112-10114 IN denotes in
T5225 10115-10124 NN denotes PKD1/3−/−
T5226 10125-10126 NN denotes B
T5227 10127-10132 NN denotes cells
T5228 10133-10141 VB denotes compared
T5229 10142-10144 TO denotes to
T5230 10145-10154 JJ denotes wild-type
T5231 10155-10156 NN denotes B
T5232 10157-10162 NN denotes cells
T5233 10163-10164 -LRB- denotes (
T5234 10164-10168 NN denotes Fig.
T5235 10169-10171 NN denotes 3B
T5236 10171-10172 -RRB- denotes )
T5237 10174-10177 CD denotes One
T5238 10178-10192 NN denotes interpretation
T5239 10193-10195 IN denotes of
T5240 10196-10200 DT denotes this
T5241 10201-10205 NN denotes data
T5242 10206-10208 VB denotes is
T5243 10209-10213 IN denotes that
T5244 10214-10217 NN denotes PKD
T5245 10218-10225 NN denotes enzymes
T5246 10226-10229 MD denotes may
T5247 10230-10238 VB denotes modulate
T5248 10239-10242 NN denotes Erk
T5249 10243-10253 NN denotes activation
T5250 10255-10261 RB denotes Indeed
T5251 10261-10262 -COMMA- denotes ,
T5252 10263-10266 NN denotes PKD
T5253 10267-10274 NN denotes enzymes
T5254 10275-10279 VB denotes have
T5255 10280-10290 RB denotes previously
T5256 10291-10295 VB denotes been
T5257 10296-10302 VB denotes linked
T5258 10303-10305 TO denotes to
T5259 10306-10309 DT denotes the
T5260 10310-10316 NN denotes growth
T5261 10317-10333 JJ denotes factor-regulated
T5262 10334-10337 NN denotes Erk
T5263 10338-10348 NN denotes signalling
T5264 10349-10351 IN denotes in
T5265 10352-10362 NN denotes fibroblast
T5266 10363-10366 CC denotes and
T5267 10367-10378 JJ denotes endothelial
T5268 10379-10383 NN denotes cell
T5269 10384-10389 NN denotes lines
T5270 10390-10391 -LRB- denotes [
T5271 10391-10396 CD denotes 33–35
T5272 10396-10397 -RRB- denotes ]
T5273 10399-10406 RB denotes However
T5274 10406-10407 -COMMA- denotes ,
T5275 10408-10419 JJ denotes BCR-induced
T5276 10420-10423 NN denotes Erk
T5277 10424-10439 NN denotes phosphorylation
T5278 10440-10443 VB denotes was
T5279 10444-10448 RB denotes also
T5280 10449-10456 VB denotes reduced
T5281 10457-10459 IN denotes in
T5282 10460-10480 JJ denotes PKD1/3−/−-Flag-PKD3+
T5283 10481-10482 NN denotes B
T5284 10483-10488 NN denotes cells
T5285 10489-10490 -LRB- denotes (
T5286 10490-10494 NN denotes data
T5287 10495-10498 RB denotes not
T5288 10499-10504 VB denotes shown
T5289 10504-10505 -RRB- denotes )
T5290 10506-10516 VB denotes suggesting
T5291 10517-10521 IN denotes that
T5292 10522-10529 VB denotes reduced
T5293 10530-10533 NN denotes BCR
T5294 10534-10540 NN denotes levels
T5295 10541-10543 IN denotes on
T5296 10544-10547 DT denotes the
T5297 10548-10555 NN denotes surface
T5298 10556-10558 IN denotes of
T5299 10559-10568 NN denotes PKD1/3−/−
T5300 10569-10570 -LRB- denotes (
T5301 10570-10573 CC denotes and
T5302 10574-10594 JJ denotes PKD1/3−/−-Flag-PKD3+
T5303 10594-10595 -RRB- denotes )
T5304 10596-10597 NN denotes B
T5305 10598-10603 NN denotes cells
T5306 10604-10607 MD denotes may
T5307 10608-10614 PRP denotes itself
T5308 10615-10621 VB denotes impact
T5309 10622-10624 IN denotes on
T5310 10625-10628 DT denotes the
T5311 10629-10637 NN denotes strength
T5312 10638-10640 IN denotes of
T5313 10641-10651 NN denotes activation
T5314 10652-10654 IN denotes of
T5315 10655-10659 DT denotes this
T5316 10660-10668 JJ denotes specific
T5317 10669-10682 JJ denotes intracellular
T5318 10683-10693 NN denotes signalling
T5319 10694-10701 NN denotes pathway
T5320 10703-10705 TO denotes To
T5321 10706-10712 VB denotes search
T5322 10713-10716 IN denotes for
T5323 10717-10722 JJ denotes other
T5324 10723-10732 JJ denotes potential
T5325 10733-10736 NN denotes PKD
T5326 10737-10744 NN denotes targets
T5327 10745-10749 WDT denotes that
T5328 10750-10753 MD denotes may
T5329 10754-10758 VB denotes show
T5330 10759-10768 JJ denotes defective
T5331 10769-10779 NN denotes regulation
T5332 10780-10782 IN denotes in
T5333 10783-10792 NN denotes PKD1/3−/−
T5334 10793-10797 NN denotes DT40
T5335 10798-10799 NN denotes B
T5336 10800-10805 NN denotes cells
T5337 10805-10806 -COMMA- denotes ,
T5338 10807-10809 PRP denotes we
T5339 10810-10814 VB denotes used
T5340 10815-10816 DT denotes a
T5341 10817-10820 NN denotes PKD
T5342 10821-10830 NN denotes substrate
T5343 10831-10847 NN denotes phospho-antibody
T5344 10848-10852 WDT denotes that
T5345 10853-10863 VB denotes recognises
T5346 10864-10873 NN denotes consensus
T5347 10874-10889 NN denotes phosphorylation
T5348 10890-10899 NN denotes sequences
T5349 10900-10908 VB denotes targeted
T5350 10909-10911 IN denotes by
T5351 10912-10915 NN denotes PKD
T5352 10916-10923 NN denotes enzymes
T5353 10924-10925 -LRB- denotes (
T5354 10925-10934 NN denotes LxRxxpS/T
T5355 10934-10935 -RRB- denotes )
T5356 10936-10937 -LRB- denotes [
T5357 10937-10939 CD denotes 36
T5358 10939-10940 -RRB- denotes ]
T5359 10942-10944 IN denotes As
T5360 10945-10950 VB denotes shown
T5361 10951-10953 IN denotes in
T5362 10954-10958 NNP denotes Fig.
T5363 10959-10961 NNP denotes 3C
T5364 10961-10962 -COMMA- denotes ,
T5365 10963-10970 NN denotes phorbol
T5366 10971-10977 NN denotes ester-
T5367 10978-10981 CC denotes and
T5368 10982-10993 JJ denotes BCR-induced
T5369 10994-11009 NN denotes phosphorylation
T5370 11010-11012 IN denotes of
T5371 11013-11021 JJ denotes cellular
T5372 11022-11032 NN denotes substrates
T5373 11033-11041 VB denotes detected
T5374 11042-11044 IN denotes by
T5375 11045-11049 DT denotes this
T5376 11050-11066 NN denotes phospho-antibody
T5377 11067-11070 VB denotes was
T5378 11071-11078 JJ denotes similar
T5379 11079-11081 IN denotes in
T5380 11082-11091 JJ denotes wild-type
T5381 11092-11095 CC denotes and
T5382 11096-11105 NN denotes PKD1/3−/−
T5383 11106-11111 NN denotes cells
T5384 11112-11115 CC denotes and
T5385 11116-11118 VB denotes is
T5386 11119-11128 RB denotes therefore
T5387 11129-11140 JJ denotes independent
T5388 11141-11143 IN denotes of
T5389 11144-11147 NN denotes PKD
T5390 11148-11155 NN denotes enzymes
T5391 11157-11164 RB denotes However
T5392 11164-11165 -COMMA- denotes ,
T5393 11166-11178 NN denotes pretreatment
T5394 11179-11181 IN denotes of
T5395 11182-11186 CC denotes both
T5396 11187-11196 JJ denotes wild-type
T5397 11197-11200 CC denotes and
T5398 11201-11210 NN denotes PKD1/3−/−
T5399 11211-11215 NN denotes DT40
T5400 11216-11217 NN denotes B
T5401 11218-11223 NN denotes cells
T5402 11224-11228 IN denotes with
T5403 11229-11238 NN denotes GF109203X
T5404 11238-11239 -COMMA- denotes ,
T5405 11240-11241 DT denotes a
T5406 11242-11260 NN denotes bisindoylmaleimide
T5407 11261-11271 NN denotes derivative
T5408 11272-11276 WDT denotes that
T5409 11277-11285 VB denotes inhibits
T5410 11286-11290 NN denotes PKCs
T5411 11291-11300 VB denotes prevented
T5412 11301-11304 DT denotes the
T5413 11305-11314 NN denotes induction
T5414 11315-11317 IN denotes of
T5415 11318-11326 NN denotes proteins
T5416 11327-11331 WDT denotes that
T5417 11332-11339 VB denotes contain
T5418 11340-11354 VB denotes phosphorylated
T5419 11355-11363 NN denotes LxRxxS/T
T5420 11364-11370 NN denotes motifs
T5421 11372-11376 RB denotes Thus
T5422 11377-11381 NN denotes loss
T5423 11382-11384 IN denotes of
T5424 11385-11391 NN denotes PKD1/3
T5425 11392-11399 NN denotes enzymes
T5426 11400-11404 VB denotes does
T5427 11405-11408 RB denotes not
T5428 11409-11417 RB denotes globally
T5429 11418-11425 VB denotes disrupt
T5430 11426-11429 DT denotes the
T5431 11430-11445 NN denotes phosphorylation
T5432 11446-11448 IN denotes of
T5433 11449-11457 JJ denotes cellular
T5434 11458-11466 NN denotes proteins
T5435 11467-11471 WDT denotes that
T5436 11472-11479 VB denotes contain
T5437 11480-11489 NN denotes LxRxxpS/T
T5438 11490-11496 NN denotes motifs
T5439 11498-11502 DT denotes This
T5440 11503-11509 NN denotes result
T5441 11510-11512 VB denotes is
T5442 11513-11520 RB denotes perhaps
T5443 11521-11524 RB denotes not
T5444 11525-11535 JJ denotes surprising
T5445 11536-11538 IN denotes as
T5446 11539-11547 NN denotes LxRxxS/T
T5447 11548-11554 NN denotes motifs
T5448 11555-11559 RB denotes also
T5449 11560-11563 VB denotes act
T5450 11564-11566 IN denotes as
T5451 11567-11571 JJ denotes good
T5452 11572-11582 NN denotes substrates
T5453 11583-11586 IN denotes for
T5454 11587-11592 JJ denotes other
T5455 11593-11609 NN denotes serine/threonine
T5456 11610-11617 NN denotes kinases
T5457 11618-11622 JJ denotes such
T5458 11623-11625 IN denotes as
T5459 11626-11634 NN denotes MAPKAPK2
T5460 11636-11643 RB denotes However
T5461 11644-11649 DT denotes these
T5462 11650-11661 NN denotes experiments
T5463 11662-11664 VB denotes do
T5464 11665-11672 VB denotes provide
T5465 11673-11680 JJ denotes further
T5466 11681-11689 NN denotes evidence
T5467 11690-11694 IN denotes that
T5468 11695-11710 JJ denotes phosphospecific
T5469 11711-11719 NN denotes antisera
T5470 11720-11723 VB denotes are
T5471 11724-11727 RB denotes not
T5472 11728-11740 RB denotes sufficiently
T5473 11741-11750 JJ denotes selective
T5474 11751-11753 TO denotes to
T5475 11754-11756 VB denotes be
T5476 11757-11767 VB denotes designated
T5477 11768-11774 NN denotes kinase
T5478 11775-11783 JJ denotes specific
T5479 11784-11793 NN denotes substrate
T5480 11794-11802 NN denotes antisera
T5481 11804-11815 JJ denotes BCR-induced
T5482 11816-11826 NN denotes signalling
T5483 11827-11835 NN denotes pathways
T5484 11836-11845 VB denotes culminate
T5485 11846-11848 IN denotes in
T5486 11849-11852 DT denotes the
T5487 11853-11863 NN denotes activation
T5488 11864-11866 IN denotes of
T5489 11867-11871 NN denotes gene
T5490 11872-11885 NN denotes transcription
T5491 11886-11892 NN denotes events
T5492 11893-11897 WDT denotes that
T5493 11898-11905 VB denotes control
T5494 11906-11907 NN denotes B
T5495 11908-11912 NN denotes cell
T5496 11913-11921 NN denotes survival
T5497 11921-11922 -COMMA- denotes ,
T5498 11923-11936 NN denotes proliferation
T5499 11937-11940 CC denotes and
T5500 11941-11949 NN denotes function
T5501 11951-11953 IN denotes In
T5502 11954-11958 DT denotes this
T5503 11959-11966 NN denotes context
T5504 11966-11967 -COMMA- denotes ,
T5505 11968-11970 PRP denotes it
T5506 11971-11974 VB denotes has
T5507 11975-11979 VB denotes been
T5508 11980-11988 VB denotes proposed
T5509 11989-11993 IN denotes that
T5510 11994-11997 NN denotes PKD
T5511 11998-12004 NN denotes family
T5512 12005-12012 NN denotes members
T5513 12013-12020 NN denotes control
T5514 12021-12023 IN denotes of
T5515 12024-12028 NN denotes gene
T5516 12029-12042 NN denotes transcription
T5517 12043-12050 IN denotes through
T5518 12051-12061 NN denotes activation
T5519 12062-12064 IN denotes of
T5520 12065-12068 DT denotes the
T5521 12069-12073 NN denotes NFκB
T5522 12074-12087 NN denotes transcription
T5523 12088-12094 NN denotes factor
T5524 12096-12100 RB denotes Thus
T5525 12100-12101 -COMMA- denotes ,
T5526 12102-12114 JJ denotes PKD-mediated
T5527 12115-12125 NN denotes activation
T5528 12126-12128 IN denotes of
T5529 12129-12133 NN denotes NFκB
T5530 12134-12140 VB denotes occurs
T5531 12141-12151 RB denotes downstream
T5532 12152-12154 IN denotes of
T5533 12155-12156 DT denotes a
T5534 12157-12164 NN denotes variety
T5535 12165-12167 IN denotes of
T5536 12168-12177 JJ denotes different
T5537 12178-12185 NN denotes signals
T5538 12185-12186 -COMMA- denotes ,
T5539 12187-12196 VB denotes including
T5540 12197-12211 JJ denotes mROS/oxidative
T5541 12212-12218 NN denotes stress
T5542 12218-12219 -COMMA- denotes ,
T5543 12220-12236 JJ denotes lysophosphatidic
T5544 12237-12241 NN denotes acid
T5545 12242-12245 CC denotes and
T5546 12246-12249 DT denotes the
T5547 12250-12257 NN denotes Bcr-Abl
T5548 12258-12266 NN denotes oncogene
T5549 12267-12268 -LRB- denotes [
T5550 12268-12282 CD denotes 17,21,23,30,37
T5551 12282-12283 -RRB- denotes ]
T5552 12285-12296 RB denotes Furthermore
T5553 12296-12297 -COMMA- denotes ,
T5554 12298-12308 NN denotes expression
T5555 12309-12311 IN denotes of
T5556 12312-12314 DT denotes an
T5557 12315-12324 VB denotes activated
T5558 12325-12329 NN denotes PKD1
T5559 12330-12336 NN denotes mutant
T5560 12337-12345 VB denotes enhances
T5561 12346-12359 JJ denotes HPK1-mediated
T5562 12360-12364 NN denotes NFκB
T5563 12365-12375 NN denotes activation
T5564 12376-12377 -LRB- denotes [
T5565 12377-12379 CD denotes 38
T5566 12379-12380 -RRB- denotes ]
T5567 12382-12384 IN denotes In
T5568 12385-12386 NN denotes B
T5569 12387-12392 NN denotes cells
T5570 12392-12393 -COMMA- denotes ,
T5571 12394-12398 NN denotes NFκB
T5572 12399-12401 VB denotes is
T5573 12402-12407 VB denotes known
T5574 12408-12410 TO denotes to
T5575 12411-12413 VB denotes be
T5576 12414-12423 VB denotes regulated
T5577 12424-12427 IN denotes via
T5578 12428-12431 NN denotes DAG
T5579 12432-12435 CC denotes and
T5580 12436-12440 NN denotes PKCβ
T5581 12441-12442 -LRB- denotes [
T5582 12442-12447 CD denotes 39,40
T5583 12447-12448 -RRB- denotes ]
T5584 12449-12452 CC denotes but
T5585 12453-12460 IN denotes whether
T5586 12461-12465 NN denotes PKDs
T5587 12466-12469 VB denotes are
T5588 12470-12473 JJ denotes key
T5589 12474-12488 NN denotes intermediaries
T5590 12489-12492 IN denotes for
T5591 12493-12497 NN denotes NFκB
T5592 12498-12508 NN denotes regulation
T5593 12509-12512 VB denotes has
T5594 12513-12516 RB denotes not
T5595 12517-12521 VB denotes been
T5596 12522-12530 VB denotes explored
T5597 12532-12535 DT denotes The
T5598 12536-12540 NN denotes data
T5599 12541-12542 -LRB- denotes (
T5600 12542-12546 NNP denotes Fig.
T5601 12547-12549 NN denotes 4A
T5602 12549-12550 -RRB- denotes )
T5603 12551-12555 VB denotes show
T5604 12556-12560 IN denotes that
T5605 12561-12565 NN denotes NFκB
T5606 12566-12581 JJ denotes transcriptional
T5607 12582-12590 NN denotes activity
T5608 12591-12594 VB denotes was
T5609 12595-12603 RB denotes strongly
T5610 12604-12611 VB denotes induced
T5611 12612-12614 IN denotes in
T5612 12615-12619 CC denotes both
T5613 12620-12629 JJ denotes wild-type
T5614 12630-12633 CC denotes and
T5615 12634-12643 NN denotes PKD1/3−/−
T5616 12644-12648 NN denotes DT40
T5617 12649-12650 NN denotes B
T5618 12651-12656 NN denotes cells
T5619 12657-12659 IN denotes in
T5620 12660-12668 NN denotes response
T5621 12669-12671 TO denotes to
T5622 12672-12678 CC denotes either
T5623 12679-12686 NN denotes phorbol
T5624 12687-12692 NN denotes ester
T5625 12693-12695 CC denotes or
T5626 12696-12699 NN denotes BCR
T5627 12700-12711 NN denotes stimulation
T5628 12713-12715 IN denotes In
T5629 12716-12724 NN denotes contrast
T5630 12724-12725 -COMMA- denotes ,
T5631 12726-12729 NN denotes BCR
T5632 12730-12733 CC denotes and
T5633 12734-12741 NN denotes phorbol
T5634 12742-12755 JJ denotes ester-induced
T5635 12756-12760 NN denotes NFκB
T5636 12761-12776 JJ denotes transcriptional
T5637 12777-12785 NN denotes activity
T5638 12786-12789 VB denotes was
T5639 12790-12799 VB denotes abolished
T5640 12800-12802 IN denotes in
T5641 12803-12810 NN denotes PKCβ−/−
T5642 12811-12815 NN denotes DT40
T5643 12816-12817 NN denotes B
T5644 12818-12823 NN denotes cells
T5645 12824-12825 -LRB- denotes (
T5646 12825-12829 NN denotes Fig.
T5647 12830-12832 NN denotes 4A
T5648 12832-12833 -RRB- denotes )
T5649 12833-12834 -COMMA- denotes ,
T5650 12835-12843 IN denotes although
T5651 12844-12850 JJ denotes strong
T5652 12851-12861 NN denotes activation
T5653 12862-12864 IN denotes of
T5654 12865-12868 NN denotes PKD
T5655 12869-12876 NN denotes kinases
T5656 12877-12878 -LRB- denotes (
T5657 12878-12880 IN denotes as
T5658 12881-12889 VB denotes assessed
T5659 12890-12892 IN denotes by
T5660 12893-12912 NN denotes autophosphorylation
T5661 12913-12915 IN denotes of
T5662 12916-12920 NN denotes PKD1
T5663 12921-12923 IN denotes at
T5664 12924-12928 NN denotes S916
T5665 12928-12929 -RRB- denotes )
T5666 12930-12933 VB denotes was
T5667 12934-12942 VB denotes observed
T5668 12943-12945 IN denotes in
T5669 12946-12949 DT denotes the
T5670 12950-12957 NN denotes PKCβ−/−
T5671 12958-12963 NN denotes cells
T5672 12964-12965 -LRB- denotes (
T5673 12965-12969 NN denotes Fig.
T5674 12970-12972 NN denotes 4B
T5675 12972-12973 -RRB- denotes )
T5676 12975-12979 RB denotes Thus
T5677 12979-12980 -COMMA- denotes ,
T5678 12981-12984 NN denotes PKD
T5679 12985-12992 NN denotes kinases
T5680 12993-12996 VB denotes are
T5681 12997-13004 DT denotes neither
T5682 13005-13014 JJ denotes essential
T5683 13015-13018 CC denotes nor
T5684 13019-13029 JJ denotes sufficient
T5685 13030-13032 TO denotes to
T5686 13033-13040 VB denotes mediate
T5687 13041-13052 JJ denotes BCR-induced
T5688 13053-13057 NN denotes NFκB
T5689 13058-13068 NN denotes activation
T5690 13069-13071 IN denotes in
T5691 13072-13076 NN denotes DT40
T5692 13077-13078 NN denotes B
T5693 13079-13084 NN denotes cells
T5694 13085-13088 CC denotes and
T5695 13089-13094 RB denotes hence
T5696 13095-13097 VB denotes do
T5697 13098-13101 RB denotes not
T5698 13102-13113 VB denotes participate
T5699 13114-13116 IN denotes in
T5700 13117-13124 NN denotes DAG/PKC
T5701 13125-13133 VB denotes mediated
T5702 13134-13141 NN denotes control
T5703 13142-13144 IN denotes of
T5704 13145-13149 NN denotes NFκB
T7043 237-13173 NN denotes 40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway. To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera. BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor. Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38]. In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored. The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB. 4 Discussion Protein
T7044 13174-13180 NN denotes kinase
T7045 13181-13182 NN denotes D
T7046 13183-13189 NN denotes serine
T7047 13190-13197 NN denotes kinases
T7048 13198-13202 VB denotes have
T7049 13203-13207 VB denotes been
T7050 13208-13216 VB denotes proposed
T7051 13217-13219 TO denotes to
T7052 13220-13228 VB denotes regulate
T7053 13229-13236 JJ denotes diverse
T7054 13237-13245 JJ denotes cellular
T7055 13246-13255 NN denotes functions
T7056 13256-13265 VB denotes including
T7057 13266-13269 DT denotes the
T7058 13270-13285 NN denotes phosphorylation
T7059 13286-13289 CC denotes and
T7060 13290-13297 JJ denotes nuclear
T7061 13298-13310 NN denotes localisation
T7062 13311-13313 IN denotes of
T7063 13314-13319 NN denotes class
T7064 13320-13322 CD denotes II
T7065 13323-13328 NN denotes HDACs
T7066 13329-13332 CC denotes and
T7067 13333-13336 DT denotes the
T7068 13337-13352 NN denotes phosphorylation
T7069 13353-13355 IN denotes of
T7070 13356-13361 NN denotes HSP27
T7071 13363-13365 PRP denotes It
T7072 13366-13369 VB denotes has
T7073 13370-13374 RB denotes also
T7074 13375-13379 VB denotes been
T7075 13380-13389 VB denotes suggested
T7076 13390-13394 IN denotes that
T7077 13395-13399 NN denotes PKDs
T7078 13400-13403 VB denotes act
T7079 13404-13406 IN denotes as
T7080 13407-13420 JJ denotes mitochondrial
T7081 13421-13428 NN denotes sensors
T7082 13429-13432 IN denotes for
T7083 13433-13442 JJ denotes oxidative
T7084 13443-13449 NN denotes stress
T7085 13450-13453 CC denotes and
T7086 13454-13458 VB denotes play
T7087 13459-13460 DT denotes a
T7088 13461-13465 NN denotes role
T7089 13466-13468 IN denotes in
T7090 13469-13479 VB denotes regulating
T7091 13480-13484 NN denotes NFκB
T7092 13485-13498 NN denotes transcription
T7093 13499-13506 NN denotes factors
T7094 13507-13508 -LRB- denotes [
T7095 13508-13510 CD denotes 41
T7096 13510-13511 -RRB- denotes ]
T7097 13513-13517 JJ denotes Most
T7098 13518-13520 IN denotes of
T7099 13521-13524 DT denotes the
T7100 13525-13529 NN denotes data
T7101 13530-13535 IN denotes about
T7102 13536-13539 DT denotes the
T7103 13540-13548 NN denotes function
T7104 13549-13551 IN denotes of
T7105 13552-13556 NN denotes PKDs
T7106 13557-13560 VB denotes has
T7107 13561-13565 VB denotes come
T7108 13566-13570 IN denotes from
T7109 13571-13582 NN denotes experiments
T7110 13583-13587 WDT denotes that
T7111 13588-13599 RB denotes ectopically
T7112 13600-13607 VB denotes express
T7113 13608-13614 JJ denotes active
T7114 13615-13617 CC denotes or
T7115 13618-13628 JJ denotes inhibitory
T7116 13629-13632 NN denotes PKD
T7117 13633-13640 NN denotes mutants
T7118 13641-13643 CC denotes or
T7119 13644-13648 WDT denotes that
T7120 13649-13652 VB denotes use
T7121 13653-13657 NN denotes RNAi
T7122 13658-13660 TO denotes to
T7123 13661-13667 VB denotes reduce
T7124 13668-13671 NN denotes PKD
T7125 13672-13682 NN denotes expression
T7126 13684-13686 PRP denotes We
T7127 13687-13691 VB denotes have
T7128 13692-13696 VB denotes used
T7129 13697-13701 NN denotes gene
T7130 13702-13711 VB denotes targeting
T7131 13712-13714 TO denotes to
T7132 13715-13727 RB denotes specifically
T7133 13728-13734 VB denotes delete
T7134 13735-13738 NN denotes PKD
T7135 13739-13746 NN denotes alleles
T7136 13747-13749 IN denotes in
T7137 13750-13754 NN denotes DT40
T7138 13755-13762 NN denotes chicken
T7139 13763-13764 NN denotes B
T7140 13765-13770 NN denotes cells
T7141 13771-13774 CC denotes and
T7142 13775-13778 MD denotes can
T7143 13779-13783 RB denotes thus
T7144 13784-13787 VB denotes use
T7145 13788-13796 JJ denotes PKD-null
T7146 13797-13801 NN denotes DT40
T7147 13802-13807 NN denotes cells
T7148 13808-13810 TO denotes to
T7149 13811-13817 VB denotes assess
T7150 13818-13821 DT denotes the
T7151 13822-13830 JJ denotes relative
T7152 13831-13843 NN denotes contribution
T7153 13844-13846 IN denotes of
T7154 13847-13857 JJ denotes individual
T7155 13858-13861 NN denotes PKD
T7156 13862-13870 NN denotes isoforms
T7157 13871-13873 IN denotes in
T7158 13874-13879 NN denotes class
T7159 13880-13882 CD denotes II
T7160 13883-13887 NN denotes HDAC
T7161 13888-13895 NN denotes control
T7162 13896-13902 IN denotes versus
T7163 13903-13912 JJ denotes oxidative
T7164 13913-13919 NN denotes stress
T7165 13920-13929 NN denotes responses
T7166 13930-13933 CC denotes and
T7167 13934-13938 NN denotes NFκB
T7168 13939-13949 NN denotes regulation
T7169 13950-13952 IN denotes in
T7170 13953-13964 NN denotes lymphocytes
T7171 13966-13968 PRP denotes We
T7172 13969-13973 VB denotes have
T7173 13974-13984 RB denotes previously
T7174 13985-13989 VB denotes used
T7175 13990-13995 DT denotes these
T7176 13996-14004 JJ denotes PKD-null
T7177 14005-14009 NN denotes DT40
T7178 14010-14015 NN denotes cells
T7179 14016-14018 TO denotes to
T7180 14019-14025 VB denotes define
T7181 14026-14028 DT denotes an
T7182 14029-14038 JJ denotes essential
T7183 14039-14043 NN denotes role
T7184 14044-14047 IN denotes for
T7185 14048-14052 NN denotes PKDs
T7186 14053-14055 IN denotes in
T7187 14056-14066 NN denotes regulation
T7188 14067-14069 IN denotes of
T7189 14070-14075 NN denotes class
T7190 14076-14078 CD denotes II
T7191 14079-14084 NN denotes HDACs
T7192 14084-14085 -COMMA- denotes ,
T7193 14086-14089 DT denotes the
T7194 14090-14097 JJ denotes present
T7195 14098-14104 NN denotes report
T7196 14105-14108 RB denotes now
T7197 14109-14118 VB denotes describes
T7198 14119-14121 DT denotes an
T7199 14122-14135 JJ denotes indispensable
T7200 14136-14140 NN denotes role
T7201 14141-14144 IN denotes for
T7202 14145-14149 NN denotes PKDs
T7203 14150-14152 IN denotes in
T7204 14153-14163 VB denotes regulating
T7205 14164-14167 DT denotes the
T7206 14168-14183 NN denotes phosphorylation
T7207 14184-14186 IN denotes of
T7208 14187-14192 NN denotes HSP27
T7209 14193-14195 IN denotes on
T7210 14196-14202 NN denotes serine
T7211 14203-14205 CD denotes 82
T7212 14205-14206 -COMMA- denotes ,
T7213 14207-14208 DT denotes a
T7214 14209-14213 NN denotes site
T7215 14214-14224 RB denotes previously
T7216 14225-14235 VB denotes identified
T7217 14236-14238 IN denotes as
T7218 14239-14240 DT denotes a
T7219 14241-14247 NN denotes target
T7220 14248-14251 IN denotes for
T7221 14252-14255 DT denotes the
T7222 14256-14268 NN denotes p38-MAPKAPK2
T7223 14269-14279 NN denotes signalling
T7224 14280-14287 NN denotes cascade
T7225 14288-14289 -LRB- denotes [
T7226 14289-14291 CD denotes 42
T7227 14291-14292 -RRB- denotes ]
T7228 14294-14301 RB denotes However
T7229 14301-14302 -COMMA- denotes ,
T7230 14303-14310 NN denotes studies
T7231 14311-14313 IN denotes of
T7232 14314-14322 JJ denotes PKD-null
T7233 14323-14327 NN denotes DT40
T7234 14328-14333 NN denotes cells
T7235 14334-14340 VB denotes reveal
T7236 14341-14345 IN denotes that
T7237 14346-14349 NN denotes PKD
T7238 14350-14356 NN denotes family
T7239 14357-14364 NN denotes kinases
T7240 14365-14368 VB denotes are
T7241 14369-14372 RB denotes not
T7242 14373-14382 JJ denotes essential
T7243 14383-14386 IN denotes for
T7244 14387-14396 JJ denotes oxidative
T7245 14397-14403 NN denotes stress
T7246 14404-14412 NN denotes survival
T7247 14413-14422 NN denotes responses
T7248 14423-14426 CC denotes nor
T7249 14427-14430 VB denotes are
T7250 14431-14435 PRP denotes they
T7251 14436-14444 VB denotes required
T7252 14445-14448 IN denotes for
T7253 14449-14459 NN denotes activation
T7254 14460-14462 IN denotes of
T7255 14463-14467 NN denotes NFκB
T7256 14468-14481 NN denotes transcription
T7257 14482-14489 NN denotes factors
T7258 14491-14496 DT denotes These
T7259 14497-14503 JJ denotes latter
T7260 14504-14512 NN denotes findings
T7261 14513-14516 VB denotes are
T7262 14517-14519 IN denotes in
T7263 14520-14528 JJ denotes striking
T7264 14529-14537 NN denotes contrast
T7265 14538-14540 TO denotes to
T7266 14541-14549 JJ denotes previous
T7267 14550-14562 NN denotes observations
T7268 14563-14565 IN denotes in
T7269 14566-14570 NN denotes HeLa
T7270 14571-14574 CC denotes and
T7271 14575-14585 JJ denotes epithelial
T7272 14586-14590 NN denotes cell
T7273 14591-14596 NN denotes lines
T7274 14597-14602 WRB denotes where
T7275 14603-14622 NN denotes overexpression/RNAi
T7276 14623-14633 NN denotes approaches
T7277 14634-14638 VB denotes have
T7278 14639-14649 VB denotes implicated
T7279 14650-14656 NN denotes PKD1/2
T7280 14657-14659 IN denotes in
T7281 14660-14663 DT denotes the
T7282 14664-14671 NN denotes control
T7283 14672-14674 IN denotes of
T7284 14675-14688 NN denotes proliferation
T7285 14688-14689 -COMMA- denotes ,
T7286 14690-14698 NN denotes survival
T7287 14699-14702 CC denotes and
T7288 14703-14707 NN denotes NFκB
T7289 14708-14718 NN denotes activation
T7290 14719-14720 -LRB- denotes [
T7291 14720-14725 CD denotes 20,23
T7292 14725-14726 -RRB- denotes ]
T7293 14728-14733 RB denotes Hence
T7294 14733-14734 -COMMA- denotes ,
T7295 14735-14738 DT denotes the
T7296 14739-14746 JJ denotes present
T7297 14747-14753 NN denotes report
T7298 14754-14759 VB denotes shows
T7299 14760-14764 IN denotes that
T7300 14765-14768 DT denotes the
T7301 14769-14777 VB denotes proposed
T7302 14778-14783 NN denotes roles
T7303 14784-14787 IN denotes for
T7304 14788-14792 NN denotes PKDs
T7305 14793-14795 IN denotes as
T7306 14796-14799 JJ denotes key
T7307 14800-14807 NN denotes sensors
T7308 14808-14812 WDT denotes that
T7309 14813-14821 VB denotes modulate
T7310 14822-14830 NN denotes survival
T7311 14831-14839 NN denotes pathways
T7312 14840-14842 IN denotes in
T7313 14843-14851 NN denotes response
T7314 14852-14854 TO denotes to
T7315 14855-14864 JJ denotes oxidative
T7316 14865-14871 NN denotes stress
T7317 14872-14875 CC denotes and
T7318 14876-14884 VB denotes regulate
T7319 14885-14889 NN denotes cell
T7320 14890-14898 NN denotes survival
T7321 14899-14902 CC denotes and
T7322 14903-14916 NN denotes proliferation
T7323 14917-14920 VB denotes are
T7324 14921-14924 RB denotes not
T7325 14925-14935 JJ denotes ubiquitous
T7326 14936-14939 CC denotes and
T7327 14940-14943 MD denotes may
T7328 14944-14946 VB denotes be
T7329 14947-14957 JJ denotes restricted
T7330 14958-14960 TO denotes to
T7331 14961-14968 JJ denotes certain
T7332 14969-14973 NN denotes cell
T7333 14974-14982 NN denotes lineages
T7334 14984-14989 VB denotes Taken
T7335 14990-14998 RB denotes together
T7336 14998-14999 -COMMA- denotes ,
T7337 15000-15005 DT denotes these
T7338 15006-15010 NN denotes data
T7339 15011-15019 VB denotes indicate
T7340 15020-15024 IN denotes that
T7341 15025-15029 NN denotes loss
T7342 15030-15032 IN denotes of
T7343 15033-15043 NN denotes expression
T7344 15044-15046 IN denotes of
T7345 15047-15050 NN denotes PKD
T7346 15051-15057 NN denotes family
T7347 15058-15065 NN denotes members
T7348 15066-15070 VB denotes does
T7349 15071-15074 RB denotes not
T7350 15075-15083 RB denotes globally
T7351 15084-15090 NN denotes impact
T7352 15091-15093 IN denotes on
T7353 15094-15099 JJ denotes early
T7354 15100-15113 JJ denotes BCR-regulated
T7355 15114-15124 NN denotes signalling
T7356 15125-15133 NN denotes pathways
T26 0-7 NN denotes Protein
T27 8-14 NN denotes kinase
T28 15-16 NN denotes D
T29 17-24 NN denotes enzymes
T30 25-28 VB denotes are
T31 29-40 JJ denotes dispensable
T32 41-44 IN denotes for
T33 45-58 NN denotes proliferation
T34 58-59 -COMMA- denotes ,
T35 60-68 NN denotes survival
T36 69-72 CC denotes and
T37 73-80 NN denotes antigen
T38 81-99 JJ denotes receptor-regulated
T39 100-104 NN denotes NFκB
T40 105-113 NN denotes activity
T41 114-116 IN denotes in
T42 117-127 NN denotes vertebrate
T43 128-135 NN denotes B-cells
T44 147-149 TO denotes To
T45 150-161 VB denotes investigate
T46 162-165 DT denotes the
T47 166-176 NN denotes importance
T48 177-179 IN denotes of
T1722 4035-4036 CD denotes 1
T1723 4036-4037 -RRB- denotes ]
T1724 4039-4054 NN denotes Chloramphenicol
T1725 4055-4061 NN denotes acetyl
T1726 4062-4073 NN denotes transferase
T1727 4074-4080 NN denotes assays
T1728 4081-4085 VB denotes have
T1729 4086-4090 VB denotes been
T1730 4091-4100 VB denotes described
T1731 4101-4111 RB denotes previously
T1732 4112-4113 -LRB- denotes [
T1733 4113-4115 CD denotes 29
T1734 4115-4116 -RRB- denotes ]
T1868 0-4128 NN denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM
T1869 4129-4137 NN denotes staining
T1870 4138-4142 NN denotes DT40
T1871 4143-4144 NN denotes B
T1872 4145-4150 NN denotes cells
R9 T51 T52 arg1Of D,(
R10 T53 T52 arg2Of PKD,(
R11 T54 T52 arg3Of ),(
R15 T56 T57 arg1Of we,generated
R17 T63 T57 arg2Of line,generated
R18 T63 T58 arg1Of line,a
R19 T63 T59 arg1Of line,PKD-null
R20 T63 T60 arg1Of line,DT40
R21 T63 T61 arg1Of line,B-lymphocyte
R22 T63 T62 arg1Of line,cell
R23 T65 T66 arg1Of we,have
R24 T65 T67 arg1Of we,shown
R25 T67 T64 arg1Of shown,Previously
R26 T67 T66 arg2Of shown,have
R27 T69 T70 arg1Of PKDs,have
R28 T70 T67 arg2Of have,shown
R29 T70 T68 arg1Of have,that
R30 T70 T74 arg1Of have,in
R31 T73 T70 arg2Of role,have
R32 T73 T71 arg1Of role,an
R33 T73 T72 arg1Of role,essential
R34 T75 T74 arg2Of regulating,in
R35 T75 T80 arg1Of regulating,in
R36 T79 T75 arg2Of deacetylases,regulating
R37 T79 T76 arg1Of deacetylases,class
R38 T79 T77 arg1Of deacetylases,II
R39 T79 T78 arg1Of deacetylases,histone
R40 T82 T80 arg2Of B-cells,in
R41 T82 T81 arg1Of B-cells,DT40
R42 T84 T85 arg1Of Matthews,","
R43 T85 T87 arg1Of ",",","
R44 T86 T85 arg2Of S.A.,","
R45 T87 T89 arg1Of ",",","
R46 T88 T87 arg2Of Liu,","
R47 T89 T91 arg1Of ",",","
R48 T90 T89 arg2Of P.,","
R49 T91 T93 arg1Of ",",","
R50 T92 T91 arg2Of Spitaler,","
R51 T93 T95 arg1Of ",",","
R52 T94 T93 arg2Of M.,","
R53 T95 T105 arg1Of ",",","
R54 T96 T97 arg1Of Olson,","
R55 T97 T99 arg1Of ",",","
R56 T98 T97 arg2Of E.N.,","
R57 T99 T101 arg1Of ",",","
R58 T100 T99 arg2Of McKinsey,","
R59 T101 T103 arg1Of ",",","
R60 T102 T101 arg2Of T.A.,","
R61 T103 T95 arg2Of ",",","
R62 T104 T103 arg2Of Cantrell,","
R63 T105 T107 arg1Of ",",and
R64 T106 T105 arg2Of D.A.,","
R65 T108 T109 arg1Of Scharenberg,","
R66 T109 T107 arg2Of ",",and
R67 T110 T109 arg2Of A.M.,","
R68 T112 T111 arg2Of 2006,(
R69 T113 T111 arg3Of ),(
R70 T115 T80 arg3Of role,in
R71 T115 T83 arg1Of role,[
R72 T115 T84 arg1Of role,Matthews
R73 T115 T86 arg1Of role,S.A.
R74 T115 T88 arg1Of role,Liu
R75 T115 T90 arg1Of role,P.
R76 T115 T92 arg1Of role,Spitaler
R77 T115 T94 arg1Of role,M.
R78 T115 T96 arg1Of role,Olson
R79 T115 T98 arg1Of role,E.N.
R80 T115 T100 arg1Of role,McKinsey
R81 T115 T102 arg1Of role,T.A.
R82 T115 T104 arg1Of role,Cantrell
R83 T115 T106 arg1Of role,D.A.
R84 T115 T108 arg1Of role,Scharenberg
R85 T115 T110 arg1Of role,A.M.
R86 T115 T111 arg1Of role,(
R87 T115 T114 arg1Of role,Essential
R88 T115 T116 arg1Of role,for
R89 T115 T122 arg1Of role,in
R90 T121 T116 arg2Of kinases,for
R91 T121 T117 arg1Of kinases,protein
R92 T121 T118 arg1Of kinases,kinase
R93 T121 T119 arg1Of kinases,D
R94 T121 T120 arg1Of kinases,family
R95 T124 T122 arg2Of regulation,in
R96 T124 T123 arg1Of regulation,the
R97 T124 T125 arg1Of regulation,of
R98 T129 T125 arg2Of deacetylases,of
R99 T129 T126 arg1Of deacetylases,class
R100 T129 T127 arg1Of deacetylases,II
R101 T129 T128 arg1Of deacetylases,histone
R102 T129 T130 arg1Of deacetylases,in
R103 T132 T130 arg2Of lymphocytes,in
R104 T132 T131 arg1Of lymphocytes,B
R105 T135 T133 arg1Of Biol.,Mol.
R106 T135 T134 arg1Of Biol.,Cell
R107 T135 T136 arg1Of Biol.,26
R108 T135 T137 arg1Of Biol.,","
R109 T138 T137 arg2Of 1569–1577,","
R110 T138 T139 arg1Of 1569–1577,]
R111 T140 T142 arg1Of We,show
R112 T142 T141 arg1Of show,now
R113 T144 T145 arg1Of PKDs,are
R114 T144 T147 arg2Of PKDs,required
R115 T144 T149 arg1Of PKDs,regulate
R116 T147 T142 arg2Of required,show
R117 T147 T143 arg1Of required,that
R118 T147 T145 arg2Of required,are
R119 T147 T146 arg1Of required,also
R120 T149 T147 arg3Of regulate,required
R121 T149 T148 arg1Of regulate,to
R122 T149 T152 arg1Of regulate,in
R123 T151 T149 arg2Of phosphorylation,regulate
R124 T151 T150 arg1Of phosphorylation,HSP27
R125 T154 T152 arg2Of B-cells,in
R126 T154 T153 arg1Of B-cells,DT40
R127 T158 T157 arg2Of contrast,in
R128 T158 T159 arg1Of contrast,to
R129 T161 T159 arg2Of observations,to
R130 T161 T160 arg1Of observations,previous
R131 T161 T162 arg1Of observations,in
R132 T165 T162 arg2Of types,in
R133 T165 T163 arg1Of types,other
R134 T165 T164 arg1Of types,cell
R135 T168 T167 arg1Of enzymes,PKD
R136 T168 T169 arg1Of enzymes,do
R137 T168 T171 arg1Of enzymes,regulate
R138 T171 T155 arg1Of regulate,However
R139 T171 T156 arg1Of regulate,","
R140 T171 T157 arg1Of regulate,in
R141 T171 T166 arg1Of regulate,","
R142 T171 T169 arg2Of regulate,do
R143 T171 T170 arg1Of regulate,not
R144 T174 T171 arg2Of processes,regulate
R145 T174 T172 arg1Of processes,basic
R146 T174 T173 arg1Of processes,cellular
R147 T174 T176 arg1Of processes,as
R148 T176 T175 arg1Of as,such
R149 T177 T178 arg1Of proliferation,or
R150 T178 T182 arg1Of or,nor
R151 T180 T178 arg2Of responses,or
R152 T180 T179 arg1Of responses,survival
R153 T182 T176 arg2Of nor,as
R154 T182 T181 arg1Of nor,","
R155 T185 T182 arg2Of activity,nor
R156 T185 T183 arg1Of activity,NFκB
R157 T185 T184 arg1Of activity,transcriptional
R158 T185 T186 arg1Of activity,downstream
R159 T186 T187 arg1Of downstream,of
R160 T192 T187 arg2Of receptor,of
R161 T192 T188 arg1Of receptor,the
R162 T192 T189 arg1Of receptor,B
R163 T192 T190 arg1Of receptor,cell
R164 T192 T191 arg1Of receptor,antigen
R165 T195 T196 arg1Of PKDs,have
R166 T196 T193 arg1Of have,Thus
R167 T196 T194 arg1Of have,","
R168 T196 T200 arg1Of have,in
R169 T199 T196 arg2Of role,have
R170 T199 T197 arg1Of role,a
R171 T199 T198 arg1Of role,selective
R172 T203 T200 arg2Of biology,in
R173 T203 T201 arg1Of biology,DT40
R174 T203 T202 arg1Of biology,B-cell
R430 T535 T532 arg1Of D,"1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The"
R431 T535 T533 arg1Of D,protein
R432 T535 T534 arg1Of D,kinase
R433 T535 T536 arg1Of D,(
R434 T537 T536 arg2Of PKD,(
R435 T538 T536 arg3Of ),(
R436 T541 T535 arg1Of family,D
R437 T541 T539 arg1Of family,serine/threonine
R438 T541 T540 arg1Of family,kinase
R439 T541 T542 arg1Of family,has
R440 T544 T542 arg2Of members,has
R441 T544 T543 arg1Of members,three
R442 T544 T545 arg1Of members,:
R443 T546 T547 arg1Of PKD1,","
R444 T547 T549 arg1Of ",",and
R445 T548 T547 arg2Of PKD2,","
R446 T549 T545 arg2Of and,:
R447 T550 T549 arg2Of PKD3,and
R448 T553 T551 arg1Of types,Most
R449 T553 T552 arg1Of types,cell
R450 T553 T554 arg1Of types,express
R451 T554 T560 arg1Of express,but
R452 T557 T555 arg1Of two,at
R453 T557 T556 arg1Of two,least
R454 T559 T554 arg2Of isoforms,express
R455 T559 T557 arg1Of isoforms,two
R456 T559 T558 arg1Of isoforms,PKD
R457 T562 T561 arg1Of enzymes,PKD
R458 T562 T563 arg1Of enzymes,are
R459 T562 T566 arg2Of enzymes,expressed
R460 T565 T564 arg1Of highly,especially
R461 T566 T560 arg2Of expressed,but
R462 T566 T563 arg2Of expressed,are
R463 T566 T565 arg1Of expressed,highly
R464 T566 T567 arg1Of expressed,in
R465 T569 T567 arg2Of cells,in
R466 T569 T568 arg1Of cells,haematopoietic
R467 T569 T570 arg1Of cells,","
R468 T569 T571 arg1Of cells,where
R469 T572 T573 arg1Of they,are
R470 T572 T574 arg2Of they,activated
R471 T574 T571 arg2Of activated,where
R472 T574 T573 arg2Of activated,are
R473 T574 T575 arg1Of activated,in
R474 T574 T581 arg1Of activated,[
R475 T575 T577 arg1Of in,to
R476 T576 T575 arg2Of response,in
R477 T580 T575 arg3Of stimulation,in
R478 T580 T578 arg1Of stimulation,antigen
R479 T580 T579 arg1Of stimulation,receptors
R480 T582 T581 arg2Of "2,3",[
R481 T583 T581 arg3Of ],[
R482 T587 T584 arg1Of pathway,A
R483 T587 T585 arg2Of pathway,conserved
R484 T587 T586 arg1Of pathway,signalling
R485 T587 T588 arg1Of pathway,linking
R486 T587 T593 arg1Of pathway,involves
R487 T590 T588 arg2Of receptors,linking
R488 T590 T589 arg1Of receptors,antigen
R489 T590 T591 arg1Of receptors,to
R490 T592 T591 arg2Of PKDs,to
R491 T595 T594 arg1Of activation,the
R492 T595 T596 arg1Of activation,of
R493 T595 T598 arg1Of activation,and
R494 T597 T596 arg2Of PLCγ,of
R495 T598 T593 arg2Of and,involves
R496 T601 T598 arg2Of production,and
R497 T601 T599 arg1Of production,the
R498 T601 T600 arg1Of production,subsequent
R499 T601 T602 arg1Of production,of
R500 T601 T607 arg1Of production,which
R501 T601 T608 arg1Of production,stimulates
R502 T603 T602 arg2Of diacylglycerol,of
R503 T603 T604 arg1Of diacylglycerol,(
R504 T605 T604 arg2Of DAG,(
R505 T606 T604 arg3Of ),(
R506 T609 T610 arg1Of classical,and/or
R507 T611 T610 arg2Of novel,and/or
R508 T614 T608 arg2Of Cs,stimulates
R509 T614 T609 arg1Of Cs,classical
R510 T614 T611 arg1Of Cs,novel
R511 T614 T612 arg1Of Cs,protein
R512 T614 T613 arg1Of Cs,kinase
R513 T614 T615 arg1Of Cs,(
R514 T614 T618 arg1Of Cs,that
R515 T614 T619 arg1Of Cs,phosphorylate
R516 T616 T615 arg2Of PKC,(
R517 T617 T615 arg3Of ),(
R518 T619 T625 arg1Of phosphorylate,in
R519 T624 T619 arg2Of residues,phosphorylate
R520 T624 T620 arg1Of residues,two
R521 T624 T621 arg1Of residues,key
R522 T624 T622 arg1Of residues,regulatory
R523 T624 T623 arg1Of residues,serine
R524 T628 T625 arg2Of loop,in
R525 T628 T626 arg1Of loop,the
R526 T628 T627 arg1Of loop,activation
R527 T628 T629 arg1Of loop,of
R528 T628 T632 arg1Of loop,[
R529 T631 T629 arg2Of kinases,of
R530 T631 T630 arg1Of kinases,PKD
R531 T633 T632 arg2Of 3–6,[
R532 T634 T632 arg3Of ],[
R533 T638 T635 arg1Of region,The
R534 T638 T636 arg1Of region,N-terminal
R535 T638 T637 arg1Of region,regulatory
R536 T638 T639 arg1Of region,of
R537 T638 T642 arg1Of region,contains
R538 T641 T639 arg2Of enzymes,of
R539 T641 T640 arg1Of enzymes,PKD
R540 T642 T647 arg1Of contains,and
R541 T646 T642 arg2Of domain,contains
R542 T646 T643 arg1Of domain,a
R543 T646 T644 arg1Of domain,DAG
R544 T646 T645 arg1Of domain,binding
R545 T649 T648 arg1Of binding,direct
R546 T649 T650 arg1Of binding,of
R547 T649 T653 arg1Of binding,contributes
R548 T651 T650 arg2Of DAG,of
R549 T653 T647 arg2Of contributes,and
R550 T653 T652 arg1Of contributes,also
R551 T653 T654 arg1Of contributes,to
R552 T653 T657 arg1Of contributes,[
R553 T653 T661 arg1Of contributes,well
R554 T653 T662 arg1Of contributes,as
R555 T656 T654 arg2Of activation,to
R556 T656 T655 arg1Of activation,PKD1
R557 T658 T657 arg2Of 7,[
R558 T659 T657 arg3Of ],[
R559 T661 T660 arg1Of well,as
R560 T663 T662 arg2Of regulating,as
R561 T666 T663 arg2Of location,regulating
R562 T666 T664 arg1Of location,the
R563 T666 T665 arg1Of location,spatial
R564 T666 T667 arg1Of location,of
R565 T666 T670 arg1Of location,within
R566 T669 T667 arg2Of enzymes,of
R567 T669 T668 arg1Of enzymes,PKD
R568 T671 T670 arg2Of cells,within
R569 T671 T672 arg1Of cells,[
R570 T673 T672 arg2Of 8–12,[
R571 T674 T672 arg3Of ],[
R572 T676 T675 arg1Of enzymes,PKD
R573 T676 T677 arg1Of enzymes,have
R574 T676 T678 arg1Of enzymes,been
R575 T676 T679 arg2Of enzymes,proposed
R576 T676 T681 arg1Of enzymes,regulate
R577 T679 T677 arg2Of proposed,have
R578 T679 T678 arg2Of proposed,been
R579 T681 T679 arg3Of regulate,proposed
R580 T681 T680 arg1Of regulate,to
R581 T684 T681 arg2Of functions,regulate
R582 T684 T682 arg1Of functions,numerous
R583 T684 T683 arg1Of functions,cellular
R584 T684 T685 arg1Of functions,","
R585 T684 T686 arg1Of functions,including
R586 T688 T687 arg1Of proliferation,cell
R587 T688 T689 arg1Of proliferation,[
R588 T688 T692 arg1Of proliferation,","
R589 T690 T689 arg2Of 13–16,[
R590 T691 T689 arg3Of ],[
R591 T692 T698 arg1Of ",",and
R592 T694 T692 arg2Of signals,","
R593 T694 T693 arg1Of signals,anti-apoptotic
R594 T694 T695 arg1Of signals,[
R595 T696 T695 arg2Of "17,18",[
R596 T697 T695 arg3Of ],[
R597 T698 T686 arg2Of and,including
R598 T700 T698 arg2Of development,and
R599 T700 T699 arg1Of development,thymocyte
R600 T700 T701 arg1Of development,[
R601 T702 T701 arg2Of 19,[
R602 T703 T701 arg3Of ],[
R603 T704 T705 arg1Of Expression,of
R604 T704 T713 arg1Of Expression,can
R605 T704 T715 arg1Of Expression,modify
R606 T708 T709 arg1Of inactive,and
R607 T710 T709 arg2Of constitutively,and
R608 T712 T705 arg2Of PKDs,of
R609 T712 T706 arg1Of PKDs,mutant
R610 T712 T707 arg1Of PKDs,catalytically
R611 T712 T708 arg1Of PKDs,inactive
R612 T712 T710 arg1Of PKDs,constitutively
R613 T712 T711 arg2Of PKDs,activated
R614 T715 T713 arg2Of modify,can
R615 T715 T714 arg1Of modify,also
R616 T717 T716 arg1Of function,Golgi
R617 T717 T718 arg1Of function,","
R618 T718 T721 arg1Of ",",and
R619 T720 T718 arg2Of adhesion,","
R620 T720 T719 arg1Of adhesion,cell
R621 T721 T715 arg2Of and,modify
R622 T723 T721 arg2Of motility,and
R623 T723 T722 arg1Of motility,cell
R624 T723 T724 arg1Of motility,(
R625 T725 T724 arg2Of reviewed,(
R626 T725 T726 arg1Of reviewed,in
R627 T725 T727 arg1Of reviewed,[
R628 T728 T727 arg2Of 20,[
R629 T729 T727 arg3Of ],[
R630 T730 T724 arg3Of ),(
R631 T732 T731 arg2Of particular,In
R632 T734 T735 arg1Of PKDs,have
R633 T734 T736 arg1Of PKDs,been
R634 T734 T738 arg2Of PKDs,linked
R635 T734 T749 arg1Of PKDs,regulating
R636 T738 T731 arg1Of linked,In
R637 T738 T733 arg1Of linked,","
R638 T738 T735 arg2Of linked,have
R639 T738 T736 arg2Of linked,been
R640 T738 T737 arg1Of linked,widely
R641 T738 T739 arg1Of linked,to
R642 T738 T748 arg1Of linked,in
R643 T739 T747 arg1Of to,and
R644 T741 T739 arg2Of activation,to
R645 T741 T740 arg1Of activation,the
R646 T741 T742 arg1Of activation,of
R647 T746 T742 arg2Of factor,of
R648 T746 T743 arg1Of factor,the
R649 T746 T744 arg1Of factor,NFκB
R650 T746 T745 arg1Of factor,transcription
R651 T748 T747 arg2Of in,and
R652 T749 T748 arg2Of regulating,in
R653 T749 T752 arg1Of regulating,during
R654 T751 T749 arg2Of survival,regulating
R655 T751 T750 arg1Of survival,cell
R656 T754 T752 arg2Of stress,during
R657 T754 T753 arg1Of stress,oxidative
R658 T754 T755 arg1Of stress,[
R659 T756 T755 arg2Of "17,21–23",[
R660 T757 T755 arg3Of ],[
R661 T760 T759 arg1Of proposed,recently
R662 T762 T758 arg1Of substrate,Another
R663 T762 T760 arg1Of substrate,proposed
R664 T762 T761 arg1Of substrate,PKD1
R665 T762 T763 arg1Of substrate,is
R666 T764 T763 arg2Of HSP27,is
R667 T764 T765 arg1Of HSP27,[
R668 T764 T768 arg1Of HSP27,","
R669 T766 T765 arg2Of 24,[
R670 T767 T765 arg3Of ],[
R671 T773 T768 arg2Of protein,","
R672 T773 T769 arg1Of protein,a
R673 T773 T770 arg1Of protein,small
R674 T773 T771 arg1Of protein,heat
R675 T773 T772 arg1Of protein,shock
R676 T773 T774 arg2Of protein,involved
R677 T774 T775 arg1Of involved,in
R678 T776 T775 arg2Of regulating,in
R679 T776 T782 arg1Of regulating,[
R680 T778 T777 arg1Of migration,cell
R681 T778 T779 arg1Of migration,and
R682 T779 T776 arg2Of and,regulating
R683 T781 T779 arg2Of survival,and
R684 T781 T780 arg1Of survival,cell
R685 T783 T782 arg2Of 25,[
R686 T784 T782 arg3Of ],[
R687 T787 T785 arg1Of role,An
R688 T787 T786 arg1Of role,essential
R689 T787 T788 arg1Of role,for
R690 T787 T808 arg1Of role,has
R691 T787 T810 arg1Of role,been
R692 T787 T811 arg2Of role,demonstrated
R693 T790 T788 arg2Of enzymes,for
R694 T790 T789 arg1Of enzymes,PKD
R695 T790 T791 arg1Of enzymes,in
R696 T792 T791 arg2Of regulating,in
R697 T796 T792 arg2Of deacetylases,regulating
R698 T796 T793 arg1Of deacetylases,class
R699 T796 T794 arg1Of deacetylases,II
R700 T796 T795 arg1Of deacetylases,histone
R701 T796 T797 arg1Of deacetylases,(
R702 T796 T800 arg1Of deacetylases,","
R703 T798 T797 arg2Of HDACs,(
R704 T799 T797 arg3Of ),(
R705 T801 T800 arg2Of enzymes,","
R706 T801 T802 arg1Of enzymes,that
R707 T801 T803 arg1Of enzymes,repress
R708 T806 T803 arg2Of transcription,repress
R709 T806 T804 arg1Of transcription,MEF2-dependent
R710 T806 T805 arg1Of transcription,gene
R711 T811 T807 arg1Of demonstrated,","
R712 T811 T808 arg2Of demonstrated,has
R713 T811 T809 arg1Of demonstrated,also
R714 T811 T810 arg2Of demonstrated,been
R715 T811 T812 arg1Of demonstrated,[
R716 T813 T812 arg2Of "1,26–28",[
R717 T814 T812 arg3Of ],[
R718 T816 T815 arg1Of investigate,To
R719 T819 T816 arg2Of role,investigate
R720 T819 T817 arg1Of role,the
R721 T819 T818 arg1Of role,biological
R722 T819 T820 arg1Of role,of
R723 T821 T820 arg2Of PKDs,of
R724 T822 T816 arg1Of we,investigate
R725 T822 T823 arg1Of we,have
R726 T822 T824 arg1Of we,generated
R727 T824 T815 modOf generated,To
R728 T824 T823 arg2Of generated,have
R729 T828 T824 arg2Of lines,generated
R730 T828 T825 arg1Of lines,DT40
R731 T828 T826 arg1Of lines,B
R732 T828 T827 arg1Of lines,cell
R733 T828 T829 arg1Of lines,that
R734 T828 T830 arg1Of lines,lack
R735 T830 T844 arg1Of lack,","
R736 T830 T845 modOf lack,allowing
R737 T830 T866 arg1Of lack,","
R738 T830 T869 modOf lack,addressing
R739 T831 T830 arg2Of expression,lack
R740 T831 T832 arg1Of expression,of
R741 T833 T834 arg1Of one,or
R742 T833 T835 arg1Of one,more
R743 T836 T832 arg2Of members,of
R744 T836 T833 arg1Of members,one
R745 T836 T837 arg1Of members,of
R746 T840 T837 arg2Of family,of
R747 T840 T838 arg1Of family,the
R748 T840 T839 arg1Of family,PKD
R749 T840 T841 arg1Of family,[
R750 T842 T841 arg2Of 1,[
R751 T843 T841 arg3Of ],[
R752 T846 T845 arg2Of us,allowing
R753 T846 T848 arg1Of us,investigate
R754 T848 T845 arg3Of investigate,allowing
R755 T848 T847 arg1Of investigate,to
R756 T850 T848 arg2Of function,investigate
R757 T850 T849 arg1Of function,the
R758 T850 T851 arg1Of function,(
R759 T850 T854 arg1Of function,of
R760 T850 T857 arg1Of function,following
R761 T852 T851 arg2Of s,(
R762 T853 T851 arg3Of ),(
R763 T856 T854 arg2Of isoforms,of
R764 T856 T855 arg1Of isoforms,PKD
R765 T863 T862 arg2Of BCR,(
R766 T864 T862 arg3Of ),(
R767 T865 T857 arg2Of stimulation,following
R768 T865 T858 arg1Of stimulation,B
R769 T865 T859 arg1Of stimulation,cell
R770 T865 T860 arg1Of stimulation,antigen
R771 T865 T861 arg1Of stimulation,receptor
R772 T865 T862 arg1Of stimulation,(
R773 T869 T867 arg1Of addressing,as
R774 T869 T868 arg1Of addressing,well
R775 T871 T869 arg2Of issue,addressing
R776 T871 T870 arg1Of issue,the
R777 T871 T872 arg1Of issue,of
R778 T874 T872 arg2Of redundancy,of
R779 T874 T873 arg1Of redundancy,functional
R780 T874 T875 arg1Of redundancy,between
R781 T880 T875 arg2Of members,between
R782 T880 T876 arg1Of members,the
R783 T880 T877 arg1Of members,different
R784 T880 T878 arg1Of members,PKD
R785 T880 T879 arg1Of members,family
R786 T882 T881 arg1Of studies,Previous
R787 T882 T883 arg1Of studies,have
R788 T882 T884 arg1Of studies,shown
R789 T884 T883 arg2Of shown,have
R790 T886 T887 arg1Of PKDs,are
R791 T886 T888 arg1Of PKDs,indispensable
R792 T887 T884 arg2Of are,shown
R793 T887 T885 arg1Of are,that
R794 T887 T895 arg1Of are,[
R795 T888 T887 arg2Of indispensable,are
R796 T888 T889 arg1Of indispensable,for
R797 T891 T889 arg2Of regulation,for
R798 T891 T890 arg1Of regulation,HDAC
R799 T891 T892 arg1Of regulation,in
R800 T894 T892 arg2Of cells,in
R801 T894 T893 arg1Of cells,B
R802 T896 T895 arg2Of 1,[
R803 T897 T895 arg3Of ],[
R804 T899 T900 arg1Of we,show
R805 T900 T898 arg1Of show,Herein
R806 T902 T903 arg1Of PKDs,are
R807 T902 T905 arg1Of PKDs,indispensable
R808 T903 T900 arg2Of are,show
R809 T903 T901 arg1Of are,that
R810 T903 T904 arg1Of are,also
R811 T905 T903 arg2Of indispensable,are
R812 T905 T906 arg1Of indispensable,for
R813 T908 T906 arg2Of phosphorylation,for
R814 T908 T907 arg1Of phosphorylation,HSP27
R815 T908 T909 arg1Of phosphorylation,in
R816 T911 T909 arg2Of cells,in
R817 T911 T910 arg1Of cells,B
R818 T917 T914 arg1Of cells,PKD-null
R819 T917 T915 arg1Of cells,DT40
R820 T917 T916 arg1Of cells,B
R821 T917 T918 arg1Of cells,are
R822 T917 T919 arg1Of cells,viable
R823 T917 T921 arg1Of cells,proliferate
R824 T918 T920 arg1Of are,and
R825 T919 T918 arg2Of viable,are
R826 T920 T912 arg1Of and,However
R827 T920 T913 arg1Of and,","
R828 T921 T920 arg2Of proliferate,and
R829 T921 T922 arg1Of proliferate,normally
R830 T925 T926 arg1Of loss,of
R831 T925 T933 arg1Of loss,does
R832 T925 T936 arg1Of loss,affect
R833 T925 T944 arg1Of loss,do
R834 T930 T926 arg2Of pool,of
R835 T930 T927 arg1Of pool,the
R836 T930 T928 arg1Of pool,entire
R837 T930 T929 arg1Of pool,cellular
R838 T930 T931 arg1Of pool,of
R839 T932 T931 arg2Of PKD,of
R840 T936 T933 arg2Of affect,does
R841 T936 T934 arg1Of affect,not
R842 T936 T935 arg1Of affect,critically
R843 T936 T943 arg1Of affect,nor
R844 T939 T936 arg2Of responses,affect
R845 T939 T937 arg1Of responses,oxidative
R846 T939 T938 arg1Of responses,stress
R847 T939 T940 arg1Of responses,in
R848 T942 T940 arg2Of cells,in
R849 T942 T941 arg1Of cells,B
R850 T943 T923 arg1Of nor,Moreover
R851 T943 T924 arg1Of nor,","
R852 T944 T943 arg2Of do,nor
R853 T945 T944 arg2Of PKD,do
R854 T946 T947 arg1Of kinases,play
R855 T946 T952 arg1Of kinases,regulating
R856 T947 T944 arg3Of play,do
R857 T947 T951 arg1Of play,in
R858 T950 T947 arg2Of role,play
R859 T950 T948 arg1Of role,an
R860 T950 T949 arg1Of role,essential
R861 T952 T951 arg2Of regulating,in
R862 T955 T952 arg2Of activity,regulating
R863 T955 T953 arg1Of activity,NFκB
R864 T955 T954 arg1Of activity,transcriptional
R865 T959 T958 arg1Of findings,these
R866 T959 T960 arg1Of findings,reveal
R867 T960 T956 arg1Of reveal,Together
R868 T960 T957 arg1Of reveal,","
R869 T964 T962 arg2Of lymphocytes,in
R870 T964 T963 arg1Of lymphocytes,B
R871 T967 T966 arg1Of kinases,PKD
R872 T967 T968 arg1Of kinases,are
R873 T968 T960 arg2Of are,reveal
R874 T968 T961 arg1Of are,that
R875 T968 T962 arg1Of are,in
R876 T968 T965 arg1Of are,","
R877 T968 T969 arg1Of are,not
R878 T971 T968 arg2Of regulators,are
R879 T971 T970 arg1Of regulators,critical
R880 T971 T972 arg1Of regulators,of
R881 T973 T972 arg2Of many,of
R882 T973 T974 arg1Of many,of
R883 T977 T974 arg2Of processes,of
R884 T977 T975 arg1Of processes,the
R885 T977 T976 arg1Of processes,cellular
R886 T977 T979 arg2Of processes,ascribed
R887 T979 T978 arg1Of ascribed,previously
R888 T979 T980 arg1Of ascribed,to
R889 T979 T982 arg1Of ascribed,in
R890 T981 T980 arg2Of them,to
R891 T985 T982 arg2Of systems,in
R892 T985 T983 arg1Of systems,other
R893 T985 T984 arg1Of systems,cellular
R1437 T1675 T1674 arg1Of culture,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell"
R1438 T1675 T1676 arg1Of culture,","
R1439 T1676 T1679 arg1Of ",",and
R1440 T1678 T1676 arg2Of transfections,","
R1441 T1678 T1677 arg1Of transfections,transient
R1442 T1681 T1679 arg2Of stimulation,and
R1443 T1681 T1680 arg1Of stimulation,cell
R1444 T1683 T1684 arg1Of generation,","
R1445 T1684 T1686 arg1Of ",",and
R1446 T1685 T1684 arg2Of culture,","
R1447 T1686 T1682 arg1Of and,The
R1448 T1686 T1688 arg1Of and,of
R1449 T1686 T1699 arg1Of and,have
R1450 T1686 T1700 arg1Of and,been
R1451 T1686 T1701 arg2Of and,described
R1452 T1687 T1686 arg2Of activation,and
R1453 T1689 T1690 arg1Of PKD1−/−,","
R1454 T1690 T1692 arg1Of ",",and
R1455 T1691 T1690 arg2Of PKD3−/−,","
R1456 T1693 T1692 arg2Of PKD1/3−/−,and
R1457 T1698 T1688 arg2Of lines,of
R1458 T1698 T1689 arg1Of lines,PKD1−/−
R1459 T1698 T1691 arg1Of lines,PKD3−/−
R1460 T1698 T1693 arg1Of lines,PKD1/3−/−
R1461 T1698 T1694 arg1Of lines,knockout
R1462 T1698 T1695 arg1Of lines,DT40
R1463 T1698 T1696 arg1Of lines,B
R1464 T1698 T1697 arg1Of lines,cell
R1465 T1701 T1699 arg2Of described,have
R1466 T1701 T1700 arg2Of described,been
R1467 T1701 T1702 arg1Of described,previously
R1468 T1701 T1703 arg1Of described,[
R1469 T1704 T1703 arg2Of 1,[
R1470 T1705 T1703 arg3Of ],[
R1471 T1706 T1707 arg1Of Cells,were
R1472 T1706 T1708 arg2Of Cells,lysed
R1473 T1708 T1707 arg2Of lysed,were
R1474 T1708 T1709 arg1Of lysed,and
R1475 T1711 T1710 arg1Of extracts,protein
R1476 T1711 T1712 arg1Of extracts,were
R1477 T1711 T1713 arg2Of extracts,analysed
R1478 T1713 T1709 arg2Of analysed,and
R1479 T1713 T1712 arg2Of analysed,were
R1480 T1713 T1714 arg1Of analysed,in
R1481 T1713 T1718 arg1Of analysed,as
R1482 T1717 T1714 arg2Of experiments,in
R1483 T1717 T1715 arg1Of experiments,Western
R1484 T1717 T1716 arg1Of experiments,blotting
R1485 T1720 T1719 arg1Of described,previously
R1486 T1722 T1718 arg2Of 1,as
R1487 T1722 T1720 arg1Of 1,described
R1488 T1722 T1721 arg2Of 1,[
R1489 T1723 T1721 arg3Of ],[
R1490 T1727 T1724 arg1Of assays,Chloramphenicol
R1491 T1727 T1725 arg1Of assays,acetyl
R1492 T1727 T1726 arg1Of assays,transferase
R1493 T1727 T1728 arg1Of assays,have
R1494 T1727 T1729 arg1Of assays,been
R1495 T1727 T1730 arg2Of assays,described
R1496 T1730 T1728 arg2Of described,have
R1497 T1730 T1729 arg2Of described,been
R1498 T1730 T1731 arg1Of described,previously
R1499 T1730 T1732 arg1Of described,[
R1500 T1733 T1732 arg2Of 29,[
R1501 T1734 T1732 arg3Of ],[
R1596 T1869 T1868 arg1Of staining,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM"
R1598 T1872 T1870 arg1Of cells,DT40
R1599 T1872 T1871 arg1Of cells,B
R1600 T1872 T1873 arg1Of cells,(
R1601 T1872 T1880 arg2Of cells,resuspended
R1604 T1875 T1873 arg2Of cells,(
R1606 T1878 T1873 arg3Of ),(
R1607 T1880 T1879 arg2Of resuspended,were
R1608 T1880 T1881 arg1Of resuspended,in
R1609 T1883 T1881 arg2Of buffer,in
R1610 T1883 T1882 arg1Of buffer,200 μl
R1611 T1883 T1884 arg1Of buffer,(
R1613 T1887 T1884 arg2Of media,(
R1614 T1887 T1885 arg1Of media,RPMI
R1615 T1887 T1886 arg1Of media,1640
R1616 T1887 T1888 arg1Of media,","
R1618 T1893 T1888 arg2Of serum,","
R1619 T1893 T1889 arg1Of serum,1
R1622 T1894 T1884 arg3Of ),(
R1653 T1924 T1923 arg2Of representative,are
R1654 T1924 T1926 arg1Of representative,at
R1655 T1926 T1925 arg1Of at,of
R1656 T1927 T1928 arg1Of two,to
R1657 T1929 T1928 arg2Of four,to
R1658 T1931 T1926 arg2Of experiments,at
R1659 T1931 T1927 arg1Of experiments,two
R1660 T1931 T1930 arg1Of experiments,independent
R1661 T1934 T1932 arg2Of indicated,unless
R1662 T1934 T1933 arg1Of indicated,otherwise
R1887 T2288 T2289 arg1Of "Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss",of
R1888 T2288 T2292 arg1Of "Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss",in
R1889 T2291 T2289 arg2Of phosphorylation,of
R1890 T2291 T2290 arg1Of phosphorylation,HSP27
R1891 T2295 T2292 arg2Of cells,in
R1892 T2295 T2293 arg1Of cells,DT40
R1893 T2295 T2294 arg1Of cells,B
R1894 T2295 T2296 arg1Of cells,lacking
R1895 T2297 T2296 arg2Of expression,lacking
R1896 T2297 T2298 arg1Of expression,of
R1897 T2301 T2298 arg2Of kinases,of
R1898 T2301 T2299 arg1Of kinases,PKD
R1899 T2301 T2300 arg1Of kinases,family
R1900 T2304 T2302 arg1Of cells,DT40
R1901 T2304 T2303 arg1Of cells,B
R1902 T2304 T2305 arg1Of cells,express
R1903 T2308 T2305 arg2Of isoforms,express
R1904 T2308 T2306 arg1Of isoforms,two
R1905 T2308 T2307 arg1Of isoforms,PKD
R1906 T2308 T2309 arg1Of isoforms,","
R1907 T2310 T2311 arg1Of PKD1,and
R1908 T2311 T2309 arg2Of and,","
R1909 T2311 T2314 arg1Of and,and
R1910 T2311 T2315 arg1Of and,as
R1911 T2312 T2311 arg2Of PKD3,and
R1912 T2314 T2313 arg1Of and,","
R1913 T2317 T2315 arg2Of described,as
R1914 T2317 T2316 arg1Of described,previously
R1915 T2318 T2319 arg1Of we,have
R1916 T2318 T2321 arg1Of we,generated
R1917 T2321 T2317 arg3Of generated,described
R1918 T2321 T2319 arg2Of generated,have
R1919 T2321 T2320 arg1Of generated,recently
R1920 T2321 T2337 arg1Of generated,[
R1921 T2325 T2321 arg2Of lines,generated
R1922 T2325 T2322 arg1Of lines,DT40
R1923 T2325 T2323 arg1Of lines,B
R1924 T2325 T2324 arg1Of lines,cell
R1925 T2325 T2326 arg1Of lines,that
R1926 T2325 T2327 arg1Of lines,lack
R1927 T2328 T2329 arg1Of expression,of
R1928 T2328 T2334 arg1Of expression,or
R1929 T2331 T2332 arg1Of PKD1,or
R1930 T2332 T2329 arg2Of or,of
R1931 T2332 T2330 arg1Of or,either
R1932 T2333 T2332 arg2Of PKD3,or
R1933 T2334 T2327 arg2Of or,lack
R1934 T2336 T2334 arg2Of enzymes,or
R1935 T2336 T2335 arg1Of enzymes,both
R1936 T2338 T2337 arg2Of 1,[
R1937 T2339 T2337 arg3Of ],[
R1938 T2341 T2340 arg2Of generating,In
R1939 T2346 T2341 arg2Of lines,generating
R1940 T2346 T2342 arg1Of lines,the
R1941 T2346 T2343 arg1Of lines,double
R1942 T2346 T2344 arg1Of lines,knockout
R1943 T2346 T2345 arg1Of lines,cell
R1944 T2347 T2348 arg1Of we,targeted
R1945 T2348 T2340 arg1Of targeted,In
R1946 T2348 T2352 arg1Of targeted,in
R1947 T2351 T2348 arg2Of loci,targeted
R1948 T2351 T2349 arg1Of loci,the
R1949 T2351 T2350 arg1Of loci,PKD1
R1950 T2356 T2352 arg2Of line,in
R1951 T2356 T2353 arg1Of line,a
R1952 T2356 T2354 arg1Of line,PKD3−/−
R1953 T2356 T2355 arg1Of line,cell
R1954 T2356 T2357 arg1Of line,that
R1955 T2356 T2358 arg1Of line,expressed
R1956 T2358 T2362 arg1Of expressed,under
R1957 T2361 T2358 arg2Of transgene,expressed
R1958 T2361 T2359 arg1Of transgene,a
R1959 T2361 T2360 arg1Of transgene,Flag-PKD3
R1960 T2364 T2362 arg2Of control,under
R1961 T2364 T2363 arg1Of control,the
R1962 T2364 T2365 arg1Of control,of
R1963 T2368 T2365 arg2Of promoter,of
R1964 T2368 T2366 arg1Of promoter,a
R1965 T2368 T2367 arg1Of promoter,doxycycline-inducible
R1966 T2373 T2371 arg2Of presence,in
R1967 T2373 T2372 arg1Of presence,the
R1968 T2373 T2374 arg1Of presence,of
R1969 T2375 T2374 arg2Of doxycycline,of
R1970 T2378 T2377 arg1Of expression,Flag-PKD3
R1971 T2378 T2379 arg1Of expression,in
R1972 T2378 T2384 arg1Of expression,is
R1973 T2378 T2385 arg1Of expression,comparable
R1974 T2383 T2379 arg2Of cells,in
R1975 T2383 T2380 arg1Of cells,PKD1/3
R1976 T2383 T2381 arg1Of cells,double
R1977 T2383 T2382 arg1Of cells,knockout
R1978 T2384 T2394 arg1Of is,and
R1979 T2385 T2384 arg2Of comparable,is
R1980 T2385 T2386 arg1Of comparable,to
R1981 T2385 T2390 arg1Of comparable,in
R1982 T2388 T2386 arg2Of PKD3,to
R1983 T2388 T2387 arg1Of PKD3,endogenous
R1984 T2388 T2389 arg1Of PKD3,present
R1985 T2393 T2390 arg2Of cells,in
R1986 T2393 T2391 arg1Of cells,wild-type
R1987 T2393 T2392 arg1Of cells,DT40
R1988 T2394 T2369 arg1Of and,Hence
R1989 T2394 T2370 arg1Of and,","
R1990 T2394 T2371 arg1Of and,in
R1991 T2394 T2376 arg1Of and,","
R1992 T2395 T2396 arg1Of removal,of
R1993 T2395 T2405 arg1Of removal,results
R1994 T2397 T2396 arg2Of doxycycline,of
R1995 T2397 T2398 arg1Of doxycycline,from
R1996 T2397 T2402 arg1Of doxycycline,for
R1997 T2401 T2398 arg2Of media,from
R1998 T2401 T2399 arg1Of media,the
R1999 T2401 T2400 arg1Of media,culture
R2000 T2404 T2402 arg2Of days,for
R2001 T2404 T2403 arg1Of days,5
R2002 T2405 T2394 arg2Of results,and
R2003 T2405 T2406 arg1Of results,in
R2004 T2409 T2408 arg1Of null,completely
R2005 T2411 T2406 arg2Of phenotype,in
R2006 T2411 T2407 arg1Of phenotype,a
R2007 T2411 T2409 arg1Of phenotype,null
R2008 T2411 T2410 arg1Of phenotype,PKD
R2009 T2411 T2412 arg1Of phenotype,(
R2010 T2414 T2412 arg2Of 1A,(
R2011 T2414 T2413 arg1Of 1A,Fig.
R2012 T2415 T2412 arg3Of ),(
R2013 T2418 T2419 arg1Of we,have
R2014 T2418 T2420 arg1Of we,demonstrated
R2015 T2420 T2416 arg1Of demonstrated,Previously
R2016 T2420 T2417 arg1Of demonstrated,","
R2017 T2420 T2419 arg2Of demonstrated,have
R2018 T2422 T2423 arg1Of phosphorylation,and
R2019 T2423 T2426 arg1Of and,of
R2020 T2423 T2437 arg1Of and,is
R2021 T2423 T2438 arg1Of and,defective
R2022 T2423 T2444 arg1Of and,can
R2023 T2423 T2445 arg1Of and,restored
R2024 T2425 T2423 arg2Of exclusion,and
R2025 T2425 T2424 arg1Of exclusion,nuclear
R2026 T2430 T2426 arg2Of deacetylases,of
R2027 T2430 T2427 arg1Of deacetylases,class
R2028 T2430 T2428 arg1Of deacetylases,II
R2029 T2430 T2429 arg1Of deacetylases,histone
R2030 T2430 T2431 arg1Of deacetylases,(
R2031 T2430 T2434 arg1Of deacetylases,during
R2032 T2432 T2431 arg2Of HDACs,(
R2033 T2433 T2431 arg3Of ),(
R2034 T2436 T2434 arg2Of engagement,during
R2035 T2436 T2435 arg1Of engagement,BCR
R2036 T2437 T2443 arg1Of is,and
R2037 T2438 T2437 arg2Of defective,is
R2038 T2438 T2439 arg1Of defective,in
R2039 T2442 T2439 arg2Of cells,in
R2040 T2442 T2440 arg1Of cells,PKD1/3−/−
R2041 T2442 T2441 arg1Of cells,B
R2042 T2443 T2420 arg2Of and,demonstrated
R2043 T2443 T2421 arg1Of and,that
R2044 T2445 T2443 arg2Of restored,and
R2045 T2445 T2444 arg2Of restored,can
R2046 T2445 T2446 arg1Of restored,upon
R2047 T2447 T2446 arg2Of re-expression,upon
R2048 T2447 T2448 arg1Of re-expression,of
R2049 T2452 T2448 arg2Of isoform,of
R2050 T2452 T2449 arg1Of isoform,a
R2051 T2452 T2450 arg1Of isoform,single
R2052 T2452 T2451 arg1Of isoform,PKD
R2053 T2452 T2453 arg1Of isoform,[
R2054 T2454 T2453 arg2Of 1,[
R2055 T2455 T2453 arg3Of ],[
R2056 T2461 T2456 arg1Of HSP27,The
R2057 T2461 T2457 arg1Of HSP27,small
R2058 T2461 T2458 arg1Of HSP27,heat
R2059 T2461 T2459 arg1Of HSP27,shock
R2060 T2461 T2460 arg1Of HSP27,protein
R2061 T2461 T2462 arg1Of HSP27,has
R2062 T2461 T2464 arg1Of HSP27,been
R2063 T2461 T2465 arg2Of HSP27,proposed
R2064 T2465 T2462 arg2Of proposed,has
R2065 T2465 T2463 arg1Of proposed,recently
R2066 T2465 T2464 arg2Of proposed,been
R2067 T2465 T2466 arg1Of proposed,as
R2068 T2465 T2470 arg1Of proposed,[
R2069 T2465 T2473 arg1Of proposed,and
R2070 T2469 T2466 arg2Of substrate,as
R2071 T2469 T2467 arg1Of substrate,a
R2072 T2469 T2468 arg1Of substrate,PKD1
R2073 T2471 T2470 arg2Of 24,[
R2074 T2472 T2470 arg3Of ],[
R2075 T2474 T2476 arg1Of we,assessed
R2076 T2476 T2473 arg2Of assessed,and
R2077 T2476 T2475 arg1Of assessed,accordingly
R2078 T2480 T2478 arg1Of cells,PKD-null
R2079 T2480 T2479 arg1Of cells,DT40
R2080 T2480 T2481 arg1Of cells,have
R2081 T2481 T2476 arg2Of have,assessed
R2082 T2481 T2477 arg1Of have,whether
R2083 T2483 T2481 arg2Of phosphorylation,have
R2084 T2483 T2482 arg1Of phosphorylation,defective
R2085 T2483 T2484 arg1Of phosphorylation,of
R2086 T2485 T2484 arg2Of HSP27,of
R2087 T2485 T2486 arg1Of HSP27,on
R2088 T2485 T2489 arg1Of HSP27,","
R2089 T2487 T2486 arg2Of serine,on
R2090 T2487 T2488 arg1Of serine,82
R2091 T2494 T2489 arg2Of sequence,","
R2092 T2494 T2490 arg1Of sequence,the
R2093 T2494 T2491 arg2Of sequence,proposed
R2094 T2494 T2492 arg1Of sequence,PKD1
R2095 T2494 T2493 arg1Of sequence,substrate
R2096 T2495 T2497 arg1Of We,investigated
R2097 T2497 T2496 arg1Of investigated,initially
R2098 T2499 T2497 arg2Of regulation,investigated
R2099 T2499 T2498 arg1Of regulation,the
R2100 T2499 T2500 arg1Of regulation,of
R2101 T2499 T2503 arg1Of regulation,in
R2102 T2502 T2500 arg2Of phosphorylation,of
R2103 T2502 T2501 arg1Of phosphorylation,HSP27
R2104 T2508 T2503 arg2Of cells,in
R2105 T2508 T2504 arg1Of cells,single
R2106 T2508 T2505 arg1Of cells,knockout
R2107 T2508 T2506 arg1Of cells,DT40
R2108 T2508 T2507 arg1Of cells,B
R2109 T2508 T2509 arg1Of cells,lacking
R2110 T2511 T2512 arg1Of PKD1,or
R2111 T2512 T2509 arg2Of or,lacking
R2112 T2512 T2510 arg1Of or,either
R2113 T2513 T2512 arg2Of PKD3,or
R2114 T2515 T2514 arg2Of shown,As
R2115 T2515 T2516 arg1Of shown,in
R2116 T2518 T2516 arg2Of 1B,in
R2117 T2518 T2517 arg1Of 1B,Fig.
R2118 T2520 T2521 arg1Of activation,of
R2119 T2520 T2524 arg1Of activation,or
R2120 T2523 T2521 arg2Of BCR,of
R2121 T2523 T2522 arg1Of BCR,the
R2122 T2524 T2530 arg1Of or,increased
R2123 T2525 T2524 arg2Of treatment,or
R2124 T2525 T2526 arg1Of treatment,with
R2125 T2529 T2526 arg2Of PdBu,with
R2126 T2529 T2527 arg1Of PdBu,the
R2127 T2529 T2528 arg1Of PdBu,DAG-mimetic
R2128 T2530 T2514 arg1Of increased,As
R2129 T2530 T2519 arg1Of increased,","
R2130 T2532 T2530 arg2Of levels,increased
R2131 T2532 T2531 arg1Of levels,the
R2132 T2532 T2533 arg1Of levels,of
R2133 T2535 T2533 arg2Of phosphorylation,of
R2134 T2535 T2534 arg1Of phosphorylation,HSP27
R2135 T2535 T2536 arg1Of phosphorylation,at
R2136 T2537 T2536 arg2Of S82,at
R2137 T2537 T2538 arg1Of S82,in
R2138 T2542 T2538 arg2Of cells,in
R2139 T2542 T2539 arg1Of cells,wild-type
R2140 T2542 T2540 arg1Of cells,DT40
R2141 T2542 T2541 arg1Of cells,B
R2142 T2543 T2544 arg1Of BCR,and
R2143 T2545 T2544 arg2Of phorbol,and
R2144 T2547 T2543 arg1Of signals,BCR
R2145 T2547 T2545 arg1Of signals,phorbol
R2146 T2547 T2546 arg1Of signals,ester
R2147 T2547 T2548 arg1Of signals,were
R2148 T2547 T2550 arg1Of signals,able
R2149 T2547 T2552 arg1Of signals,increase
R2150 T2548 T2549 arg1Of were,also
R2151 T2550 T2548 arg2Of able,were
R2152 T2552 T2550 arg2Of increase,able
R2153 T2552 T2551 arg1Of increase,to
R2154 T2554 T2552 arg2Of phosphorylation,increase
R2155 T2554 T2553 arg1Of phosphorylation,HSP27
R2156 T2554 T2555 arg1Of phosphorylation,in
R2157 T2556 T2557 arg1Of PKD1,or
R2158 T2558 T2557 arg2Of PKD3,or
R2159 T2563 T2555 arg2Of cells,in
R2160 T2563 T2556 arg1Of cells,PKD1
R2161 T2563 T2558 arg1Of cells,PKD3
R2162 T2563 T2559 arg1Of cells,single
R2163 T2563 T2560 arg1Of cells,knockout
R2164 T2563 T2561 arg1Of cells,DT40
R2165 T2563 T2562 arg1Of cells,B
R2166 T2563 T2564 arg1Of cells,(
R2167 T2566 T2564 arg2Of 1B,(
R2168 T2566 T2565 arg1Of 1B,Fig.
R2169 T2567 T2564 arg3Of ),(
R2170 T2570 T2571 arg1Of BCR-,and
R2171 T2571 T2577 arg1Of and,on
R2172 T2571 T2579 arg1Of and,was
R2173 T2571 T2580 arg2Of and,abolished
R2174 T2573 T2572 arg1Of ester-induced,phorbol
R2175 T2574 T2571 arg2Of phosphorylation,and
R2176 T2574 T2573 arg1Of phosphorylation,ester-induced
R2177 T2574 T2575 arg1Of phosphorylation,of
R2178 T2576 T2575 arg2Of HSP27,of
R2179 T2578 T2577 arg2Of S82,on
R2180 T2580 T2568 arg1Of abolished,However
R2181 T2580 T2569 arg1Of abolished,","
R2182 T2580 T2579 arg2Of abolished,was
R2183 T2580 T2581 arg1Of abolished,in
R2184 T2583 T2581 arg2Of cells,in
R2185 T2583 T2582 arg1Of cells,B
R2186 T2583 T2584 arg1Of cells,that
R2187 T2583 T2585 arg1Of cells,lacked
R2188 T2587 T2588 arg1Of PKD1,and
R2189 T2588 T2585 arg2Of and,lacked
R2190 T2588 T2586 arg1Of and,both
R2191 T2588 T2590 arg1Of and,(
R2192 T2589 T2588 arg2Of PKD3,and
R2193 T2592 T2590 arg2Of 1C,(
R2194 T2592 T2591 arg1Of 1C,Fig.
R2195 T2593 T2590 arg3Of ),(
R2196 T2597 T2596 arg1Of expression,doxycycline-induced
R2197 T2597 T2598 arg1Of expression,of
R2198 T2597 T2602 arg1Of expression,in
R2199 T2597 T2607 arg1Of expression,was
R2200 T2597 T2608 arg1Of expression,sufficient
R2201 T2601 T2598 arg2Of transgene,of
R2202 T2601 T2599 arg1Of transgene,the
R2203 T2601 T2600 arg1Of transgene,Flag-PKD3
R2204 T2606 T2602 arg2Of cells,in
R2205 T2606 T2603 arg1Of cells,the
R2206 T2606 T2604 arg1Of cells,double
R2207 T2606 T2605 arg1Of cells,knockout
R2208 T2607 T2594 arg1Of was,Significantly
R2209 T2607 T2595 arg1Of was,","
R2210 T2607 T2609 modOf was,to
R2211 T2608 T2607 arg2Of sufficient,was
R2212 T2610 T2609 arg1Of restore,to
R2213 T2612 T2610 arg2Of regulation,restore
R2214 T2612 T2611 arg1Of regulation,normal
R2215 T2612 T2613 arg1Of regulation,of
R2216 T2615 T2613 arg2Of phosphorylation,of
R2217 T2615 T2614 arg1Of phosphorylation,HSP27
R2218 T2615 T2616 arg1Of phosphorylation,(
R2219 T2618 T2616 arg2Of 1C,(
R2220 T2618 T2617 arg1Of 1C,Fig.
R2221 T2619 T2616 arg3Of ),(
R2222 T2621 T2620 arg2Of contrast,In
R2223 T2623 T2624 arg1Of expression,of
R2224 T2623 T2630 arg1Of expression,in
R2225 T2623 T2635 arg1Of expression,was
R2226 T2623 T2637 arg1Of expression,able
R2227 T2623 T2639 arg1Of expression,restore
R2228 T2629 T2624 arg2Of protein,of
R2229 T2629 T2625 arg1Of protein,a
R2230 T2629 T2626 arg1Of protein,kinase-deficient
R2231 T2629 T2627 arg1Of protein,PKD3
R2232 T2629 T2628 arg1Of protein,mutant
R2233 T2634 T2630 arg2Of cells,in
R2234 T2634 T2631 arg1Of cells,the
R2235 T2634 T2632 arg1Of cells,double
R2236 T2634 T2633 arg1Of cells,knockout
R2237 T2635 T2620 arg1Of was,In
R2238 T2635 T2622 arg1Of was,","
R2239 T2635 T2636 arg1Of was,not
R2240 T2637 T2635 arg2Of able,was
R2241 T2639 T2637 arg2Of restore,able
R2242 T2639 T2638 arg1Of restore,to
R2243 T2640 T2641 arg1Of BCR-,or
R2244 T2641 T2639 arg2Of or,restore
R2245 T2645 T2641 arg2Of phosphorylation,or
R2246 T2645 T2642 arg1Of phosphorylation,phorbol
R2247 T2645 T2643 arg1Of phosphorylation,ester-induced
R2248 T2645 T2644 arg1Of phosphorylation,HSP27
R2249 T2645 T2646 arg1Of phosphorylation,(
R2250 T2648 T2646 arg2Of 1D,(
R2251 T2648 T2647 arg1Of 1D,Fig.
R2252 T2649 T2646 arg3Of ),(
R2253 T2652 T2655 arg1Of PKD3,as
R2254 T2655 T2653 arg1Of as,as
R2255 T2655 T2654 arg1Of as,well
R2256 T2655 T2657 arg1Of as,can
R2257 T2655 T2658 arg1Of as,regulate
R2258 T2656 T2655 arg2Of PKD1,as
R2259 T2658 T2657 arg2Of regulate,can
R2260 T2658 T2661 arg1Of regulate,and
R2261 T2660 T2658 arg2Of phosphorylation,regulate
R2262 T2660 T2659 arg1Of phosphorylation,HSP27
R2263 T2661 T2650 arg1Of and,Hence
R2264 T2661 T2651 arg1Of and,","
R2265 T2665 T2662 arg2Of cells,in
R2266 T2665 T2663 arg1Of cells,DT40
R2267 T2665 T2664 arg1Of cells,B
R2268 T2666 T2667 arg1Of they,are
R2269 T2666 T2669 arg2Of they,redundant
R2270 T2669 T2661 arg2Of redundant,and
R2271 T2669 T2662 arg1Of redundant,in
R2272 T2669 T2667 arg2Of redundant,are
R2273 T2669 T2668 arg1Of redundant,functionally
R2274 T2669 T2670 arg1Of redundant,as
R2275 T2672 T2670 arg2Of kinases,as
R2276 T2672 T2671 arg1Of kinases,HSP27
R3056 T3837 T3838 arg1Of proliferation,and
R3057 T3838 T3836 arg1Of and,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular"
R3058 T3838 T3840 arg1Of and,in
R3059 T3839 T3838 arg2Of survival,and
R3060 T3843 T3840 arg2Of cells,in
R3061 T3843 T3841 arg1Of cells,DT40
R3062 T3843 T3842 arg1Of cells,B
R3063 T3843 T3844 arg1Of cells,lacking
R3064 T3845 T3844 arg2Of expression,lacking
R3065 T3845 T3846 arg1Of expression,of
R3066 T3849 T3846 arg2Of kinases,of
R3067 T3849 T3847 arg1Of kinases,PKD
R3068 T3849 T3848 arg1Of kinases,family
R3069 T3851 T3850 arg1Of enzymes,PKD
R3070 T3851 T3852 arg1Of enzymes,have
R3071 T3851 T3854 arg1Of enzymes,been
R3072 T3851 T3855 arg2Of enzymes,linked
R3073 T3855 T3852 arg2Of linked,have
R3074 T3855 T3853 arg1Of linked,previously
R3075 T3855 T3854 arg2Of linked,been
R3076 T3855 T3856 arg1Of linked,to
R3077 T3858 T3856 arg2Of regulation,to
R3078 T3858 T3857 arg1Of regulation,the
R3079 T3858 T3859 arg1Of regulation,of
R3080 T3861 T3862 arg1Of proliferation,and
R3081 T3862 T3859 arg2Of and,of
R3082 T3862 T3860 arg1Of and,cell
R3083 T3862 T3864 arg1Of and,(
R3084 T3863 T3862 arg2Of survival,and
R3085 T3865 T3864 arg2Of reviewed,(
R3086 T3865 T3866 arg1Of reviewed,in
R3087 T3865 T3867 arg1Of reviewed,[
R3088 T3868 T3867 arg2Of 20,[
R3089 T3869 T3867 arg3Of ],[
R3090 T3870 T3864 arg3Of ),(
R3091 T3872 T3871 arg1Of investigate,To
R3092 T3874 T3872 arg2Of effect,investigate
R3093 T3874 T3873 arg1Of effect,the
R3094 T3876 T3877 arg1Of loss,of
R3095 T3876 T3880 arg1Of loss,had
R3096 T3879 T3877 arg2Of kinases,of
R3097 T3879 T3878 arg1Of kinases,PKD
R3098 T3880 T3874 arg2Of had,effect
R3099 T3880 T3875 arg1Of had,that
R3100 T3880 T3881 arg1Of had,on
R3101 T3884 T3885 arg1Of survival,and/or
R3102 T3885 T3881 arg2Of and/or,on
R3103 T3885 T3882 arg1Of and/or,B
R3104 T3885 T3883 arg1Of and/or,cell
R3105 T3886 T3885 arg2Of proliferation,and/or
R3106 T3887 T3872 arg1Of we,investigate
R3107 T3887 T3888 arg1Of we,cultured
R3108 T3888 T3871 modOf cultured,To
R3109 T3888 T3893 arg1Of cultured,in
R3110 T3889 T3890 arg1Of wild-type,and
R3111 T3891 T3890 arg2Of PKD-null,and
R3112 T3892 T3888 arg2Of cells,cultured
R3113 T3892 T3889 arg1Of cells,wild-type
R3114 T3892 T3891 arg1Of cells,PKD-null
R3115 T3895 T3893 arg2Of presence,in
R3116 T3895 T3894 arg1Of presence,the
R3117 T3895 T3896 arg1Of presence,(
R3118 T3897 T3896 arg2Of PKD1/3−/−,(
R3119 T3898 T3896 arg3Of :,(
R3120 T3899 T3900 arg1Of Flag-PKD3+ve,)
R3121 T3899 T3901 arg1Of Flag-PKD3+ve,or
R3122 T3901 T3909 arg1Of or,monitored
R3123 T3902 T3901 arg2Of absence,or
R3124 T3902 T3903 arg1Of absence,(
R3125 T3902 T3906 arg1Of absence,of
R3126 T3904 T3903 arg2Of PKD1/3−/−,(
R3127 T3905 T3903 arg3Of ),(
R3128 T3907 T3906 arg2Of doxycycline,of
R3129 T3909 T3908 arg1Of monitored,and
R3130 T3911 T3909 arg2Of growth,monitored
R3131 T3911 T3910 arg1Of growth,exponential
R3132 T3913 T3912 arg2Of shown,As
R3133 T3913 T3914 arg1Of shown,in
R3134 T3916 T3914 arg2Of 2A,in
R3135 T3916 T3915 arg1Of 2A,Fig.
R3136 T3919 T3918 arg1Of cells,PKD1/3−/−
R3137 T3919 T3920 arg1Of cells,proliferated
R3138 T3920 T3921 arg1Of proliferated,exponentially
R3139 T3920 T3922 arg1Of proliferated,and
R3140 T3922 T3912 arg1Of and,As
R3141 T3922 T3917 arg1Of and,","
R3142 T3923 T3924 arg1Of re-expression,of
R3143 T3923 T3926 arg1Of re-expression,in
R3144 T3923 T3929 arg1Of re-expression,had
R3145 T3925 T3924 arg2Of Flag-PKD3,of
R3146 T3928 T3926 arg2Of cells,in
R3147 T3928 T3927 arg1Of cells,these
R3148 T3929 T3922 arg2Of had,and
R3149 T3931 T3929 arg2Of impact,had
R3150 T3931 T3930 arg1Of impact,no
R3151 T3931 T3932 arg1Of impact,on
R3152 T3934 T3932 arg2Of rate,on
R3153 T3934 T3933 arg1Of rate,the
R3154 T3934 T3935 arg1Of rate,of
R3155 T3936 T3935 arg2Of proliferation,of
R3156 T3940 T3939 arg1Of viability,the
R3157 T3940 T3941 arg1Of viability,of
R3158 T3940 T3945 arg1Of viability,during
R3159 T3940 T3948 arg1Of viability,was
R3160 T3940 T3951 arg1Of viability,different
R3161 T3944 T3941 arg2Of cells,of
R3162 T3944 T3942 arg1Of cells,PKD1/3−/−
R3163 T3944 T3943 arg1Of cells,B
R3164 T3947 T3945 arg2Of culturing,during
R3165 T3947 T3946 arg1Of culturing,routine
R3166 T3948 T3937 arg1Of was,Furthermore
R3167 T3948 T3938 arg1Of was,","
R3168 T3948 T3949 arg1Of was,not
R3169 T3951 T3948 arg2Of different,was
R3170 T3951 T3950 arg1Of different,significantly
R3171 T3951 T3952 arg1Of different,from
R3172 T3953 T3952 arg2Of that,from
R3173 T3953 T3954 arg1Of that,of
R3174 T3957 T3954 arg2Of cells,of
R3175 T3957 T3955 arg1Of cells,wild-type
R3176 T3957 T3956 arg1Of cells,B
R3177 T3957 T3958 arg1Of cells,(
R3178 T3959 T3958 arg2Of data,(
R3179 T3959 T3961 arg2Of data,shown
R3180 T3961 T3960 arg1Of shown,not
R3181 T3962 T3958 arg3Of ),(
R3182 T3963 T3964 arg1Of It,was
R3183 T3963 T3965 arg2Of It,noted
R3184 T3965 T3964 arg2Of noted,was
R3185 T3970 T3967 arg1Of time,the
R3186 T3970 T3968 arg1Of time,population
R3187 T3970 T3969 arg1Of time,doubling
R3188 T3970 T3971 arg1Of time,of
R3189 T3970 T3974 arg1Of time,was
R3190 T3970 T3976 arg1Of time,slower
R3191 T3973 T3971 arg2Of cells,of
R3192 T3973 T3972 arg1Of cells,PKD1/3−/−
R3193 T3974 T3989 arg1Of was,but
R3194 T3976 T3974 arg2Of slower,was
R3195 T3976 T3975 arg1Of slower,slightly
R3196 T3976 T3977 arg1Of slower,than
R3197 T3978 T3977 arg2Of that,than
R3198 T3978 T3979 arg1Of that,of
R3199 T3983 T3979 arg2Of cells,of
R3200 T3983 T3980 arg1Of cells,wild
R3201 T3983 T3981 arg1Of cells,type
R3202 T3983 T3982 arg1Of cells,DT40
R3203 T3983 T3984 arg1Of cells,(
R3204 T3985 T3984 arg2Of 12.7 ± 2.8 h,(
R3205 T3985 T3986 arg1Of 12.7 ± 2.8 h,versus
R3206 T3987 T3986 arg2Of 10.2 ± 0.4 h,versus
R3207 T3988 T3984 arg3Of ),(
R3208 T3989 T3965 arg3Of but,noted
R3209 T3989 T3966 arg1Of but,that
R3210 T3991 T3990 arg1Of failure,the
R3211 T3991 T3992 arg1Of failure,of
R3212 T3991 T3995 modOf failure,to
R3213 T3991 T4003 arg1Of failure,suggests
R3214 T3994 T3992 arg2Of re-expression,of
R3215 T3994 T3993 arg1Of re-expression,PKD3
R3216 T3996 T3995 arg1Of modify,to
R3217 T3999 T3996 arg2Of rate,modify
R3218 T3999 T3997 arg1Of rate,the
R3219 T3999 T3998 arg1Of rate,proliferation
R3220 T3999 T4000 arg1Of rate,of
R3221 T4002 T4000 arg2Of cells,of
R3222 T4002 T4001 arg1Of cells,PKD1/3−/−
R3223 T4003 T3989 arg2Of suggests,but
R3224 T4007 T4005 arg1Of differences,these
R3225 T4007 T4006 arg1Of differences,small
R3226 T4007 T4008 arg1Of differences,were
R3227 T4007 T4017 arg1Of differences,were
R3228 T4007 T4019 arg2Of differences,caused
R3229 T4008 T4010 arg1Of were,likely
R3230 T4008 T4016 arg1Of were,and
R3231 T4010 T4009 arg1Of likely,most
R3232 T4012 T4008 arg2Of result,were
R3233 T4012 T4011 arg1Of result,the
R3234 T4012 T4013 arg1Of result,of
R3235 T4015 T4013 arg2Of variation,of
R3236 T4015 T4014 arg1Of variation,clonal
R3237 T4016 T4003 arg2Of and,suggests
R3238 T4016 T4004 arg1Of and,that
R3239 T4019 T4016 arg2Of caused,and
R3240 T4019 T4017 arg2Of caused,were
R3241 T4019 T4018 arg1Of caused,not
R3242 T4019 T4020 arg1Of caused,specifically
R3243 T4022 T4019 arg1Of loss,caused
R3244 T4022 T4021 arg2Of loss,by
R3245 T4022 T4023 arg1Of loss,of
R3246 T4025 T4023 arg2Of enzymes,of
R3247 T4025 T4024 arg1Of enzymes,PKD
R3248 T4030 T4028 arg1Of enzymes,PKD
R3249 T4030 T4029 arg1Of enzymes,family
R3250 T4030 T4031 arg1Of enzymes,are
R3251 T4030 T4033 arg1Of enzymes,essential
R3252 T4031 T4026 arg1Of are,Thus
R3253 T4031 T4027 arg1Of are,","
R3254 T4031 T4032 arg1Of are,not
R3255 T4033 T4031 arg2Of essential,are
R3256 T4033 T4034 arg1Of essential,for
R3257 T4035 T4034 arg2Of regulating,for
R3258 T4037 T4036 arg1Of survival,basal
R3259 T4037 T4038 arg1Of survival,and
R3260 T4038 T4035 arg2Of and,regulating
R3261 T4039 T4038 arg2Of proliferation,and
R3262 T4039 T4040 arg1Of proliferation,of
R3263 T4043 T4040 arg2Of cells,of
R3264 T4043 T4041 arg1Of cells,DT40
R3265 T4043 T4042 arg1Of cells,B
R3266 T4045 T4044 arg1Of enzymes,PKD
R3267 T4045 T4046 arg1Of enzymes,","
R3268 T4045 T4052 arg1Of enzymes,have
R3269 T4045 T4054 arg1Of enzymes,been
R3270 T4045 T4055 arg2Of enzymes,linked
R3271 T4048 T4049 arg1Of PKD1,and
R3272 T4049 T4046 arg2Of and,","
R3273 T4049 T4047 arg1Of and,specifically
R3274 T4050 T4049 arg2Of PKD2,and
R3275 T4055 T4051 arg1Of linked,","
R3276 T4055 T4052 arg2Of linked,have
R3277 T4055 T4053 arg1Of linked,previously
R3278 T4055 T4054 arg2Of linked,been
R3279 T4055 T4056 arg1Of linked,to
R3280 T4059 T4056 arg2Of role,to
R3281 T4059 T4057 arg1Of role,a
R3282 T4059 T4058 arg1Of role,protective
R3283 T4059 T4060 arg1Of role,against
R3284 T4062 T4061 arg1Of stress-induced,oxidative
R3285 T4063 T4060 arg2Of injury,against
R3286 T4063 T4062 arg1Of injury,stress-induced
R3287 T4063 T4064 arg1Of injury,in
R3288 T4066 T4067 arg1Of fibroblast,","
R3289 T4068 T4067 arg2Of HeLa,","
R3290 T4072 T4064 arg2Of lines,in
R3291 T4072 T4065 arg1Of lines,3T3
R3292 T4072 T4066 arg1Of lines,fibroblast
R3293 T4072 T4068 arg1Of lines,HeLa
R3294 T4072 T4069 arg1Of lines,and
R3295 T4072 T4070 arg1Of lines,epithelial
R3296 T4072 T4071 arg1Of lines,cell
R3297 T4072 T4073 arg1Of lines,[
R3298 T4074 T4073 arg2Of "17,30–32",[
R3299 T4075 T4073 arg3Of ],[
R3300 T4076 T4078 arg1Of We,addressed
R3301 T4078 T4077 arg1Of addressed,therefore
R3302 T4080 T4078 arg2Of role,addressed
R3303 T4080 T4079 arg1Of role,the
R3304 T4080 T4081 arg1Of role,of
R3305 T4080 T4085 arg1Of role,in
R3306 T4084 T4081 arg2Of kinases,of
R3307 T4084 T4082 arg1Of kinases,PKD
R3308 T4084 T4083 arg1Of kinases,family
R3309 T4086 T4085 arg2Of regulating,in
R3310 T4086 T4090 arg1Of regulating,in
R3311 T4089 T4086 arg2Of survival,regulating
R3312 T4089 T4087 arg1Of survival,B
R3313 T4089 T4088 arg1Of survival,cell
R3314 T4090 T4092 arg1Of in,to
R3315 T4091 T4090 arg2Of response,in
R3316 T4094 T4093 arg1Of stress,oxidative
R3317 T4094 T4095 arg1Of stress,and
R3318 T4095 T4090 arg3Of and,in
R3319 T4098 T4095 arg2Of stimuli,and
R3320 T4098 T4096 arg1Of stimuli,other
R3321 T4098 T4097 arg1Of stimuli,stress
R3322 T4100 T4099 arg2Of shown,As
R3323 T4100 T4101 arg1Of shown,in
R3324 T4103 T4101 arg2Of 2B,in
R3325 T4103 T4102 arg1Of 2B,Fig.
R3326 T4105 T4106 arg1Of loss,of
R3327 T4105 T4109 arg1Of loss,had
R3328 T4108 T4106 arg2Of expression,of
R3329 T4108 T4107 arg1Of expression,PKD1/3
R3330 T4109 T4099 arg1Of had,As
R3331 T4109 T4104 arg1Of had,","
R3332 T4109 T4113 arg1Of had,on
R3333 T4109 T4120 arg1Of had,in
R3334 T4109 T4153 arg1Of had,following
R3335 T4112 T4109 arg2Of impact,had
R3336 T4112 T4110 arg1Of impact,no
R3337 T4112 T4111 arg1Of impact,significant
R3338 T4115 T4113 arg2Of survival,on
R3339 T4115 T4114 arg1Of survival,the
R3340 T4115 T4116 arg1Of survival,of
R3341 T4119 T4116 arg2Of cells,of
R3342 T4119 T4117 arg1Of cells,DT40
R3343 T4119 T4118 arg1Of cells,B
R3344 T4120 T4122 arg1Of in,to
R3345 T4120 T4152 arg1Of in,or
R3346 T4121 T4120 arg2Of response,in
R3347 T4125 T4123 arg1Of stimuli,mitochondrial
R3348 T4125 T4124 arg1Of stimuli,stress
R3349 T4125 T4126 arg1Of stimuli,(
R3350 T4125 T4132 arg1Of stimuli,;
R3351 T4127 T4128 arg1Of H2O2,or
R3352 T4128 T4126 arg2Of or,(
R3353 T4130 T4128 arg2Of deprivation,or
R3354 T4130 T4129 arg1Of deprivation,serum
R3355 T4131 T4126 arg3Of ),(
R3356 T4132 T4141 arg1Of ;,;
R3357 T4135 T4132 arg2Of agents,;
R3358 T4135 T4133 arg1Of agents,DNA
R3359 T4135 T4134 arg1Of agents,damaging
R3360 T4135 T4136 arg1Of agents,(
R3361 T4137 T4138 arg1Of etoposide,or
R3362 T4138 T4136 arg2Of or,(
R3363 T4139 T4138 arg2Of doxorubicin,or
R3364 T4140 T4136 arg3Of ),(
R3365 T4141 T4120 arg3Of ;,in
R3366 T4144 T4141 arg2Of stress,;
R3367 T4144 T4142 arg1Of stress,ER
R3368 T4144 T4143 arg1Of stress,pathway
R3369 T4144 T4145 arg1Of stress,due
R3370 T4145 T4146 arg1Of due,to
R3371 T4148 T4145 arg2Of overload,due
R3372 T4148 T4147 arg1Of overload,calcium
R3373 T4148 T4149 arg1Of overload,(
R3374 T4150 T4149 arg2Of thapsigargin,(
R3375 T4151 T4149 arg3Of ),(
R3376 T4153 T4152 arg2Of following,or
R3377 T4155 T4153 arg2Of treatment,following
R3378 T4155 T4154 arg2Of treatment,prolonged
R3379 T4155 T4156 arg1Of treatment,with
R3380 T4158 T4157 arg1Of esters,phorbol
R3381 T4158 T4159 arg1Of esters,or
R3382 T4159 T4156 arg2Of or,with
R3383 T4159 T4162 arg1Of or,","
R3384 T4161 T4159 arg2Of A,or
R3385 T4161 T4160 arg1Of A,Trichostatin
R3386 T4164 T4162 arg2Of inhibitor,","
R3387 T4164 T4163 arg1Of inhibitor,an
R3388 T4164 T4165 arg1Of inhibitor,of
R3389 T4168 T4165 arg2Of HDACs,of
R3390 T4168 T4166 arg1Of HDACs,class
R3391 T4168 T4167 arg1Of HDACs,I/II
R3392 T4172 T4171 arg1Of kinases,PKD
R3393 T4172 T4173 arg1Of kinases,do
R3394 T4172 T4175 arg1Of kinases,play
R3395 T4175 T4169 arg1Of play,Thus
R3396 T4175 T4170 arg1Of play,","
R3397 T4175 T4173 arg2Of play,do
R3398 T4175 T4174 arg1Of play,not
R3399 T4175 T4179 arg1Of play,in
R3400 T4178 T4175 arg2Of role,play
R3401 T4178 T4176 arg1Of role,an
R3402 T4178 T4177 arg1Of role,essential
R3403 T4180 T4179 arg2Of regulating,in
R3404 T4180 T4184 arg1Of regulating,in
R3405 T4183 T4180 arg2Of survival,regulating
R3406 T4183 T4181 arg1Of survival,B
R3407 T4183 T4182 arg1Of survival,cell
R3408 T4184 T4186 arg1Of in,to
R3409 T4185 T4184 arg2Of response,in
R3410 T4188 T4184 arg3Of range,in
R3411 T4188 T4187 arg1Of range,a
R3412 T4188 T4189 arg1Of range,of
R3413 T4192 T4189 arg2Of stimuli,of
R3414 T4192 T4190 arg1Of stimuli,different
R3415 T4192 T4191 arg1Of stimuli,stress
R3931 T5032 T5031 arg1Of receptor,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen"
R3932 T5032 T5033 arg1Of receptor,regulated
R3933 T5035 T5033 arg2Of pathways,regulated
R3934 T5035 T5034 arg1Of pathways,signalling
R3935 T5035 T5036 arg1Of pathways,in
R3936 T5040 T5036 arg2Of cells,in
R3937 T5040 T5037 arg1Of cells,PKD-null
R3938 T5040 T5038 arg1Of cells,DT40
R3939 T5040 T5039 arg1Of cells,B
R3940 T5043 T5041 arg1Of explore,To
R3941 T5043 T5042 arg1Of explore,further
R3942 T5045 T5043 arg2Of contribution,explore
R3943 T5045 T5044 arg1Of contribution,the
R3944 T5045 T5046 arg1Of contribution,of
R3945 T5045 T5049 arg1Of contribution,to
R3946 T5048 T5046 arg2Of kinases,of
R3947 T5048 T5047 arg1Of kinases,PKD
R3948 T5053 T5049 arg2Of biology,to
R3949 T5053 T5050 arg1Of biology,DT40
R3950 T5053 T5051 arg1Of biology,B
R3951 T5053 T5052 arg1Of biology,cell
R3952 T5054 T5043 arg1Of we,explore
R3953 T5054 T5055 arg1Of we,investigated
R3954 T5055 T5041 modOf investigated,To
R3955 T5058 T5057 arg1Of BCR-regulated,specific
R3956 T5060 T5058 arg1Of events,BCR-regulated
R3957 T5060 T5059 arg1Of events,signalling
R3958 T5060 T5061 arg1Of events,were
R3959 T5060 T5062 arg1Of events,defective
R3960 T5061 T5055 arg2Of were,investigated
R3961 T5061 T5056 arg1Of were,whether
R3962 T5062 T5061 arg2Of defective,were
R3963 T5062 T5063 arg1Of defective,in
R3964 T5067 T5063 arg2Of cells,in
R3965 T5067 T5064 arg1Of cells,the
R3966 T5067 T5065 arg1Of cells,PKD-null
R3967 T5067 T5066 arg1Of cells,B
R3968 T5069 T5068 arg1Of experiments,Initial
R3969 T5069 T5070 arg1Of experiments,revealed
R3970 T5073 T5072 arg1Of expression,surface
R3971 T5073 T5074 arg1Of expression,of
R3972 T5073 T5077 arg1Of expression,was
R3973 T5073 T5078 arg2Of expression,reduced
R3974 T5076 T5074 arg2Of BCR,of
R3975 T5076 T5075 arg1Of BCR,the
R3976 T5078 T5070 arg2Of reduced,revealed
R3977 T5078 T5071 arg1Of reduced,that
R3978 T5078 T5077 arg2Of reduced,was
R3979 T5078 T5079 arg1Of reduced,in
R3980 T5078 T5089 arg1Of reduced,compared
R3981 T5080 T5081 arg1Of PKD1/3−/−,(
R3982 T5083 T5081 arg2Of in,(
R3983 T5083 T5082 arg1Of in,and
R3984 T5084 T5083 arg2Of PKD1/3−/−,in
R3985 T5084 T5085 arg1Of PKD1/3−/−,:
R3986 T5084 T5086 arg1Of PKD1/3−/−,Flag-PKD3+ve
R3987 T5087 T5081 arg3Of ),(
R3988 T5088 T5079 arg2Of cells,in
R3989 T5088 T5080 arg1Of cells,PKD1/3−/−
R3990 T5090 T5089 arg2Of to,compared
R3991 T5094 T5090 arg2Of cells,to
R3992 T5094 T5091 arg1Of cells,wild-type
R3993 T5094 T5092 arg1Of cells,DT40
R3994 T5094 T5093 arg1Of cells,B
R3995 T5094 T5095 arg1Of cells,(
R3996 T5097 T5096 arg1Of 3A,Fig.
R3997 T5097 T5098 arg1Of 3A,and
R3998 T5098 T5095 arg2Of and,(
R3999 T5099 T5098 arg2Of data,and
R4000 T5099 T5101 arg2Of data,shown
R4001 T5101 T5100 arg1Of shown,not
R4002 T5102 T5095 arg3Of ),(
R4003 T5105 T5106 arg1Of BCR-crosslinking,of
R4004 T5105 T5109 arg1Of BCR-crosslinking,was
R4005 T5105 T5110 arg1Of BCR-crosslinking,sufficient
R4006 T5105 T5112 arg1Of BCR-crosslinking,induce
R4007 T5108 T5106 arg2Of cells,of
R4008 T5108 T5107 arg1Of cells,PKD1/3−/−
R4009 T5109 T5103 arg1Of was,Nevertheless
R4010 T5109 T5104 arg1Of was,","
R4011 T5110 T5109 arg2Of sufficient,was
R4012 T5112 T5110 arg2Of induce,sufficient
R4013 T5112 T5111 arg1Of induce,to
R4014 T5114 T5112 arg2Of activation,induce
R4015 T5114 T5113 arg1Of activation,the
R4016 T5114 T5115 arg1Of activation,of
R4017 T5117 T5115 arg2Of number,of
R4018 T5117 T5116 arg1Of number,a
R4019 T5117 T5118 arg1Of number,of
R4020 T5117 T5121 arg1Of number,","
R4021 T5117 T5122 arg1Of number,similar
R4022 T5120 T5118 arg2Of cascades,of
R4023 T5120 T5119 arg1Of cascades,signalling
R4024 T5122 T5123 arg1Of similar,to
R4025 T5124 T5123 arg2Of that,to
R4026 T5124 T5125 arg2Of that,observed
R4027 T5125 T5126 arg1Of observed,in
R4028 T5128 T5126 arg2Of cells,in
R4029 T5128 T5127 arg1Of cells,wild-type
R4030 T5128 T5129 arg1Of cells,(
R4031 T5131 T5129 arg2Of 3B,(
R4032 T5131 T5130 arg1Of 3B,Fig.
R4033 T5132 T5129 arg3Of ),(
R4034 T5136 T5135 arg1Of activation,BCR-induced
R4035 T5136 T5137 arg1Of activation,of
R4036 T5136 T5140 arg1Of activation,","
R4037 T5139 T5137 arg2Of Akt,of
R4038 T5139 T5138 arg1Of Akt,the
R4039 T5140 T5153 arg1Of ",",and
R4040 T5143 T5140 arg2Of kinase,","
R4041 T5143 T5141 arg1Of kinase,mTOR/p70
R4042 T5143 T5142 arg1Of kinase,S6
R4043 T5143 T5144 arg1Of kinase,(
R4044 T5143 T5145 arg1Of kinase,as
R4045 T5143 T5152 arg1Of kinase,)
R4046 T5146 T5145 arg2Of shown,as
R4047 T5151 T5146 arg1Of phosphorylation,shown
R4048 T5151 T5147 arg2Of phosphorylation,by
R4049 T5151 T5148 arg1Of phosphorylation,S6
R4050 T5151 T5149 arg1Of phosphorylation,ribosomal
R4051 T5151 T5150 arg1Of phosphorylation,protein
R4052 T5153 T5157 arg1Of and,was
R4053 T5153 T5159 arg1Of and,detectable
R4054 T5156 T5153 arg2Of pathways,and
R4055 T5156 T5154 arg1Of pathways,MAPK
R4056 T5156 T5155 arg1Of pathways,signalling
R4057 T5157 T5133 arg1Of was,Hence
R4058 T5157 T5134 arg1Of was,","
R4059 T5159 T5157 arg2Of detectable,was
R4060 T5159 T5158 arg1Of detectable,clearly
R4061 T5159 T5160 arg1Of detectable,in
R4062 T5163 T5160 arg2Of cells,in
R4063 T5163 T5161 arg1Of cells,PKD1/3-null
R4064 T5163 T5162 arg1Of cells,B
R4065 T5163 T5164 arg1Of cells,(
R4066 T5166 T5164 arg2Of 3B,(
R4067 T5166 T5165 arg1Of 3B,Fig.
R4068 T5167 T5164 arg3Of ),(
R4069 T5172 T5170 arg2Of phosphorylation,enhanced
R4070 T5172 T5171 arg1Of phosphorylation,tyrosine
R4071 T5172 T5173 arg1Of phosphorylation,of
R4072 T5172 T5179 arg1Of phosphorylation,as
R4073 T5176 T5173 arg2Of proteins,of
R4074 T5176 T5174 arg1Of proteins,multiple
R4075 T5176 T5175 arg1Of proteins,cellular
R4076 T5179 T5177 arg1Of as,as
R4077 T5179 T5178 arg1Of as,well
R4078 T5179 T5186 arg1Of as,was
R4079 T5179 T5188 arg2Of as,observed
R4080 T5181 T5179 arg2Of increase,as
R4081 T5181 T5180 arg1Of increase,an
R4082 T5181 T5182 arg1Of increase,in
R4083 T5185 T5182 arg2Of levels,in
R4084 T5185 T5183 arg1Of levels,intracellular
R4085 T5185 T5184 arg1Of levels,calcium
R4086 T5188 T5168 arg1Of observed,Furthermore
R4087 T5188 T5169 arg1Of observed,","
R4088 T5188 T5186 arg2Of observed,was
R4089 T5188 T5187 arg1Of observed,also
R4090 T5188 T5189 arg1Of observed,following
R4091 T5191 T5189 arg2Of stimulation,following
R4092 T5191 T5190 arg1Of stimulation,BCR
R4093 T5191 T5192 arg1Of stimulation,of
R4094 T5195 T5192 arg2Of cells,of
R4095 T5195 T5193 arg1Of cells,PKD1/3-null
R4096 T5195 T5194 arg1Of cells,B
R4097 T5195 T5196 arg1Of cells,(
R4098 T5197 T5196 arg2Of data,(
R4099 T5197 T5199 arg2Of data,shown
R4100 T5199 T5198 arg1Of shown,not
R4101 T5200 T5196 arg3Of ),(
R4102 T5201 T5202 arg1Of We,did
R4103 T5201 T5203 arg1Of We,observe
R4104 T5203 T5202 arg2Of observe,did
R4105 T5206 T5205 arg1Of strength,the
R4106 T5206 T5207 arg1Of strength,of
R4107 T5206 T5222 arg1Of strength,was
R4108 T5206 T5223 arg2Of strength,reduced
R4109 T5208 T5209 arg1Of BCR,(
R4110 T5210 T5211 arg1Of but,not
R4111 T5213 T5209 arg2Of ester,(
R4112 T5213 T5210 arg1Of ester,but
R4113 T5213 T5212 arg1Of ester,phorbol
R4114 T5214 T5209 arg3Of ),(
R4115 T5216 T5207 arg2Of regulation,of
R4116 T5216 T5208 arg1Of regulation,BCR
R4117 T5216 T5215 arg1Of regulation,-induced
R4118 T5216 T5217 arg1Of regulation,of
R4119 T5221 T5217 arg2Of pathway,of
R4120 T5221 T5218 arg1Of pathway,the
R4121 T5221 T5219 arg1Of pathway,Erk1-RSK1
R4122 T5221 T5220 arg1Of pathway,signalling
R4123 T5223 T5203 arg2Of reduced,observe
R4124 T5223 T5204 arg1Of reduced,that
R4125 T5223 T5222 arg2Of reduced,was
R4126 T5223 T5224 arg1Of reduced,in
R4127 T5223 T5228 arg1Of reduced,compared
R4128 T5227 T5224 arg2Of cells,in
R4129 T5227 T5225 arg1Of cells,PKD1/3−/−
R4130 T5227 T5226 arg1Of cells,B
R4131 T5229 T5228 arg2Of to,compared
R4132 T5232 T5229 arg2Of cells,to
R4133 T5232 T5230 arg1Of cells,wild-type
R4134 T5232 T5231 arg1Of cells,B
R4135 T5232 T5233 arg1Of cells,(
R4136 T5235 T5233 arg2Of 3B,(
R4137 T5235 T5234 arg1Of 3B,Fig.
R4138 T5236 T5233 arg3Of ),(
R4139 T5238 T5237 arg1Of interpretation,One
R4140 T5238 T5239 arg1Of interpretation,of
R4141 T5238 T5242 arg1Of interpretation,is
R4142 T5241 T5239 arg2Of data,of
R4143 T5241 T5240 arg1Of data,this
R4144 T5245 T5244 arg1Of enzymes,PKD
R4145 T5245 T5246 arg1Of enzymes,may
R4146 T5245 T5247 arg1Of enzymes,modulate
R4147 T5247 T5242 arg2Of modulate,is
R4148 T5247 T5243 arg1Of modulate,that
R4149 T5247 T5246 arg2Of modulate,may
R4150 T5249 T5247 arg2Of activation,modulate
R4151 T5249 T5248 arg1Of activation,Erk
R4152 T5253 T5252 arg1Of enzymes,PKD
R4153 T5253 T5254 arg1Of enzymes,have
R4154 T5253 T5256 arg1Of enzymes,been
R4155 T5253 T5257 arg2Of enzymes,linked
R4156 T5257 T5250 arg1Of linked,Indeed
R4157 T5257 T5251 arg1Of linked,","
R4158 T5257 T5254 arg2Of linked,have
R4159 T5257 T5255 arg1Of linked,previously
R4160 T5257 T5256 arg2Of linked,been
R4161 T5257 T5258 arg1Of linked,to
R4162 T5257 T5264 arg1Of linked,in
R4163 T5263 T5258 arg2Of signalling,to
R4164 T5263 T5259 arg1Of signalling,the
R4165 T5263 T5260 arg1Of signalling,growth
R4166 T5263 T5261 arg1Of signalling,factor-regulated
R4167 T5263 T5262 arg1Of signalling,Erk
R4168 T5265 T5266 arg1Of fibroblast,and
R4169 T5266 T5264 arg2Of and,in
R4170 T5269 T5266 arg2Of lines,and
R4171 T5269 T5267 arg1Of lines,endothelial
R4172 T5269 T5268 arg1Of lines,cell
R4173 T5269 T5270 arg1Of lines,[
R4174 T5271 T5270 arg2Of 33–35,[
R4175 T5272 T5270 arg3Of ],[
R4176 T5277 T5275 arg1Of phosphorylation,BCR-induced
R4177 T5277 T5276 arg1Of phosphorylation,Erk
R4178 T5277 T5278 arg1Of phosphorylation,was
R4179 T5277 T5280 arg2Of phosphorylation,reduced
R4180 T5280 T5273 arg1Of reduced,However
R4181 T5280 T5274 arg1Of reduced,","
R4182 T5280 T5278 arg2Of reduced,was
R4183 T5280 T5279 arg1Of reduced,also
R4184 T5280 T5281 arg1Of reduced,in
R4185 T5280 T5290 modOf reduced,suggesting
R4186 T5284 T5281 arg2Of cells,in
R4187 T5284 T5282 arg1Of cells,PKD1/3−/−-Flag-PKD3+
R4188 T5284 T5283 arg1Of cells,B
R4189 T5284 T5285 arg1Of cells,(
R4190 T5286 T5285 arg2Of data,(
R4191 T5286 T5288 arg2Of data,shown
R4192 T5288 T5287 arg1Of shown,not
R4193 T5289 T5285 arg3Of ),(
R4194 T5294 T5292 arg2Of levels,reduced
R4195 T5294 T5293 arg1Of levels,BCR
R4196 T5294 T5295 arg1Of levels,on
R4197 T5294 T5306 arg1Of levels,may
R4198 T5294 T5308 arg1Of levels,impact
R4199 T5297 T5295 arg2Of surface,on
R4200 T5297 T5296 arg1Of surface,the
R4201 T5297 T5298 arg1Of surface,of
R4202 T5299 T5300 arg1Of PKD1/3−/−,(
R4203 T5302 T5300 arg2Of PKD1/3−/−-Flag-PKD3+,(
R4204 T5302 T5301 arg1Of PKD1/3−/−-Flag-PKD3+,and
R4205 T5303 T5300 arg3Of ),(
R4206 T5305 T5298 arg2Of cells,of
R4207 T5305 T5299 arg1Of cells,PKD1/3−/−
R4208 T5305 T5304 arg1Of cells,B
R4209 T5308 T5290 arg2Of impact,suggesting
R4210 T5308 T5291 arg1Of impact,that
R4211 T5308 T5306 arg2Of impact,may
R4212 T5308 T5307 arg1Of impact,itself
R4213 T5308 T5309 arg1Of impact,on
R4214 T5311 T5309 arg2Of strength,on
R4215 T5311 T5310 arg1Of strength,the
R4216 T5311 T5312 arg1Of strength,of
R4217 T5313 T5312 arg2Of activation,of
R4218 T5313 T5314 arg1Of activation,of
R4219 T5319 T5314 arg2Of pathway,of
R4220 T5319 T5315 arg1Of pathway,this
R4221 T5319 T5316 arg1Of pathway,specific
R4222 T5319 T5317 arg1Of pathway,intracellular
R4223 T5319 T5318 arg1Of pathway,signalling
R4224 T5321 T5320 arg1Of search,To
R4225 T5321 T5322 arg1Of search,for
R4226 T5326 T5322 arg2Of targets,for
R4227 T5326 T5323 arg1Of targets,other
R4228 T5326 T5324 arg1Of targets,potential
R4229 T5326 T5325 arg1Of targets,PKD
R4230 T5326 T5327 arg1Of targets,that
R4231 T5326 T5328 arg1Of targets,may
R4232 T5326 T5329 arg1Of targets,show
R4233 T5329 T5328 arg2Of show,may
R4234 T5329 T5332 arg1Of show,in
R4235 T5331 T5329 arg2Of regulation,show
R4236 T5331 T5330 arg1Of regulation,defective
R4237 T5336 T5332 arg2Of cells,in
R4238 T5336 T5333 arg1Of cells,PKD1/3−/−
R4239 T5336 T5334 arg1Of cells,DT40
R4240 T5336 T5335 arg1Of cells,B
R4241 T5338 T5321 arg1Of we,search
R4242 T5338 T5339 arg1Of we,used
R4243 T5339 T5320 modOf used,To
R4244 T5339 T5337 arg1Of used,","
R4245 T5343 T5339 arg2Of phospho-antibody,used
R4246 T5343 T5340 arg1Of phospho-antibody,a
R4247 T5343 T5341 arg1Of phospho-antibody,PKD
R4248 T5343 T5342 arg1Of phospho-antibody,substrate
R4249 T5343 T5344 arg1Of phospho-antibody,that
R4250 T5343 T5345 arg1Of phospho-antibody,recognises
R4251 T5348 T5345 arg2Of sequences,recognises
R4252 T5348 T5346 arg1Of sequences,consensus
R4253 T5348 T5347 arg1Of sequences,phosphorylation
R4254 T5348 T5349 arg2Of sequences,targeted
R4255 T5349 T5356 arg1Of targeted,[
R4256 T5352 T5349 arg1Of enzymes,targeted
R4257 T5352 T5350 arg2Of enzymes,by
R4258 T5352 T5351 arg1Of enzymes,PKD
R4259 T5352 T5353 arg1Of enzymes,(
R4260 T5354 T5353 arg2Of LxRxxpS/T,(
R4261 T5355 T5353 arg3Of ),(
R4262 T5357 T5356 arg2Of 36,[
R4263 T5358 T5356 arg3Of ],[
R4264 T5360 T5359 arg2Of shown,As
R4265 T5360 T5361 arg1Of shown,in
R4266 T5363 T5361 arg2Of 3C,in
R4267 T5363 T5362 arg1Of 3C,Fig.
R4268 T5366 T5365 arg1Of ester-,phorbol
R4269 T5366 T5367 arg1Of ester-,and
R4270 T5368 T5367 arg2Of BCR-induced,and
R4271 T5369 T5366 arg1Of phosphorylation,ester-
R4272 T5369 T5368 arg1Of phosphorylation,BCR-induced
R4273 T5369 T5370 arg1Of phosphorylation,of
R4274 T5369 T5377 arg1Of phosphorylation,was
R4275 T5369 T5378 arg1Of phosphorylation,similar
R4276 T5369 T5385 arg1Of phosphorylation,is
R4277 T5369 T5387 arg1Of phosphorylation,independent
R4278 T5372 T5370 arg2Of substrates,of
R4279 T5372 T5371 arg1Of substrates,cellular
R4280 T5372 T5373 arg2Of substrates,detected
R4281 T5376 T5373 arg1Of phospho-antibody,detected
R4282 T5376 T5374 arg2Of phospho-antibody,by
R4283 T5376 T5375 arg1Of phospho-antibody,this
R4284 T5377 T5384 arg1Of was,and
R4285 T5378 T5377 arg2Of similar,was
R4286 T5378 T5379 arg1Of similar,in
R4287 T5380 T5381 arg1Of wild-type,and
R4288 T5381 T5379 arg2Of and,in
R4289 T5383 T5381 arg2Of cells,and
R4290 T5383 T5382 arg1Of cells,PKD1/3−/−
R4291 T5384 T5359 arg1Of and,As
R4292 T5384 T5364 arg1Of and,","
R4293 T5385 T5384 arg2Of is,and
R4294 T5385 T5386 arg1Of is,therefore
R4295 T5387 T5385 arg2Of independent,is
R4296 T5387 T5388 arg1Of independent,of
R4297 T5390 T5388 arg2Of enzymes,of
R4298 T5390 T5389 arg1Of enzymes,PKD
R4299 T5393 T5394 arg1Of pretreatment,of
R4300 T5393 T5402 arg1Of pretreatment,with
R4301 T5393 T5411 arg1Of pretreatment,prevented
R4302 T5401 T5394 arg2Of cells,of
R4303 T5401 T5395 arg1Of cells,both
R4304 T5401 T5396 arg1Of cells,wild-type
R4305 T5401 T5397 arg1Of cells,and
R4306 T5401 T5398 arg1Of cells,PKD1/3−/−
R4307 T5401 T5399 arg1Of cells,DT40
R4308 T5401 T5400 arg1Of cells,B
R4309 T5403 T5402 arg2Of GF109203X,with
R4310 T5403 T5404 arg1Of GF109203X,","
R4311 T5407 T5404 arg2Of derivative,","
R4312 T5407 T5405 arg1Of derivative,a
R4313 T5407 T5406 arg1Of derivative,bisindoylmaleimide
R4314 T5407 T5408 arg1Of derivative,that
R4315 T5407 T5409 arg1Of derivative,inhibits
R4316 T5410 T5409 arg2Of PKCs,inhibits
R4317 T5411 T5391 arg1Of prevented,However
R4318 T5411 T5392 arg1Of prevented,","
R4319 T5413 T5411 arg2Of induction,prevented
R4320 T5413 T5412 arg1Of induction,the
R4321 T5413 T5414 arg1Of induction,of
R4322 T5415 T5414 arg2Of proteins,of
R4323 T5415 T5416 arg1Of proteins,that
R4324 T5415 T5417 arg1Of proteins,contain
R4325 T5420 T5417 arg2Of motifs,contain
R4326 T5420 T5418 arg2Of motifs,phosphorylated
R4327 T5420 T5419 arg1Of motifs,LxRxxS/T
R4328 T5422 T5423 arg1Of loss,of
R4329 T5422 T5426 arg1Of loss,does
R4330 T5422 T5429 arg1Of loss,disrupt
R4331 T5425 T5423 arg2Of enzymes,of
R4332 T5425 T5424 arg1Of enzymes,PKD1/3
R4333 T5429 T5421 arg1Of disrupt,Thus
R4334 T5429 T5426 arg2Of disrupt,does
R4335 T5429 T5427 arg1Of disrupt,not
R4336 T5429 T5428 arg1Of disrupt,globally
R4337 T5431 T5429 arg2Of phosphorylation,disrupt
R4338 T5431 T5430 arg1Of phosphorylation,the
R4339 T5431 T5432 arg1Of phosphorylation,of
R4340 T5434 T5432 arg2Of proteins,of
R4341 T5434 T5433 arg1Of proteins,cellular
R4342 T5434 T5435 arg1Of proteins,that
R4343 T5434 T5436 arg1Of proteins,contain
R4344 T5438 T5436 arg2Of motifs,contain
R4345 T5438 T5437 arg1Of motifs,LxRxxpS/T
R4346 T5440 T5439 arg1Of result,This
R4347 T5440 T5441 arg1Of result,is
R4348 T5440 T5444 arg1Of result,surprising
R4349 T5441 T5443 arg1Of is,not
R4350 T5441 T5445 arg1Of is,as
R4351 T5443 T5442 arg1Of not,perhaps
R4352 T5444 T5441 arg2Of surprising,is
R4353 T5447 T5446 arg1Of motifs,LxRxxS/T
R4354 T5447 T5449 arg1Of motifs,act
R4355 T5449 T5445 arg2Of act,as
R4356 T5449 T5448 arg1Of act,also
R4357 T5449 T5450 arg1Of act,as
R4358 T5452 T5450 arg2Of substrates,as
R4359 T5452 T5451 arg1Of substrates,good
R4360 T5452 T5453 arg1Of substrates,for
R4361 T5456 T5453 arg2Of kinases,for
R4362 T5456 T5454 arg1Of kinases,other
R4363 T5456 T5455 arg1Of kinases,serine/threonine
R4364 T5456 T5458 arg1Of kinases,as
R4365 T5458 T5457 arg1Of as,such
R4366 T5459 T5458 arg2Of MAPKAPK2,as
R4367 T5462 T5461 arg1Of experiments,these
R4368 T5462 T5463 arg1Of experiments,do
R4369 T5462 T5464 arg1Of experiments,provide
R4370 T5464 T5460 arg1Of provide,However
R4371 T5464 T5463 arg2Of provide,do
R4372 T5466 T5464 arg2Of evidence,provide
R4373 T5466 T5465 arg1Of evidence,further
R4374 T5469 T5468 arg1Of antisera,phosphospecific
R4375 T5469 T5470 arg1Of antisera,are
R4376 T5469 T5473 arg1Of antisera,selective
R4377 T5470 T5466 arg2Of are,evidence
R4378 T5470 T5467 arg1Of are,that
R4379 T5470 T5471 arg1Of are,not
R4380 T5473 T5470 arg2Of selective,are
R4381 T5473 T5472 arg1Of selective,sufficiently
R4382 T5473 T5474 modOf selective,to
R4383 T5476 T5474 arg1Of designated,to
R4384 T5476 T5475 arg2Of designated,be
R4385 T5478 T5477 arg1Of specific,kinase
R4386 T5480 T5476 arg3Of antisera,designated
R4387 T5480 T5478 arg1Of antisera,specific
R4388 T5480 T5479 arg1Of antisera,substrate
R4389 T5483 T5481 arg1Of pathways,BCR-induced
R4390 T5483 T5482 arg1Of pathways,signalling
R4391 T5483 T5484 arg1Of pathways,culminate
R4392 T5484 T5485 arg1Of culminate,in
R4393 T5487 T5485 arg2Of activation,in
R4394 T5487 T5486 arg1Of activation,the
R4395 T5487 T5488 arg1Of activation,of
R4396 T5491 T5488 arg2Of events,of
R4397 T5491 T5489 arg1Of events,gene
R4398 T5491 T5490 arg1Of events,transcription
R4399 T5491 T5492 arg1Of events,that
R4400 T5491 T5493 arg1Of events,control
R4401 T5496 T5497 arg1Of survival,","
R4402 T5497 T5499 arg1Of ",",and
R4403 T5498 T5497 arg2Of proliferation,","
R4404 T5499 T5493 arg2Of and,control
R4405 T5499 T5494 arg1Of and,B
R4406 T5499 T5495 arg1Of and,cell
R4407 T5500 T5499 arg2Of function,and
R4408 T5503 T5501 arg2Of context,In
R4409 T5503 T5502 arg1Of context,this
R4410 T5508 T5501 arg1Of proposed,In
R4411 T5508 T5504 arg1Of proposed,","
R4412 T5508 T5506 arg2Of proposed,has
R4413 T5508 T5507 arg2Of proposed,been
R4414 T5508 T5517 arg1Of proposed,through
R4415 T5513 T5505 arg1Of control,it
R4416 T5513 T5506 arg1Of control,has
R4417 T5513 T5507 arg1Of control,been
R4418 T5513 T5508 arg2Of control,proposed
R4419 T5513 T5509 arg1Of control,that
R4420 T5513 T5510 arg1Of control,PKD
R4421 T5513 T5511 arg1Of control,family
R4422 T5513 T5512 arg1Of control,members
R4423 T5513 T5514 arg1Of control,of
R4424 T5516 T5514 arg2Of transcription,of
R4425 T5516 T5515 arg1Of transcription,gene
R4426 T5518 T5517 arg2Of activation,through
R4427 T5518 T5519 arg1Of activation,of
R4428 T5523 T5519 arg2Of factor,of
R4429 T5523 T5520 arg1Of factor,the
R4430 T5523 T5521 arg1Of factor,NFκB
R4431 T5523 T5522 arg1Of factor,transcription
R4432 T5527 T5526 arg1Of activation,PKD-mediated
R4433 T5527 T5528 arg1Of activation,of
R4434 T5527 T5530 arg1Of activation,occurs
R4435 T5529 T5528 arg2Of NFκB,of
R4436 T5530 T5524 arg1Of occurs,Thus
R4437 T5530 T5525 arg1Of occurs,","
R4438 T5530 T5531 arg1Of occurs,downstream
R4439 T5531 T5532 arg1Of downstream,of
R4440 T5534 T5532 arg2Of variety,of
R4441 T5534 T5533 arg1Of variety,a
R4442 T5534 T5535 arg1Of variety,of
R4443 T5537 T5535 arg2Of signals,of
R4444 T5537 T5536 arg1Of signals,different
R4445 T5537 T5538 arg1Of signals,","
R4446 T5537 T5539 arg1Of signals,including
R4447 T5541 T5540 arg1Of stress,mROS/oxidative
R4448 T5541 T5542 arg1Of stress,","
R4449 T5542 T5545 arg1Of ",",and
R4450 T5544 T5542 arg2Of acid,","
R4451 T5544 T5543 arg1Of acid,lysophosphatidic
R4452 T5545 T5539 arg2Of and,including
R4453 T5547 T5546 arg1Of Bcr-Abl,the
R4454 T5548 T5545 arg2Of oncogene,and
R4455 T5548 T5547 arg1Of oncogene,Bcr-Abl
R4456 T5548 T5549 arg1Of oncogene,[
R4457 T5550 T5549 arg2Of "17,21,23,30,37",[
R4458 T5551 T5549 arg3Of ],[
R4459 T5554 T5555 arg1Of expression,of
R4460 T5554 T5560 arg1Of expression,enhances
R4461 T5559 T5555 arg2Of mutant,of
R4462 T5559 T5556 arg1Of mutant,an
R4463 T5559 T5557 arg2Of mutant,activated
R4464 T5559 T5558 arg1Of mutant,PKD1
R4465 T5560 T5552 arg1Of enhances,Furthermore
R4466 T5560 T5553 arg1Of enhances,","
R4467 T5560 T5564 arg1Of enhances,[
R4468 T5563 T5560 arg2Of activation,enhances
R4469 T5563 T5561 arg1Of activation,HPK1-mediated
R4470 T5563 T5562 arg1Of activation,NFκB
R4471 T5565 T5564 arg2Of 38,[
R4472 T5566 T5564 arg3Of ],[
R4473 T5569 T5567 arg2Of cells,In
R4474 T5569 T5568 arg1Of cells,B
R4475 T5571 T5572 arg1Of NFκB,is
R4476 T5571 T5573 arg2Of NFκB,known
R4477 T5571 T5575 arg1Of NFκB,be
R4478 T5571 T5576 arg2Of NFκB,regulated
R4479 T5573 T5572 arg2Of known,is
R4480 T5573 T5584 arg1Of known,but
R4481 T5576 T5573 arg3Of regulated,known
R4482 T5576 T5574 arg1Of regulated,to
R4483 T5576 T5575 arg2Of regulated,be
R4484 T5576 T5577 arg1Of regulated,via
R4485 T5576 T5581 arg1Of regulated,[
R4486 T5578 T5579 arg1Of DAG,and
R4487 T5579 T5577 arg2Of and,via
R4488 T5580 T5579 arg2Of PKCβ,and
R4489 T5582 T5581 arg2Of "39,40",[
R4490 T5583 T5581 arg3Of ],[
R4491 T5584 T5567 arg1Of but,In
R4492 T5584 T5570 arg1Of but,","
R4493 T5586 T5587 arg1Of PKDs,are
R4494 T5587 T5585 arg1Of are,whether
R4495 T5587 T5593 arg1Of are,has
R4496 T5587 T5595 arg1Of are,been
R4497 T5587 T5596 arg2Of are,explored
R4498 T5589 T5587 arg2Of intermediaries,are
R4499 T5589 T5588 arg1Of intermediaries,key
R4500 T5589 T5590 arg1Of intermediaries,for
R4501 T5592 T5590 arg2Of regulation,for
R4502 T5592 T5591 arg1Of regulation,NFκB
R4503 T5596 T5584 arg2Of explored,but
R4504 T5596 T5593 arg2Of explored,has
R4505 T5596 T5594 arg1Of explored,not
R4506 T5596 T5595 arg2Of explored,been
R4507 T5598 T5597 arg1Of data,The
R4508 T5598 T5599 arg1Of data,(
R4509 T5598 T5603 arg1Of data,show
R4510 T5601 T5599 arg2Of 4A,(
R4511 T5601 T5600 arg1Of 4A,Fig.
R4512 T5602 T5599 arg3Of ),(
R4513 T5607 T5605 arg1Of activity,NFκB
R4514 T5607 T5606 arg1Of activity,transcriptional
R4515 T5607 T5608 arg1Of activity,was
R4516 T5607 T5610 arg2Of activity,induced
R4517 T5610 T5603 arg2Of induced,show
R4518 T5610 T5604 arg1Of induced,that
R4519 T5610 T5608 arg2Of induced,was
R4520 T5610 T5609 arg1Of induced,strongly
R4521 T5610 T5611 arg1Of induced,in
R4522 T5610 T5619 arg1Of induced,in
R4523 T5618 T5611 arg2Of cells,in
R4524 T5618 T5612 arg1Of cells,both
R4525 T5618 T5613 arg1Of cells,wild-type
R4526 T5618 T5614 arg1Of cells,and
R4527 T5618 T5615 arg1Of cells,PKD1/3−/−
R4528 T5618 T5616 arg1Of cells,DT40
R4529 T5618 T5617 arg1Of cells,B
R4530 T5620 T5619 arg2Of response,in
R4531 T5620 T5621 arg1Of response,to
R4532 T5624 T5623 arg1Of ester,phorbol
R4533 T5624 T5625 arg1Of ester,or
R4534 T5625 T5621 arg2Of or,to
R4535 T5625 T5622 arg1Of or,either
R4536 T5627 T5625 arg2Of stimulation,or
R4537 T5627 T5626 arg1Of stimulation,BCR
R4538 T5629 T5628 arg2Of contrast,In
R4539 T5631 T5666 arg1Of BCR,was
R4540 T5631 T5667 arg2Of BCR,observed
R4541 T5634 T5632 arg1Of ester-induced,and
R4542 T5634 T5633 arg1Of ester-induced,phorbol
R4543 T5637 T5634 arg1Of activity,ester-induced
R4544 T5637 T5635 arg1Of activity,NFκB
R4545 T5637 T5636 arg1Of activity,transcriptional
R4546 T5637 T5638 arg1Of activity,was
R4547 T5637 T5639 arg2Of activity,abolished
R4548 T5639 T5638 arg2Of abolished,was
R4549 T5639 T5640 arg1Of abolished,in
R4550 T5639 T5649 arg1Of abolished,","
R4551 T5639 T5650 arg1Of abolished,although
R4552 T5639 T5656 arg1Of abolished,(
R4553 T5644 T5640 arg2Of cells,in
R4554 T5644 T5641 arg1Of cells,PKCβ−/−
R4555 T5644 T5642 arg1Of cells,DT40
R4556 T5644 T5643 arg1Of cells,B
R4557 T5644 T5645 arg1Of cells,(
R4558 T5647 T5645 arg2Of 4A,(
R4559 T5647 T5646 arg1Of 4A,Fig.
R4560 T5648 T5645 arg3Of ),(
R4561 T5652 T5650 arg2Of activation,although
R4562 T5652 T5651 arg1Of activation,strong
R4563 T5652 T5653 arg1Of activation,of
R4564 T5655 T5653 arg2Of kinases,of
R4565 T5655 T5654 arg1Of kinases,PKD
R4566 T5657 T5656 arg2Of as,(
R4567 T5658 T5657 arg2Of assessed,as
R4568 T5658 T5659 arg1Of assessed,by
R4569 T5660 T5659 arg2Of autophosphorylation,by
R4570 T5660 T5661 arg1Of autophosphorylation,of
R4571 T5660 T5663 arg1Of autophosphorylation,at
R4572 T5662 T5661 arg2Of PKD1,of
R4573 T5664 T5663 arg2Of S916,at
R4574 T5665 T5656 arg3Of ),(
R4575 T5667 T5628 arg1Of observed,In
R4576 T5667 T5630 arg1Of observed,","
R4577 T5667 T5638 modOf observed,was
R4578 T5667 T5666 arg2Of observed,was
R4579 T5667 T5668 arg1Of observed,in
R4580 T5671 T5668 arg2Of cells,in
R4581 T5671 T5669 arg1Of cells,the
R4582 T5671 T5670 arg1Of cells,PKCβ−/−
R4583 T5671 T5672 arg1Of cells,(
R4584 T5674 T5672 arg2Of 4B,(
R4585 T5674 T5673 arg1Of 4B,Fig.
R4586 T5675 T5672 arg3Of ),(
R4587 T5679 T5678 arg1Of kinases,PKD
R4588 T5679 T5680 arg1Of kinases,are
R4589 T5679 T5682 arg1Of kinases,essential
R4590 T5679 T5684 arg1Of kinases,sufficient
R4591 T5679 T5686 arg1Of kinases,mediate
R4592 T5679 T5696 arg1Of kinases,do
R4593 T5679 T5698 arg1Of kinases,participate
R4594 T5680 T5694 arg1Of are,and
R4595 T5682 T5683 arg1Of essential,nor
R4596 T5683 T5680 arg2Of nor,are
R4597 T5683 T5681 arg1Of nor,neither
R4598 T5684 T5683 arg2Of sufficient,nor
R4599 T5686 T5682 arg2Of mediate,essential
R4600 T5686 T5684 arg2Of mediate,sufficient
R4601 T5686 T5685 arg1Of mediate,to
R4602 T5689 T5686 arg2Of activation,mediate
R4603 T5689 T5687 arg1Of activation,BCR-induced
R4604 T5689 T5688 arg1Of activation,NFκB
R4605 T5689 T5690 arg1Of activation,in
R4606 T5693 T5690 arg2Of cells,in
R4607 T5693 T5691 arg1Of cells,DT40
R4608 T5693 T5692 arg1Of cells,B
R4609 T5694 T5676 arg1Of and,Thus
R4610 T5694 T5677 arg1Of and,","
R4611 T5698 T5694 arg2Of participate,and
R4612 T5698 T5695 arg1Of participate,hence
R4613 T5698 T5696 arg2Of participate,do
R4614 T5698 T5697 arg1Of participate,not
R4615 T5698 T5699 arg1Of participate,in
R4616 T5702 T5699 arg2Of control,in
R4617 T5702 T5700 arg1Of control,DAG/PKC
R4618 T5702 T5701 arg2Of control,mediated
R4619 T5702 T5703 arg1Of control,of
R4620 T5704 T5703 arg2Of NFκB,of
R5531 T7047 T7043 arg1Of kinases,"40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway. To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera. BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor. Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38]. In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored. The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB. 4 Discussion Protein"
R5532 T7047 T7044 arg1Of kinases,kinase
R5533 T7047 T7045 arg1Of kinases,D
R5534 T7047 T7046 arg1Of kinases,serine
R5535 T7047 T7048 arg1Of kinases,have
R5536 T7047 T7049 arg1Of kinases,been
R5537 T7047 T7050 arg2Of kinases,proposed
R5538 T7047 T7052 arg1Of kinases,regulate
R5539 T7050 T7048 arg2Of proposed,have
R5540 T7050 T7049 arg2Of proposed,been
R5541 T7052 T7050 arg3Of regulate,proposed
R5542 T7052 T7051 arg1Of regulate,to
R5543 T7055 T7052 arg2Of functions,regulate
R5544 T7055 T7053 arg1Of functions,diverse
R5545 T7055 T7054 arg1Of functions,cellular
R5546 T7055 T7056 arg1Of functions,including
R5547 T7058 T7059 arg1Of phosphorylation,and
R5548 T7059 T7057 arg1Of and,the
R5549 T7059 T7062 arg1Of and,of
R5550 T7059 T7066 arg1Of and,and
R5551 T7061 T7059 arg2Of localisation,and
R5552 T7061 T7060 arg1Of localisation,nuclear
R5553 T7065 T7062 arg2Of HDACs,of
R5554 T7065 T7063 arg1Of HDACs,class
R5555 T7065 T7064 arg1Of HDACs,II
R5556 T7066 T7056 arg2Of and,including
R5557 T7068 T7066 arg2Of phosphorylation,and
R5558 T7068 T7067 arg1Of phosphorylation,the
R5559 T7068 T7069 arg1Of phosphorylation,of
R5560 T7070 T7069 arg2Of HSP27,of
R5561 T7071 T7072 arg1Of It,has
R5562 T7071 T7074 arg1Of It,been
R5563 T7071 T7075 arg2Of It,suggested
R5564 T7075 T7072 arg2Of suggested,has
R5565 T7075 T7073 arg1Of suggested,also
R5566 T7075 T7074 arg2Of suggested,been
R5567 T7077 T7078 arg1Of PKDs,act
R5568 T7077 T7086 arg1Of PKDs,play
R5569 T7077 T7090 arg1Of PKDs,regulating
R5570 T7078 T7079 arg1Of act,as
R5571 T7078 T7085 arg1Of act,and
R5572 T7081 T7079 arg2Of sensors,as
R5573 T7081 T7080 arg1Of sensors,mitochondrial
R5574 T7081 T7082 arg1Of sensors,for
R5575 T7084 T7082 arg2Of stress,for
R5576 T7084 T7083 arg1Of stress,oxidative
R5577 T7085 T7075 arg3Of and,suggested
R5578 T7085 T7076 arg1Of and,that
R5579 T7086 T7085 arg2Of play,and
R5580 T7086 T7089 arg1Of play,in
R5581 T7088 T7086 arg2Of role,play
R5582 T7088 T7087 arg1Of role,a
R5583 T7090 T7089 arg2Of regulating,in
R5584 T7093 T7090 arg2Of factors,regulating
R5585 T7093 T7091 arg1Of factors,NFκB
R5586 T7093 T7092 arg1Of factors,transcription
R5587 T7093 T7094 arg1Of factors,[
R5588 T7095 T7094 arg2Of 41,[
R5589 T7096 T7094 arg3Of ],[
R5590 T7097 T7098 arg1Of Most,of
R5591 T7097 T7101 arg1Of Most,about
R5592 T7097 T7106 arg1Of Most,has
R5593 T7097 T7107 arg1Of Most,come
R5594 T7100 T7098 arg2Of data,of
R5595 T7100 T7099 arg1Of data,the
R5596 T7103 T7101 arg2Of function,about
R5597 T7103 T7102 arg1Of function,the
R5598 T7103 T7104 arg1Of function,of
R5599 T7105 T7104 arg2Of PKDs,of
R5600 T7107 T7106 arg2Of come,has
R5601 T7107 T7108 arg1Of come,from
R5602 T7109 T7108 arg2Of experiments,from
R5603 T7109 T7110 arg1Of experiments,that
R5604 T7109 T7112 arg1Of experiments,express
R5605 T7109 T7119 arg1Of experiments,that
R5606 T7109 T7120 arg1Of experiments,use
R5607 T7109 T7123 arg1Of experiments,reduce
R5608 T7112 T7111 arg1Of express,ectopically
R5609 T7112 T7118 arg1Of express,or
R5610 T7113 T7114 arg1Of active,or
R5611 T7115 T7114 arg2Of inhibitory,or
R5612 T7117 T7112 arg2Of mutants,express
R5613 T7117 T7113 arg1Of mutants,active
R5614 T7117 T7115 arg1Of mutants,inhibitory
R5615 T7117 T7116 arg1Of mutants,PKD
R5616 T7120 T7118 arg2Of use,or
R5617 T7120 T7122 modOf use,to
R5618 T7121 T7120 arg2Of RNAi,use
R5619 T7123 T7122 arg1Of reduce,to
R5620 T7125 T7123 arg2Of expression,reduce
R5621 T7125 T7124 arg1Of expression,PKD
R5622 T7126 T7127 arg1Of We,have
R5623 T7126 T7128 arg1Of We,used
R5624 T7126 T7133 arg1Of We,delete
R5625 T7126 T7142 arg1Of We,can
R5626 T7126 T7144 arg1Of We,use
R5627 T7126 T7149 arg1Of We,assess
R5628 T7128 T7127 arg2Of used,have
R5629 T7128 T7141 arg1Of used,and
R5630 T7130 T7128 arg2Of targeting,used
R5631 T7130 T7129 arg1Of targeting,gene
R5632 T7133 T7128 arg3Of delete,used
R5633 T7133 T7131 arg1Of delete,to
R5634 T7133 T7132 arg1Of delete,specifically
R5635 T7135 T7133 arg2Of alleles,delete
R5636 T7135 T7134 arg1Of alleles,PKD
R5637 T7135 T7136 arg1Of alleles,in
R5638 T7140 T7136 arg2Of cells,in
R5639 T7140 T7137 arg1Of cells,DT40
R5640 T7140 T7138 arg1Of cells,chicken
R5641 T7140 T7139 arg1Of cells,B
R5642 T7144 T7141 arg2Of use,and
R5643 T7144 T7142 arg2Of use,can
R5644 T7144 T7143 arg1Of use,thus
R5645 T7144 T7148 modOf use,to
R5646 T7147 T7144 arg2Of cells,use
R5647 T7147 T7145 arg1Of cells,PKD-null
R5648 T7147 T7146 arg1Of cells,DT40
R5649 T7149 T7148 arg1Of assess,to
R5650 T7152 T7149 arg2Of contribution,assess
R5651 T7152 T7150 arg1Of contribution,the
R5652 T7152 T7151 arg1Of contribution,relative
R5653 T7152 T7153 arg1Of contribution,of
R5654 T7152 T7157 arg1Of contribution,in
R5655 T7156 T7153 arg2Of isoforms,of
R5656 T7156 T7154 arg1Of isoforms,individual
R5657 T7156 T7155 arg1Of isoforms,PKD
R5658 T7161 T7158 arg1Of control,class
R5659 T7161 T7159 arg1Of control,II
R5660 T7161 T7160 arg1Of control,HDAC
R5661 T7161 T7162 arg1Of control,versus
R5662 T7161 T7166 arg1Of control,and
R5663 T7165 T7162 arg2Of responses,versus
R5664 T7165 T7163 arg1Of responses,oxidative
R5665 T7165 T7164 arg1Of responses,stress
R5666 T7166 T7157 arg2Of and,in
R5667 T7168 T7166 arg2Of regulation,and
R5668 T7168 T7167 arg1Of regulation,NFκB
R5669 T7168 T7169 arg1Of regulation,in
R5670 T7170 T7169 arg2Of lymphocytes,in
R5671 T7171 T7172 arg1Of We,have
R5672 T7171 T7174 arg1Of We,used
R5673 T7171 T7180 arg1Of We,define
R5674 T7171 T7204 arg1Of We,regulating
R5675 T7174 T7172 arg2Of used,have
R5676 T7174 T7173 arg1Of used,previously
R5677 T7174 T7179 modOf used,to
R5678 T7174 T7192 arg1Of used,","
R5679 T7178 T7174 arg2Of cells,used
R5680 T7178 T7175 arg1Of cells,these
R5681 T7178 T7176 arg1Of cells,PKD-null
R5682 T7178 T7177 arg1Of cells,DT40
R5683 T7180 T7179 arg1Of define,to
R5684 T7183 T7180 arg2Of role,define
R5685 T7183 T7181 arg1Of role,an
R5686 T7183 T7182 arg1Of role,essential
R5687 T7183 T7184 arg1Of role,for
R5688 T7183 T7186 arg1Of role,in
R5689 T7185 T7184 arg2Of PKDs,for
R5690 T7187 T7186 arg2Of regulation,in
R5691 T7187 T7188 arg1Of regulation,of
R5692 T7191 T7188 arg2Of HDACs,of
R5693 T7191 T7189 arg1Of HDACs,class
R5694 T7191 T7190 arg1Of HDACs,II
R5695 T7192 T7203 arg1Of ",",in
R5696 T7195 T7193 arg1Of report,the
R5697 T7195 T7194 arg1Of report,present
R5698 T7195 T7197 arg1Of report,describes
R5699 T7197 T7192 arg2Of describes,","
R5700 T7197 T7196 arg1Of describes,now
R5701 T7200 T7197 arg2Of role,describes
R5702 T7200 T7198 arg1Of role,an
R5703 T7200 T7199 arg1Of role,indispensable
R5704 T7200 T7201 arg1Of role,for
R5705 T7202 T7201 arg2Of PKDs,for
R5706 T7204 T7203 arg2Of regulating,in
R5707 T7206 T7204 arg2Of phosphorylation,regulating
R5708 T7206 T7205 arg1Of phosphorylation,the
R5709 T7206 T7207 arg1Of phosphorylation,of
R5710 T7206 T7209 arg1Of phosphorylation,on
R5711 T7206 T7212 arg1Of phosphorylation,","
R5712 T7208 T7207 arg2Of HSP27,of
R5713 T7210 T7209 arg2Of serine,on
R5714 T7210 T7211 arg1Of serine,82
R5715 T7214 T7212 arg2Of site,","
R5716 T7214 T7213 arg1Of site,a
R5717 T7214 T7216 arg2Of site,identified
R5718 T7216 T7215 arg1Of identified,previously
R5719 T7216 T7217 arg1Of identified,as
R5720 T7219 T7217 arg2Of target,as
R5721 T7219 T7218 arg1Of target,a
R5722 T7219 T7220 arg1Of target,for
R5723 T7224 T7220 arg2Of cascade,for
R5724 T7224 T7221 arg1Of cascade,the
R5725 T7224 T7222 arg1Of cascade,p38-MAPKAPK2
R5726 T7224 T7223 arg1Of cascade,signalling
R5727 T7224 T7225 arg1Of cascade,[
R5728 T7226 T7225 arg2Of 42,[
R5729 T7227 T7225 arg3Of ],[
R5730 T7230 T7231 arg1Of studies,of
R5731 T7230 T7235 arg1Of studies,reveal
R5732 T7234 T7231 arg2Of cells,of
R5733 T7234 T7232 arg1Of cells,PKD-null
R5734 T7234 T7233 arg1Of cells,DT40
R5735 T7235 T7228 arg1Of reveal,However
R5736 T7235 T7229 arg1Of reveal,","
R5737 T7239 T7237 arg1Of kinases,PKD
R5738 T7239 T7238 arg1Of kinases,family
R5739 T7239 T7240 arg1Of kinases,are
R5740 T7239 T7242 arg1Of kinases,essential
R5741 T7240 T7241 arg1Of are,not
R5742 T7240 T7248 arg1Of are,nor
R5743 T7242 T7240 arg2Of essential,are
R5744 T7242 T7243 arg1Of essential,for
R5745 T7247 T7243 arg2Of responses,for
R5746 T7247 T7244 arg1Of responses,oxidative
R5747 T7247 T7245 arg1Of responses,stress
R5748 T7247 T7246 arg1Of responses,survival
R5749 T7248 T7235 arg2Of nor,reveal
R5750 T7248 T7236 arg1Of nor,that
R5751 T7249 T7248 arg2Of are,nor
R5752 T7250 T7249 arg1Of they,are
R5753 T7250 T7251 arg2Of they,required
R5754 T7251 T7252 arg1Of required,for
R5755 T7253 T7252 arg2Of activation,for
R5756 T7253 T7254 arg1Of activation,of
R5757 T7257 T7254 arg2Of factors,of
R5758 T7257 T7255 arg1Of factors,NFκB
R5759 T7257 T7256 arg1Of factors,transcription
R5760 T7260 T7258 arg1Of findings,These
R5761 T7260 T7259 arg1Of findings,latter
R5762 T7260 T7261 arg1Of findings,are
R5763 T7260 T7262 arg1Of findings,in
R5764 T7262 T7261 arg2Of in,are
R5765 T7264 T7262 arg2Of contrast,in
R5766 T7264 T7263 arg1Of contrast,striking
R5767 T7264 T7265 arg1Of contrast,to
R5768 T7267 T7266 arg1Of observations,previous
R5769 T7267 T7268 arg1Of observations,in
R5770 T7267 T7270 arg1Of observations,and
R5771 T7269 T7268 arg2Of HeLa,in
R5772 T7270 T7265 arg2Of and,to
R5773 T7273 T7270 arg2Of lines,and
R5774 T7273 T7271 arg1Of lines,epithelial
R5775 T7273 T7272 arg1Of lines,cell
R5776 T7273 T7274 arg1Of lines,where
R5777 T7276 T7275 arg1Of approaches,overexpression/RNAi
R5778 T7276 T7277 arg1Of approaches,have
R5779 T7276 T7278 arg1Of approaches,implicated
R5780 T7278 T7274 arg2Of implicated,where
R5781 T7278 T7277 arg2Of implicated,have
R5782 T7278 T7280 arg1Of implicated,in
R5783 T7279 T7278 arg2Of PKD1/2,implicated
R5784 T7282 T7280 arg2Of control,in
R5785 T7282 T7281 arg1Of control,the
R5786 T7282 T7283 arg1Of control,of
R5787 T7284 T7285 arg1Of proliferation,","
R5788 T7285 T7287 arg1Of ",",and
R5789 T7286 T7285 arg2Of survival,","
R5790 T7287 T7283 arg2Of and,of
R5791 T7289 T7287 arg2Of activation,and
R5792 T7289 T7288 arg1Of activation,NFκB
R5793 T7289 T7290 arg1Of activation,[
R5794 T7291 T7290 arg2Of "20,23",[
R5795 T7292 T7290 arg3Of ],[
R5796 T7297 T7295 arg1Of report,the
R5797 T7297 T7296 arg1Of report,present
R5798 T7297 T7298 arg1Of report,shows
R5799 T7298 T7293 arg1Of shows,Hence
R5800 T7298 T7294 arg1Of shows,","
R5801 T7302 T7300 arg1Of roles,the
R5802 T7302 T7301 arg2Of roles,proposed
R5803 T7302 T7303 arg1Of roles,for
R5804 T7302 T7305 arg1Of roles,as
R5805 T7302 T7308 arg1Of roles,that
R5806 T7302 T7309 arg1Of roles,modulate
R5807 T7302 T7318 arg1Of roles,regulate
R5808 T7302 T7323 arg1Of roles,are
R5809 T7302 T7325 arg1Of roles,ubiquitous
R5810 T7302 T7327 arg1Of roles,may
R5811 T7302 T7328 arg1Of roles,be
R5812 T7302 T7329 arg1Of roles,restricted
R5813 T7304 T7303 arg2Of PKDs,for
R5814 T7307 T7305 arg2Of sensors,as
R5815 T7307 T7306 arg1Of sensors,key
R5816 T7309 T7312 arg1Of modulate,in
R5817 T7309 T7317 arg1Of modulate,and
R5818 T7311 T7309 arg2Of pathways,modulate
R5819 T7311 T7310 arg1Of pathways,survival
R5820 T7312 T7314 arg1Of in,to
R5821 T7313 T7312 arg2Of response,in
R5822 T7316 T7312 arg3Of stress,in
R5823 T7316 T7315 arg1Of stress,oxidative
R5824 T7318 T7317 arg2Of regulate,and
R5825 T7320 T7321 arg1Of survival,and
R5826 T7321 T7318 arg2Of and,regulate
R5827 T7321 T7319 arg1Of and,cell
R5828 T7322 T7321 arg2Of proliferation,and
R5829 T7323 T7324 arg1Of are,not
R5830 T7323 T7326 arg1Of are,and
R5831 T7325 T7323 arg2Of ubiquitous,are
R5832 T7326 T7298 arg2Of and,shows
R5833 T7326 T7299 arg1Of and,that
R5834 T7328 T7326 arg2Of be,and
R5835 T7328 T7327 arg2Of be,may
R5836 T7329 T7328 arg2Of restricted,be
R5837 T7329 T7330 arg1Of restricted,to
R5838 T7333 T7330 arg2Of lineages,to
R5839 T7333 T7331 arg1Of lineages,certain
R5840 T7333 T7332 arg1Of lineages,cell
R5841 T7334 T7335 arg1Of Taken,together
R5842 T7338 T7337 arg1Of data,these
R5843 T7338 T7339 arg1Of data,indicate
R5844 T7339 T7334 modOf indicate,Taken
R5845 T7339 T7336 arg1Of indicate,","
R5846 T7341 T7342 arg1Of loss,of
R5847 T7341 T7348 arg1Of loss,does
R5848 T7343 T7342 arg2Of expression,of
R5849 T7343 T7344 arg1Of expression,of
R5850 T7347 T7344 arg2Of members,of
R5851 T7347 T7345 arg1Of members,PKD
R5852 T7347 T7346 arg1Of members,family
R5853 T7348 T7339 arg2Of does,indicate
R5854 T7348 T7340 arg1Of does,that
R5855 T7348 T7349 arg1Of does,not
R5856 T7348 T7350 arg1Of does,globally
R5857 T7348 T7352 arg1Of does,on
R5858 T7351 T7350 arg2Of impact,globally
R5859 T7356 T7352 arg2Of pathways,on
R5860 T7356 T7353 arg1Of pathways,early
R5861 T7356 T7354 arg1Of pathways,BCR-regulated
R5862 T7356 T7355 arg1Of pathways,signalling
R1597 T1872 T1879 arg1Of cells,were
R1602 T1875 T1874 arg1Of cells,2 × 106
R1603 T1875 T1876 arg1Of cells,per
R1605 T1877 T1876 arg2Of point,per
R7 T51 T49 arg1Of D,protein
R8 T51 T50 arg1Of D,kinase
R12 T55 T48 arg2Of enzymes,of
R13 T55 T51 arg1Of enzymes,D
R14 T56 T45 arg1Of we,investigate
R16 T57 T44 modOf generated,To
R374 T29 T26 arg1Of enzymes,Protein
R375 T29 T27 arg1Of enzymes,kinase
R376 T29 T28 arg1Of enzymes,D
R377 T29 T30 arg1Of enzymes,are
R378 T29 T31 arg1Of enzymes,dispensable
R379 T31 T30 arg2Of dispensable,are
R380 T31 T32 arg1Of dispensable,for
R381 T33 T34 arg1Of proliferation,","
R382 T34 T36 arg1Of ",",and
R383 T35 T34 arg2Of survival,","
R384 T36 T32 arg2Of and,for
R385 T36 T41 arg1Of and,in
R386 T40 T36 arg2Of activity,and
R387 T40 T37 arg1Of activity,antigen
R388 T40 T38 arg1Of activity,receptor-regulated
R389 T40 T39 arg1Of activity,NFκB
R390 T43 T41 arg2Of B-cells,in
R391 T43 T42 arg1Of B-cells,vertebrate
R392 T45 T44 arg1Of investigate,To
R393 T47 T45 arg2Of importance,investigate
R394 T47 T46 arg1Of importance,the
R395 T47 T48 arg1Of importance,of
R1612 T1883 T1895 arg1Of buffer,containing
R1617 T1889 T1890 arg1Of 1,%
R1620 T1893 T1891 arg1Of serum,foetal
R1621 T1893 T1892 arg1Of serum,calf
R1623 T1899 T1895 arg2Of antibody,containing
R1624 T1899 T1896 arg1Of antibody,anti-chicken
R1625 T1899 T1897 arg1Of antibody,M1
R1626 T1899 T1898 arg1Of antibody,monoclonal
R1627 T1899 T1900 arg2Of antibody,conjugated
R1628 T1900 T1901 arg1Of conjugated,to
R1629 T1902 T1901 arg2Of FITC,to
R1630 T1902 T1903 arg1Of FITC,for
R1631 T1904 T1903 arg2Of 20 min,for
R1632 T1904 T1905 arg1Of 20 min,on
R1633 T1906 T1905 arg2Of ice,on
R1634 T1908 T1907 arg1Of cells,The
R1635 T1908 T1909 arg1Of cells,were
R1636 T1908 T1910 arg2Of cells,washed
R1637 T1910 T1909 arg2Of washed,were
R1638 T1910 T1911 arg1Of washed,twice
R1639 T1910 T1912 arg1Of washed,and
R1640 T1914 T1913 arg1Of intensity,fluorescent
R1641 T1914 T1915 arg1Of intensity,was
R1642 T1914 T1916 arg2Of intensity,analysed
R1643 T1916 T1912 arg2Of analysed,and
R1644 T1916 T1915 arg2Of analysed,was
R1645 T1919 T1916 arg1Of cytometry,analysed
R1646 T1919 T1917 arg2Of cytometry,by
R1647 T1919 T1918 arg1Of cytometry,flow
R1648 T1921 T1920 arg1Of results,All
R1649 T1921 T1922 arg2Of results,shown
R1650 T1921 T1923 arg1Of results,are
R1651 T1921 T1924 arg1Of results,representative
R1652 T1923 T1932 arg1Of are,unless

bionlp-st-ge-2016-coref

Id Subject Object Predicate Lexical cue
T2673 4655-4671 Anaphor denotes two PKD isoforms
T2674 4673-4677 Antecedent denotes PKD1
T2675 4682-4686 Antecedent denotes PKD3
T7357 13229-13255 Anaphor denotes diverse cellular functions
T7358 13337-13352 Antecedent denotes phosphorylation
R2277 T2673 T2674 boundBy two PKD isoforms,PKD1
R2278 T2673 T2675 boundBy two PKD isoforms,PKD3
R5863 T7357 T7358 boundBy diverse cellular functions,phosphorylation

bionlp-st-ge-2016-spacy-parsed

Id Subject Object Predicate Lexical cue
T212 0-7 NN denotes Protein
T213 8-14 NN denotes kinase
T214 15-16 NNP denotes D
T215 17-24 NNS denotes enzymes
T216 25-28 VBP denotes are
T217 29-40 JJ denotes dispensable
T218 41-44 IN denotes for
T219 45-58 NN denotes proliferation
T220 58-59 , denotes ,
T221 60-68 NN denotes survival
T222 69-72 CC denotes and
T223 73-80 NN denotes antigen
T224 81-99 JJ denotes receptor-regulated
T225 100-104 NN denotes NFκB
T226 105-113 NN denotes activity
T227 114-116 IN denotes in
T228 117-127 JJ denotes vertebrate
T229 128-135 NNPS denotes B-cells
T230 147-149 TO denotes To
T231 150-161 VB denotes investigate
T232 162-165 DT denotes the
T233 166-176 NN denotes importance
T234 177-179 IN denotes of
T235 180-187 NN denotes protein
T236 188-194 NN denotes kinase
T237 195-196 NNP denotes D
T238 197-198 -LRB- denotes (
T239 198-201 NNP denotes PKD
T240 201-202 -RRB- denotes )
T241 203-210 VBZ denotes enzymes
T242 211-213 PRP denotes we
T243 214-223 VBD denotes generated
T244 224-225 DT denotes a
T245 226-234 NNP denotes PKD-null
T246 235-239 NNP denotes DT40
T247 240-252 NNP denotes B-lymphocyte
T248 253-257 NN denotes cell
T249 258-262 NN denotes line
T250 262-263 . denotes .
T251 264-274 RB denotes Previously
T252 275-277 PRP denotes we
T253 278-282 VBP denotes have
T254 283-288 VBN denotes shown
T255 289-293 IN denotes that
T256 294-298 NNS denotes PKDs
T257 299-303 VBP denotes have
T258 304-306 DT denotes an
T259 307-316 JJ denotes essential
T260 317-321 NN denotes role
T261 322-324 IN denotes in
T262 325-335 VBG denotes regulating
T263 336-341 NN denotes class
T264 342-344 NNP denotes II
T265 345-352 NN denotes histone
T266 353-365 NNS denotes deacetylases
T267 366-368 IN denotes in
T268 369-373 NNP denotes DT40
T269 374-381 NNPS denotes B-cells
T270 382-383 NNP denotes [
T271 383-391 NNP denotes Matthews
T272 391-392 , denotes ,
T273 393-397 NNP denotes S.A.
T274 397-398 , denotes ,
T275 399-402 NNP denotes Liu
T276 402-403 , denotes ,
T277 404-406 NNP denotes P.
T278 406-407 , denotes ,
T279 408-416 NNP denotes Spitaler
T280 416-417 , denotes ,
T281 418-420 NNP denotes M.
T282 420-421 , denotes ,
T283 422-427 NNP denotes Olson
T284 427-428 , denotes ,
T285 429-433 NNP denotes E.N.
T286 433-434 , denotes ,
T287 435-443 NNP denotes McKinsey
T288 443-444 , denotes ,
T289 445-449 NNP denotes T.A.
T290 449-450 , denotes ,
T291 451-459 NNP denotes Cantrell
T292 459-460 , denotes ,
T293 461-465 NNP denotes D.A.
T294 466-469 CC denotes and
T295 470-481 NNP denotes Scharenberg
T296 481-482 , denotes ,
T297 483-487 NNP denotes A.M.
T298 488-489 -LRB- denotes (
T299 489-493 CD denotes 2006
T300 493-494 -RRB- denotes )
T301 495-504 JJ denotes Essential
T302 505-509 NN denotes role
T303 510-513 IN denotes for
T304 514-521 NN denotes protein
T305 522-528 NN denotes kinase
T306 529-530 NNP denotes D
T307 531-537 NN denotes family
T308 538-545 NNS denotes kinases
T309 546-548 IN denotes in
T310 549-552 DT denotes the
T311 553-563 NN denotes regulation
T312 564-566 IN denotes of
T313 567-572 NN denotes class
T314 573-575 NNP denotes II
T315 576-583 NN denotes histone
T316 584-596 NNS denotes deacetylases
T317 597-599 IN denotes in
T318 600-601 NNP denotes B
T319 602-613 NNS denotes lymphocytes
T320 613-614 . denotes .
T321 615-618 NNP denotes Mol
T322 618-619 . denotes .
T323 620-624 NNP denotes Cell
T324 625-629 NNP denotes Biol
T325 629-630 . denotes .
T326 631-633 CD denotes 26
T327 633-634 , denotes ,
T328 635-639 CD denotes 1569
T329 640-644 CD denotes 1577
T330 644-645 NNP denotes ]
T331 645-646 . denotes .
T332 647-649 PRP denotes We
T333 650-653 RB denotes now
T334 654-658 VBP denotes show
T335 659-663 IN denotes that
T336 664-668 NNS denotes PKDs
T337 669-672 VBP denotes are
T338 673-677 RB denotes also
T339 678-686 VBN denotes required
T340 687-689 TO denotes to
T341 690-698 VB denotes regulate
T342 699-704 NNP denotes HSP27
T343 705-720 NN denotes phosphorylation
T344 721-723 IN denotes in
T345 724-728 NNP denotes DT40
T346 729-736 NNPS denotes B-cells
T347 736-737 . denotes .
T348 738-745 RB denotes However
T349 745-746 , denotes ,
T350 747-749 IN denotes in
T351 750-758 NN denotes contrast
T352 759-761 TO denotes to
T353 762-770 JJ denotes previous
T354 771-783 NNS denotes observations
T355 784-786 IN denotes in
T356 787-792 JJ denotes other
T357 793-797 NN denotes cell
T358 798-803 NNS denotes types
T359 803-804 , denotes ,
T360 805-808 NNP denotes PKD
T361 809-816 VBZ denotes enzymes
T362 817-819 VB denotes do
T363 820-823 RB denotes not
T364 824-832 VB denotes regulate
T365 833-838 JJ denotes basic
T366 839-847 JJ denotes cellular
T367 848-857 NNS denotes processes
T368 858-862 JJ denotes such
T369 863-865 IN denotes as
T370 866-879 NN denotes proliferation
T371 880-882 CC denotes or
T372 883-891 NN denotes survival
T373 892-901 NNS denotes responses
T374 901-902 , denotes ,
T375 903-906 CC denotes nor
T376 907-911 NNP denotes NFκB
T377 912-927 JJ denotes transcriptional
T378 928-936 NN denotes activity
T379 937-947 JJ denotes downstream
T380 948-950 IN denotes of
T381 951-954 DT denotes the
T382 955-956 NNP denotes B
T383 957-961 NN denotes cell
T384 962-969 NN denotes antigen
T385 970-978 NN denotes receptor
T386 978-979 . denotes .
T387 980-984 RB denotes Thus
T388 984-985 , denotes ,
T389 986-990 NNS denotes PKDs
T390 991-995 VBP denotes have
T391 996-997 DT denotes a
T392 998-1007 JJ denotes selective
T393 1008-1012 NN denotes role
T394 1013-1015 IN denotes in
T395 1016-1020 NNP denotes DT40
T396 1021-1027 NNP denotes B-cell
T397 1028-1035 NN denotes biology
T398 1035-1036 . denotes .
T1008 635-1057 JJ denotes 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The
T1009 1058-1065 NN denotes protein
T1010 1066-1072 NN denotes kinase
T1011 1073-1074 NNP denotes D
T1012 1075-1076 -LRB- denotes (
T1013 1076-1079 NNP denotes PKD
T1014 1079-1080 -RRB- denotes )
T1015 1081-1097 JJ denotes serine/threonine
T1016 1098-1104 NN denotes kinase
T1017 1105-1111 NN denotes family
T1018 1112-1115 VBZ denotes has
T1019 1116-1121 CD denotes three
T1020 1122-1129 NNS denotes members
T1021 1129-1130 : denotes :
T1022 1131-1135 NNP denotes PKD1
T1023 1135-1136 , denotes ,
T1024 1137-1141 NNP denotes PKD2
T1025 1142-1145 CC denotes and
T1026 1146-1150 NNP denotes PKD3
T1027 1150-1151 . denotes .
T1028 1152-1156 JJS denotes Most
T1029 1157-1161 NN denotes cell
T1030 1162-1167 NNS denotes types
T1031 1168-1175 VBP denotes express
T1032 1176-1178 IN denotes at
T1033 1179-1184 JJS denotes least
T1034 1185-1188 CD denotes two
T1035 1189-1192 NNP denotes PKD
T1036 1193-1201 NNS denotes isoforms
T1037 1202-1205 CC denotes but
T1038 1206-1209 NNP denotes PKD
T1039 1210-1217 NNS denotes enzymes
T1040 1218-1221 VBP denotes are
T1041 1222-1232 RB denotes especially
T1042 1233-1239 RB denotes highly
T1043 1240-1249 VBN denotes expressed
T1044 1250-1252 IN denotes in
T1045 1253-1267 JJ denotes haematopoietic
T1046 1268-1273 NNS denotes cells
T1047 1273-1274 , denotes ,
T1048 1275-1280 WRB denotes where
T1049 1281-1285 PRP denotes they
T1050 1286-1289 VBP denotes are
T1051 1290-1299 VBN denotes activated
T1052 1300-1302 IN denotes in
T1053 1303-1311 NN denotes response
T1054 1312-1314 TO denotes to
T1055 1315-1322 NN denotes antigen
T1056 1323-1332 NNS denotes receptors
T1057 1333-1344 NN denotes stimulation
T1058 1345-1346 NNP denotes [
T1059 1346-1349 CD denotes 2,3
T1060 1349-1350 NNP denotes ]
T1061 1350-1351 . denotes .
T1062 1352-1353 DT denotes A
T1063 1354-1363 JJ denotes conserved
T1064 1364-1374 JJ denotes signalling
T1065 1375-1382 NN denotes pathway
T1066 1383-1390 VBG denotes linking
T1067 1391-1398 NN denotes antigen
T1068 1399-1408 NNS denotes receptors
T1069 1409-1411 TO denotes to
T1070 1412-1416 NNS denotes PKDs
T1071 1417-1425 VBZ denotes involves
T1072 1426-1429 DT denotes the
T1073 1430-1440 NN denotes activation
T1074 1441-1443 IN denotes of
T1075 1444-1448 NNP denotes PLCγ
T1076 1449-1452 CC denotes and
T1077 1453-1456 DT denotes the
T1078 1457-1467 JJ denotes subsequent
T1079 1468-1478 NN denotes production
T1080 1479-1481 IN denotes of
T1081 1482-1496 NN denotes diacylglycerol
T1082 1497-1498 -LRB- denotes (
T1083 1498-1501 NNP denotes DAG
T1084 1501-1502 -RRB- denotes )
T1085 1503-1508 WDT denotes which
T1086 1509-1519 VBZ denotes stimulates
T1087 1520-1529 JJ denotes classical
T1088 1530-1536 CC denotes and/or
T1089 1537-1542 JJ denotes novel
T1090 1543-1550 NN denotes protein
T1091 1551-1557 NN denotes kinase
T1092 1558-1560 NNS denotes Cs
T1093 1561-1562 -LRB- denotes (
T1094 1562-1565 NNP denotes PKC
T1095 1565-1566 -RRB- denotes )
T1096 1567-1571 WDT denotes that
T1097 1572-1585 VBP denotes phosphorylate
T1098 1586-1589 CD denotes two
T1099 1590-1593 JJ denotes key
T1100 1594-1604 JJ denotes regulatory
T1101 1605-1611 NN denotes serine
T1102 1612-1620 NNS denotes residues
T1103 1621-1623 IN denotes in
T1104 1624-1627 DT denotes the
T1105 1628-1638 NN denotes activation
T1106 1639-1643 NN denotes loop
T1107 1644-1646 IN denotes of
T1108 1647-1650 NNP denotes PKD
T1109 1651-1658 VBZ denotes kinases
T1110 1659-1660 NNP denotes [
T1111 1660-1661 CD denotes 3
T1112 1662-1663 CD denotes 6
T1113 1663-1664 NNP denotes ]
T1114 1664-1665 . denotes .
T1115 1666-1669 DT denotes The
T1116 1670-1680 JJ denotes N-terminal
T1117 1681-1691 JJ denotes regulatory
T1118 1692-1698 NN denotes region
T1119 1699-1701 IN denotes of
T1120 1702-1705 NNP denotes PKD
T1121 1706-1713 VBZ denotes enzymes
T1122 1714-1722 VBZ denotes contains
T1123 1723-1724 DT denotes a
T1124 1725-1728 NNP denotes DAG
T1125 1729-1736 JJ denotes binding
T1126 1737-1743 NN denotes domain
T1127 1744-1747 CC denotes and
T1128 1748-1754 JJ denotes direct
T1129 1755-1762 JJ denotes binding
T1130 1763-1765 IN denotes of
T1131 1766-1769 NNP denotes DAG
T1132 1770-1774 RB denotes also
T1133 1775-1786 VBZ denotes contributes
T1134 1787-1789 TO denotes to
T1135 1790-1794 CD denotes PKD1
T1136 1795-1805 NN denotes activation
T1137 1806-1807 NN denotes [
T1138 1807-1808 CD denotes 7
T1139 1808-1809 NNP denotes ]
T1140 1810-1812 RB denotes as
T1141 1813-1817 RB denotes well
T1142 1818-1820 IN denotes as
T1143 1821-1831 VBG denotes regulating
T1144 1832-1835 DT denotes the
T1145 1836-1843 JJ denotes spatial
T1146 1844-1852 NN denotes location
T1147 1853-1855 IN denotes of
T1148 1856-1859 NNP denotes PKD
T1149 1860-1867 VBZ denotes enzymes
T1150 1868-1874 IN denotes within
T1151 1875-1880 NNS denotes cells
T1152 1881-1882 VBP denotes [
T1153 1882-1883 CD denotes 8
T1154 1884-1886 CD denotes 12
T1155 1886-1887 NN denotes ]
T1156 1887-1888 . denotes .
T1157 1889-1892 NNP denotes PKD
T1158 1893-1900 NNS denotes enzymes
T1159 1901-1905 VBP denotes have
T1160 1906-1910 VBN denotes been
T1161 1911-1919 VBN denotes proposed
T1162 1920-1922 TO denotes to
T1163 1923-1931 VB denotes regulate
T1164 1932-1940 JJ denotes numerous
T1165 1941-1949 JJ denotes cellular
T1166 1950-1959 NNS denotes functions
T1167 1959-1960 , denotes ,
T1168 1961-1970 VBG denotes including
T1169 1971-1975 NN denotes cell
T1170 1976-1989 NN denotes proliferation
T1171 1990-1991 NNP denotes [
T1172 1991-1993 CD denotes 13
T1173 1994-1996 CD denotes 16
T1174 1996-1997 NNP denotes ]
T1175 1997-1998 , denotes ,
T1176 1999-2013 JJ denotes anti-apoptotic
T1177 2014-2021 NNS denotes signals
T1178 2022-2023 NNP denotes [
T1179 2023-2028 CD denotes 17,18
T1180 2028-2029 NNP denotes ]
T1181 2030-2033 CC denotes and
T1182 2034-2043 JJ denotes thymocyte
T1183 2044-2055 NN denotes development
T1184 2056-2057 NNP denotes [
T1185 2057-2059 CD denotes 19
T1186 2059-2060 NNP denotes ]
T1187 2060-2061 . denotes .
T1188 2062-2072 NN denotes Expression
T1189 2073-2075 IN denotes of
T1190 2076-2082 JJ denotes mutant
T1191 2083-2096 RB denotes catalytically
T1192 2097-2105 JJ denotes inactive
T1193 2106-2109 CC denotes and
T1194 2110-2124 RB denotes constitutively
T1195 2125-2134 VBN denotes activated
T1196 2135-2139 NNS denotes PKDs
T1197 2140-2143 MD denotes can
T1198 2144-2148 RB denotes also
T1199 2149-2155 VB denotes modify
T1200 2156-2161 NNP denotes Golgi
T1201 2162-2170 NN denotes function
T1202 2170-2171 , denotes ,
T1203 2172-2176 NN denotes cell
T1204 2177-2185 NN denotes adhesion
T1205 2186-2189 CC denotes and
T1206 2190-2194 NN denotes cell
T1207 2195-2203 NN denotes motility
T1208 2204-2205 -LRB- denotes (
T1209 2205-2213 VBN denotes reviewed
T1210 2214-2216 IN denotes in
T1211 2217-2218 NNP denotes [
T1212 2218-2220 CD denotes 20
T1213 2220-2221 NNP denotes ]
T1214 2221-2222 -RRB- denotes )
T1215 2222-2223 . denotes .
T1216 2224-2226 IN denotes In
T1217 2227-2237 JJ denotes particular
T1218 2237-2238 , denotes ,
T1219 2239-2243 NNS denotes PKDs
T1220 2244-2248 VBP denotes have
T1221 2249-2253 VBN denotes been
T1222 2254-2260 RB denotes widely
T1223 2261-2267 VBN denotes linked
T1224 2268-2270 TO denotes to
T1225 2271-2274 DT denotes the
T1226 2275-2285 NN denotes activation
T1227 2286-2288 IN denotes of
T1228 2289-2292 DT denotes the
T1229 2293-2297 NNP denotes NFκB
T1230 2298-2311 NN denotes transcription
T1231 2312-2318 NN denotes factor
T1232 2319-2322 CC denotes and
T1233 2323-2325 IN denotes in
T1234 2326-2336 VBG denotes regulating
T1235 2337-2341 NN denotes cell
T1236 2342-2350 NN denotes survival
T1237 2351-2357 IN denotes during
T1238 2358-2367 JJ denotes oxidative
T1239 2368-2374 NN denotes stress
T1240 2375-2376 NNP denotes [
T1241 2376-2381 CD denotes 17,21
T1242 2382-2384 CD denotes 23
T1243 2384-2385 NNP denotes ]
T1244 2385-2386 . denotes .
T1245 2387-2394 DT denotes Another
T1246 2395-2403 RB denotes recently
T1247 2404-2412 VBN denotes proposed
T1248 2413-2417 NNP denotes PKD1
T1249 2418-2427 NN denotes substrate
T1250 2428-2430 VBZ denotes is
T1251 2431-2436 NNP denotes HSP27
T1252 2437-2438 NNP denotes [
T1253 2438-2440 CD denotes 24
T1254 2440-2441 NNP denotes ]
T1255 2441-2442 , denotes ,
T1256 2443-2444 DT denotes a
T1257 2445-2450 JJ denotes small
T1258 2451-2455 NN denotes heat
T1259 2456-2461 NN denotes shock
T1260 2462-2469 NN denotes protein
T1261 2470-2478 VBN denotes involved
T1262 2479-2481 IN denotes in
T1263 2482-2492 VBG denotes regulating
T1264 2493-2497 NN denotes cell
T1265 2498-2507 NN denotes migration
T1266 2508-2511 CC denotes and
T1267 2512-2516 NN denotes cell
T1268 2517-2525 NN denotes survival
T1269 2526-2527 NNP denotes [
T1270 2527-2529 CD denotes 25
T1271 2529-2530 NNP denotes ]
T1272 2530-2531 . denotes .
T1273 2532-2534 DT denotes An
T1274 2535-2544 JJ denotes essential
T1275 2545-2549 NN denotes role
T1276 2550-2553 IN denotes for
T1277 2554-2557 NNP denotes PKD
T1278 2558-2565 VBZ denotes enzymes
T1279 2566-2568 IN denotes in
T1280 2569-2579 VBG denotes regulating
T1281 2580-2585 NN denotes class
T1282 2586-2588 NNP denotes II
T1283 2589-2596 NN denotes histone
T1284 2597-2609 NNS denotes deacetylases
T1285 2610-2611 -LRB- denotes (
T1286 2611-2616 NNS denotes HDACs
T1287 2616-2617 -RRB- denotes )
T1288 2617-2618 , denotes ,
T1289 2619-2626 VBZ denotes enzymes
T1290 2627-2631 IN denotes that
T1291 2632-2639 VBZ denotes repress
T1292 2640-2654 JJ denotes MEF2-dependent
T1293 2655-2659 NN denotes gene
T1294 2660-2673 NN denotes transcription
T1295 2673-2674 , denotes ,
T1296 2675-2678 VBZ denotes has
T1297 2679-2683 RB denotes also
T1298 2684-2688 VBN denotes been
T1299 2689-2701 VBN denotes demonstrated
T1300 2702-2703 NNP denotes [
T1301 2703-2707 CD denotes 1,26
T1302 2708-2710 CD denotes 28
T1303 2710-2711 NN denotes ]
T1304 2711-2712 . denotes .
T1305 2713-2715 TO denotes To
T1306 2716-2727 VB denotes investigate
T1307 2728-2731 DT denotes the
T1308 2732-2742 JJ denotes biological
T1309 2743-2747 NN denotes role
T1310 2748-2750 IN denotes of
T1311 2751-2755 NNS denotes PKDs
T1312 2756-2758 PRP denotes we
T1313 2759-2763 VBP denotes have
T1314 2764-2773 VBN denotes generated
T1315 2774-2778 NNP denotes DT40
T1316 2779-2780 NNP denotes B
T1317 2781-2785 NN denotes cell
T1318 2786-2791 NNS denotes lines
T1319 2792-2796 WDT denotes that
T1320 2797-2801 VBP denotes lack
T1321 2802-2812 NN denotes expression
T1322 2813-2815 IN denotes of
T1323 2816-2819 CD denotes one
T1324 2820-2822 CC denotes or
T1325 2823-2827 JJR denotes more
T1326 2828-2835 NNS denotes members
T1327 2836-2838 IN denotes of
T1328 2839-2842 DT denotes the
T1329 2843-2846 NNP denotes PKD
T1330 2847-2853 NN denotes family
T1331 2854-2855 NNP denotes [
T1332 2855-2856 CD denotes 1
T1333 2856-2857 NNP denotes ]
T1334 2857-2858 , denotes ,
T1335 2859-2867 VBG denotes allowing
T1336 2868-2870 PRP denotes us
T1337 2871-2873 TO denotes to
T1338 2874-2885 VB denotes investigate
T1339 2886-2889 DT denotes the
T1340 2890-2898 NN denotes function
T1341 2898-2899 -LRB- denotes (
T1342 2899-2900 PRP denotes s
T1343 2900-2901 -RRB- denotes )
T1344 2902-2904 IN denotes of
T1345 2905-2908 NNP denotes PKD
T1346 2909-2917 VBZ denotes isoforms
T1347 2918-2927 VBG denotes following
T1348 2928-2929 NNP denotes B
T1349 2930-2934 NN denotes cell
T1350 2935-2942 NN denotes antigen
T1351 2943-2951 NN denotes receptor
T1352 2952-2953 -LRB- denotes (
T1353 2953-2956 NNP denotes BCR
T1354 2956-2957 -RRB- denotes )
T1355 2958-2969 NN denotes stimulation
T1356 2969-2970 , denotes ,
T1357 2971-2973 RB denotes as
T1358 2974-2978 RB denotes well
T1359 2979-2989 VBG denotes addressing
T1360 2990-2993 DT denotes the
T1361 2994-2999 NN denotes issue
T1362 3000-3002 IN denotes of
T1363 3003-3013 JJ denotes functional
T1364 3014-3024 NN denotes redundancy
T1365 3025-3032 IN denotes between
T1366 3033-3036 DT denotes the
T1367 3037-3046 JJ denotes different
T1368 3047-3050 NNP denotes PKD
T1369 3051-3057 NN denotes family
T1370 3058-3065 NNS denotes members
T1371 3065-3066 . denotes .
T1372 3067-3075 JJ denotes Previous
T1373 3076-3083 NNS denotes studies
T1374 3084-3088 VBP denotes have
T1375 3089-3094 VBN denotes shown
T1376 3095-3099 IN denotes that
T1377 3100-3104 NNS denotes PKDs
T1378 3105-3108 VBP denotes are
T1379 3109-3122 JJ denotes indispensable
T1380 3123-3126 IN denotes for
T1381 3127-3131 NNP denotes HDAC
T1382 3132-3142 NN denotes regulation
T1383 3143-3145 IN denotes in
T1384 3146-3147 NNP denotes B
T1385 3148-3153 NNS denotes cells
T1386 3154-3155 NNP denotes [
T1387 3155-3156 CD denotes 1
T1388 3156-3157 NNP denotes ]
T1389 3157-3158 . denotes .
T1390 3159-3165 NNP denotes Herein
T1391 3166-3168 PRP denotes we
T1392 3169-3173 VBP denotes show
T1393 3174-3178 IN denotes that
T1394 3179-3183 NNS denotes PKDs
T1395 3184-3187 VBP denotes are
T1396 3188-3192 RB denotes also
T1397 3193-3206 JJ denotes indispensable
T1398 3207-3210 IN denotes for
T1399 3211-3216 NNP denotes HSP27
T1400 3217-3232 NN denotes phosphorylation
T1401 3233-3235 IN denotes in
T1402 3236-3237 NNP denotes B
T1403 3238-3243 NNS denotes cells
T1404 3243-3244 . denotes .
T1405 3245-3252 RB denotes However
T1406 3252-3253 , denotes ,
T1407 3254-3262 NNP denotes PKD-null
T1408 3263-3267 NNP denotes DT40
T1409 3268-3269 NNP denotes B
T1410 3270-3275 NNS denotes cells
T1411 3276-3279 VBP denotes are
T1412 3280-3286 JJ denotes viable
T1413 3287-3290 CC denotes and
T1414 3291-3302 VB denotes proliferate
T1415 3303-3311 RB denotes normally
T1416 3311-3312 . denotes .
T1417 3313-3321 RB denotes Moreover
T1418 3321-3322 , denotes ,
T1419 3323-3327 NN denotes loss
T1420 3328-3330 IN denotes of
T1421 3331-3334 DT denotes the
T1422 3335-3341 JJ denotes entire
T1423 3342-3350 JJ denotes cellular
T1424 3351-3355 NN denotes pool
T1425 3356-3358 IN denotes of
T1426 3359-3362 NNP denotes PKD
T1427 3363-3367 VBZ denotes does
T1428 3368-3371 RB denotes not
T1429 3372-3382 RB denotes critically
T1430 3383-3389 VB denotes affect
T1431 3390-3399 JJ denotes oxidative
T1432 3400-3406 NN denotes stress
T1433 3407-3416 NNS denotes responses
T1434 3417-3419 IN denotes in
T1435 3420-3421 NNP denotes B
T1436 3422-3427 NNS denotes cells
T1437 3428-3431 CC denotes nor
T1438 3432-3434 VBP denotes do
T1439 3435-3438 NNP denotes PKD
T1440 3439-3446 VBZ denotes kinases
T1441 3447-3451 VB denotes play
T1442 3452-3454 DT denotes an
T1443 3455-3464 JJ denotes essential
T1444 3465-3469 NN denotes role
T1445 3470-3472 IN denotes in
T1446 3473-3483 VBG denotes regulating
T1447 3484-3488 NNP denotes NFκB
T1448 3489-3504 JJ denotes transcriptional
T1449 3505-3513 NN denotes activity
T1450 3513-3514 . denotes .
T1451 3515-3523 RB denotes Together
T1452 3523-3524 , denotes ,
T1453 3525-3530 DT denotes these
T1454 3531-3539 NNS denotes findings
T1455 3540-3546 VBP denotes reveal
T1456 3547-3551 IN denotes that
T1457 3552-3554 IN denotes in
T1458 3555-3556 NNP denotes B
T1459 3557-3568 NNS denotes lymphocytes
T1460 3568-3569 , denotes ,
T1461 3570-3573 NNP denotes PKD
T1462 3574-3581 NNS denotes kinases
T1463 3582-3585 VBP denotes are
T1464 3586-3589 RB denotes not
T1465 3590-3598 JJ denotes critical
T1466 3599-3609 NNS denotes regulators
T1467 3610-3612 IN denotes of
T1468 3613-3617 JJ denotes many
T1469 3618-3620 IN denotes of
T1470 3621-3624 DT denotes the
T1471 3625-3633 JJ denotes cellular
T1472 3634-3643 NNS denotes processes
T1473 3644-3654 RB denotes previously
T1474 3655-3663 VBD denotes ascribed
T1475 3664-3666 TO denotes to
T1476 3667-3671 PRP denotes them
T1477 3672-3674 IN denotes in
T1478 3675-3680 JJ denotes other
T1479 3681-3689 JJ denotes cellular
T1480 3690-3697 NNS denotes systems
T1481 3697-3698 . denotes .
T1742 0-3731 CD denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1
T1743 3731-3735 NNP denotes Cell
T1744 3736-3743 NN denotes culture
T1745 3743-3744 , denotes ,
T1746 3745-3754 NN denotes transient
T1747 3755-3768 NNS denotes transfections
T1748 3769-3772 CC denotes and
T1749 3773-3777 NN denotes cell
T1750 3778-3789 NN denotes stimulation
T1751 3790-3793 DT denotes The
T1752 3794-3804 NN denotes generation
T1753 3804-3805 , denotes ,
T1754 3806-3813 NN denotes culture
T1755 3814-3817 CC denotes and
T1756 3818-3828 NN denotes activation
T1757 3829-3831 IN denotes of
T1758 3832-3836 CD denotes PKD1
T1759 3836-3837 CD denotes −
T1760 3837-3838 NN denotes /
T1761 3838-3839 NN denotes −
T1762 3839-3840 , denotes ,
T1763 3841-3845 NNP denotes PKD3
T1764 3845-3846 NNP denotes −
T1765 3846-3847 VBD denotes /
T1766 3847-3848 CD denotes −
T1767 3849-3852 CC denotes and
T1768 3853-3859 CD denotes PKD1/3
T1769 3859-3860 CD denotes −
T1770 3860-3861 NN denotes /
T1771 3861-3862 NN denotes −
T1772 3863-3871 NN denotes knockout
T1773 3872-3876 NNP denotes DT40
T1774 3877-3878 NNP denotes B
T1775 3879-3883 NN denotes cell
T1776 3884-3889 NNS denotes lines
T1777 3890-3894 VBP denotes have
T1778 3895-3899 VBN denotes been
T1779 3900-3909 VBN denotes described
T1780 3910-3920 RB denotes previously
T1781 3921-3922 NNP denotes [
T1782 3922-3923 CD denotes 1
T1783 3923-3924 NNP denotes ]
T1784 3924-3925 . denotes .
T1785 3926-3931 NNS denotes Cells
T1786 3932-3936 VBD denotes were
T1787 3937-3942 VBN denotes lysed
T1788 3943-3946 CC denotes and
T1789 3947-3954 NN denotes protein
T1790 3955-3963 NNS denotes extracts
T1791 3964-3968 VBD denotes were
T1792 3969-3977 VBN denotes analysed
T1793 3978-3980 IN denotes in
T1794 3981-3988 JJ denotes Western
T1795 3989-3997 VBG denotes blotting
T1796 3998-4009 NNS denotes experiments
T1797 4010-4012 IN denotes as
T1798 4013-4023 RB denotes previously
T1799 4024-4033 VBN denotes described
T1800 4034-4035 NNP denotes [
T1801 4035-4036 CD denotes 1
T1802 4036-4037 NNP denotes ]
T1803 4037-4038 . denotes .
T1804 4039-4054 NNP denotes Chloramphenicol
T1805 4055-4061 NN denotes acetyl
T1806 4062-4073 NN denotes transferase
T1807 4074-4080 NNS denotes assays
T1808 4081-4085 VBP denotes have
T1809 4086-4090 VBN denotes been
T1810 4091-4100 VBN denotes described
T1811 4101-4111 RB denotes previously
T1812 4112-4113 NNP denotes [
T1813 4113-4115 CD denotes 29
T1814 4115-4116 NNP denotes ]
T1815 4116-4117 . denotes .
T1939 0-4124 CD denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2
T1940 4124-4128 JJ denotes sIgM
T1941 4129-4137 VBG denotes staining
T1942 4138-4142 NNP denotes DT40
T1943 4143-4144 NNP denotes B
T1944 4145-4150 NNS denotes cells
T1945 4151-4152 -LRB- denotes (
T1946 4152-4153 CD denotes 2
T1947 4154-4155 CD denotes ×
T1948 4156-4159 CD denotes 106
T1949 4160-4165 NNS denotes cells
T1950 4166-4169 IN denotes per
T1951 4170-4175 NN denotes point
T1952 4175-4176 -RRB- denotes )
T1953 4177-4181 VBD denotes were
T1954 4182-4193 VBN denotes resuspended
T1955 4194-4196 IN denotes in
T1956 4197-4200 CD denotes 200
T1957 4201-4203 NN denotes μl
T1958 4204-4210 NN denotes buffer
T1959 4211-4212 -LRB- denotes (
T1960 4212-4216 NNP denotes RPMI
T1961 4217-4221 CD denotes 1640
T1962 4222-4227 NNS denotes media
T1963 4227-4228 , denotes ,
T1964 4229-4230 CD denotes 1
T1965 4230-4231 NN denotes %
T1966 4232-4238 JJ denotes foetal
T1967 4239-4243 NN denotes calf
T1968 4244-4249 NN denotes serum
T1969 4249-4250 -RRB- denotes )
T1970 4251-4261 VBG denotes containing
T1971 4262-4274 JJ denotes anti-chicken
T1972 4275-4277 CD denotes M1
T1973 4278-4288 JJ denotes monoclonal
T1974 4289-4297 NN denotes antibody
T1975 4298-4308 VBN denotes conjugated
T1976 4309-4311 TO denotes to
T1977 4312-4316 NNP denotes FITC
T1978 4317-4320 IN denotes for
T1979 4321-4323 CD denotes 20
T1980 4324-4327 NN denotes min
T1981 4328-4330 IN denotes on
T1982 4331-4334 NN denotes ice
T1983 4334-4335 . denotes .
T1984 4336-4339 DT denotes The
T1985 4340-4345 NNS denotes cells
T1986 4346-4350 VBD denotes were
T1987 4351-4357 VBN denotes washed
T1988 4358-4363 RB denotes twice
T1989 4364-4367 CC denotes and
T1990 4368-4379 JJ denotes fluorescent
T1991 4380-4389 NN denotes intensity
T1992 4390-4393 VBD denotes was
T1993 4394-4402 VBN denotes analysed
T1994 4403-4405 IN denotes by
T1995 4406-4410 NN denotes flow
T1996 4411-4420 NN denotes cytometry
T1997 4420-4421 . denotes .
T1998 4422-4425 DT denotes All
T1999 4426-4433 NNS denotes results
T2000 4434-4439 VBN denotes shown
T2001 4440-4443 VBP denotes are
T2002 4444-4458 NN denotes representative
T2003 4459-4461 IN denotes of
T2004 4462-4464 IN denotes at
T2005 4465-4468 CD denotes two
T2006 4469-4471 TO denotes to
T2007 4472-4476 CD denotes four
T2008 4477-4488 JJ denotes independent
T2009 4489-4500 NNS denotes experiments
T2010 4501-4507 IN denotes unless
T2011 4508-4517 RB denotes otherwise
T2012 4518-4527 VBN denotes indicated
T2013 4527-4528 . denotes .
T2730 0-4547 CD denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1
T2731 4547-4551 NN denotes Loss
T2732 4552-4554 IN denotes of
T2733 4555-4560 NNP denotes HSP27
T2734 4561-4576 NN denotes phosphorylation
T2735 4577-4579 IN denotes in
T2736 4580-4584 NNP denotes DT40
T2737 4585-4586 NNP denotes B
T2738 4587-4592 NNS denotes cells
T2739 4593-4600 VBG denotes lacking
T2740 4601-4611 NN denotes expression
T2741 4612-4614 IN denotes of
T2742 4615-4618 NNP denotes PKD
T2743 4619-4625 NN denotes family
T2744 4626-4633 VBZ denotes kinases
T2745 4634-4638 NNP denotes DT40
T2746 4639-4640 NNP denotes B
T2747 4641-4646 NNS denotes cells
T2748 4647-4654 VBP denotes express
T2749 4655-4658 CD denotes two
T2750 4659-4662 NNP denotes PKD
T2751 4663-4671 NNS denotes isoforms
T2752 4671-4672 , denotes ,
T2753 4673-4677 NNP denotes PKD1
T2754 4678-4681 CC denotes and
T2755 4682-4686 NNP denotes PKD3
T2756 4686-4687 , denotes ,
T2757 4688-4691 CC denotes and
T2758 4692-4694 IN denotes as
T2759 4695-4705 RB denotes previously
T2760 4706-4715 VBN denotes described
T2761 4716-4718 PRP denotes we
T2762 4719-4723 VBP denotes have
T2763 4724-4732 RB denotes recently
T2764 4733-4742 VBN denotes generated
T2765 4743-4747 NNP denotes DT40
T2766 4748-4749 NNP denotes B
T2767 4750-4754 NN denotes cell
T2768 4755-4760 NNS denotes lines
T2769 4761-4765 WDT denotes that
T2770 4766-4770 VBP denotes lack
T2771 4771-4781 NN denotes expression
T2772 4782-4784 IN denotes of
T2773 4785-4791 DT denotes either
T2774 4792-4796 CD denotes PKD1
T2775 4797-4799 CC denotes or
T2776 4800-4804 CD denotes PKD3
T2777 4805-4807 CC denotes or
T2778 4808-4812 DT denotes both
T2779 4813-4820 NNS denotes enzymes
T2780 4821-4822 VBP denotes [
T2781 4822-4823 CD denotes 1
T2782 4823-4824 NNP denotes ]
T2783 4824-4825 . denotes .
T2784 4826-4828 IN denotes In
T2785 4829-4839 VBG denotes generating
T2786 4840-4843 DT denotes the
T2787 4844-4850 JJ denotes double
T2788 4851-4859 NN denotes knockout
T2789 4860-4864 NN denotes cell
T2790 4865-4870 NNS denotes lines
T2791 4871-4873 PRP denotes we
T2792 4874-4882 VBD denotes targeted
T2793 4883-4886 DT denotes the
T2794 4887-4891 NNP denotes PKD1
T2795 4892-4896 NN denotes loci
T2796 4897-4899 IN denotes in
T2797 4900-4901 DT denotes a
T2798 4902-4906 NNP denotes PKD3
T2799 4906-4907 NNP denotes −
T2800 4907-4908 VBD denotes /
T2801 4908-4909 CD denotes −
T2802 4910-4914 NN denotes cell
T2803 4915-4919 NN denotes line
T2804 4920-4924 WDT denotes that
T2805 4925-4934 VBD denotes expressed
T2806 4935-4936 DT denotes a
T2807 4937-4946 NN denotes Flag-PKD3
T2808 4947-4956 NN denotes transgene
T2809 4957-4962 IN denotes under
T2810 4963-4966 DT denotes the
T2811 4967-4974 NN denotes control
T2812 4975-4977 IN denotes of
T2813 4978-4979 DT denotes a
T2814 4980-5001 JJ denotes doxycycline-inducible
T2815 5002-5010 NN denotes promoter
T2816 5010-5011 . denotes .
T2817 5012-5017 RB denotes Hence
T2818 5017-5018 , denotes ,
T2819 5019-5021 IN denotes in
T2820 5022-5025 DT denotes the
T2821 5026-5034 NN denotes presence
T2822 5035-5037 IN denotes of
T2823 5038-5049 NN denotes doxycycline
T2824 5049-5050 , denotes ,
T2825 5051-5060 NN denotes Flag-PKD3
T2826 5061-5071 NN denotes expression
T2827 5072-5074 IN denotes in
T2828 5075-5081 CD denotes PKD1/3
T2829 5082-5088 JJ denotes double
T2830 5089-5097 NN denotes knockout
T2831 5098-5103 NNS denotes cells
T2832 5104-5106 VBZ denotes is
T2833 5107-5117 JJ denotes comparable
T2834 5118-5120 TO denotes to
T2835 5121-5131 JJ denotes endogenous
T2836 5132-5136 JJ denotes PKD3
T2837 5137-5144 NN denotes present
T2838 5145-5147 IN denotes in
T2839 5148-5157 JJ denotes wild-type
T2840 5158-5162 NNP denotes DT40
T2841 5163-5168 NNS denotes cells
T2842 5169-5172 CC denotes and
T2843 5173-5180 NN denotes removal
T2844 5181-5183 IN denotes of
T2845 5184-5195 NN denotes doxycycline
T2846 5196-5200 IN denotes from
T2847 5201-5204 DT denotes the
T2848 5205-5212 NN denotes culture
T2849 5213-5218 NNS denotes media
T2850 5219-5222 IN denotes for
T2851 5223-5224 CD denotes 5
T2852 5225-5229 NNS denotes days
T2853 5230-5237 NNS denotes results
T2854 5238-5240 IN denotes in
T2855 5241-5242 DT denotes a
T2856 5243-5253 RB denotes completely
T2857 5254-5258 JJ denotes null
T2858 5259-5262 NNP denotes PKD
T2859 5263-5272 NN denotes phenotype
T2860 5273-5274 -LRB- denotes (
T2861 5274-5278 NNP denotes Fig.
T2862 5279-5281 NNP denotes 1A
T2863 5281-5282 -RRB- denotes )
T2864 5282-5283 . denotes .
T2865 5284-5294 RB denotes Previously
T2866 5294-5295 , denotes ,
T2867 5296-5298 PRP denotes we
T2868 5299-5303 VBP denotes have
T2869 5304-5316 VBN denotes demonstrated
T2870 5317-5321 IN denotes that
T2871 5322-5337 NN denotes phosphorylation
T2872 5338-5341 CC denotes and
T2873 5342-5349 JJ denotes nuclear
T2874 5350-5359 NN denotes exclusion
T2875 5360-5362 IN denotes of
T2876 5363-5368 NN denotes class
T2877 5369-5371 NNP denotes II
T2878 5372-5379 NN denotes histone
T2879 5380-5392 NNS denotes deacetylases
T2880 5393-5394 -LRB- denotes (
T2881 5394-5399 NNS denotes HDACs
T2882 5399-5400 -RRB- denotes )
T2883 5401-5407 IN denotes during
T2884 5408-5411 NNP denotes BCR
T2885 5412-5422 NN denotes engagement
T2886 5423-5425 VBZ denotes is
T2887 5426-5435 JJ denotes defective
T2888 5436-5438 IN denotes in
T2889 5439-5445 CD denotes PKD1/3
T2890 5445-5446 CD denotes −
T2891 5446-5447 NN denotes /
T2892 5447-5448 NN denotes −
T2893 5449-5450 NNP denotes B
T2894 5451-5456 NNS denotes cells
T2895 5457-5460 CC denotes and
T2896 5461-5464 MD denotes can
T2897 5465-5473 VBD denotes restored
T2898 5474-5478 IN denotes upon
T2899 5479-5492 NN denotes re-expression
T2900 5493-5495 IN denotes of
T2901 5496-5497 DT denotes a
T2902 5498-5504 JJ denotes single
T2903 5505-5508 NNP denotes PKD
T2904 5509-5516 NN denotes isoform
T2905 5517-5518 NNP denotes [
T2906 5518-5519 CD denotes 1
T2907 5519-5520 NNP denotes ]
T2908 5520-5521 . denotes .
T2909 5522-5525 DT denotes The
T2910 5526-5531 JJ denotes small
T2911 5532-5536 NN denotes heat
T2912 5537-5542 NN denotes shock
T2913 5543-5550 NN denotes protein
T2914 5551-5556 NNP denotes HSP27
T2915 5557-5560 VBZ denotes has
T2916 5561-5569 RB denotes recently
T2917 5570-5574 VBN denotes been
T2918 5575-5583 VBN denotes proposed
T2919 5584-5586 IN denotes as
T2920 5587-5588 DT denotes a
T2921 5589-5593 NNP denotes PKD1
T2922 5594-5603 VB denotes substrate
T2923 5604-5605 NNP denotes [
T2924 5605-5607 CD denotes 24
T2925 5607-5608 NNP denotes ]
T2926 5609-5612 CC denotes and
T2927 5613-5615 PRP denotes we
T2928 5616-5627 RB denotes accordingly
T2929 5628-5636 VBD denotes assessed
T2930 5637-5644 IN denotes whether
T2931 5645-5653 NNP denotes PKD-null
T2932 5654-5658 NNP denotes DT40
T2933 5659-5664 NNS denotes cells
T2934 5665-5669 VBP denotes have
T2935 5670-5679 JJ denotes defective
T2936 5680-5695 NN denotes phosphorylation
T2937 5696-5698 IN denotes of
T2938 5699-5704 NNP denotes HSP27
T2939 5705-5707 IN denotes on
T2940 5708-5714 NN denotes serine
T2941 5715-5717 CD denotes 82
T2942 5717-5718 , denotes ,
T2943 5719-5722 DT denotes the
T2944 5723-5731 VBN denotes proposed
T2945 5732-5736 NNP denotes PKD1
T2946 5737-5746 VB denotes substrate
T2947 5747-5755 NN denotes sequence
T2948 5755-5756 . denotes .
T2949 5757-5759 PRP denotes We
T2950 5760-5769 RB denotes initially
T2951 5770-5782 VBD denotes investigated
T2952 5783-5786 DT denotes the
T2953 5787-5797 NN denotes regulation
T2954 5798-5800 IN denotes of
T2955 5801-5806 NNP denotes HSP27
T2956 5807-5822 NN denotes phosphorylation
T2957 5823-5825 IN denotes in
T2958 5826-5832 JJ denotes single
T2959 5833-5841 NN denotes knockout
T2960 5842-5846 NNP denotes DT40
T2961 5847-5848 NNP denotes B
T2962 5849-5854 NNS denotes cells
T2963 5855-5862 VBG denotes lacking
T2964 5863-5869 CC denotes either
T2965 5870-5874 CD denotes PKD1
T2966 5875-5877 CC denotes or
T2967 5878-5882 CD denotes PKD3
T2968 5882-5883 . denotes .
T2969 5884-5886 IN denotes As
T2970 5887-5892 VBN denotes shown
T2971 5893-5895 IN denotes in
T2972 5896-5900 NNP denotes Fig.
T2973 5901-5903 NNP denotes 1B
T2974 5903-5904 , denotes ,
T2975 5905-5915 NN denotes activation
T2976 5916-5918 IN denotes of
T2977 5919-5922 DT denotes the
T2978 5923-5926 NNP denotes BCR
T2979 5927-5929 CC denotes or
T2980 5930-5939 NN denotes treatment
T2981 5940-5944 IN denotes with
T2982 5945-5948 DT denotes the
T2983 5949-5960 JJ denotes DAG-mimetic
T2984 5961-5965 NNP denotes PdBu
T2985 5966-5975 VBD denotes increased
T2986 5976-5979 DT denotes the
T2987 5980-5986 NNS denotes levels
T2988 5987-5989 IN denotes of
T2989 5990-5995 NNP denotes HSP27
T2990 5996-6011 NN denotes phosphorylation
T2991 6012-6014 IN denotes at
T2992 6015-6018 CD denotes S82
T2993 6019-6021 IN denotes in
T2994 6022-6031 JJ denotes wild-type
T2995 6032-6036 NNP denotes DT40
T2996 6037-6038 NNP denotes B
T2997 6039-6044 NNS denotes cells
T2998 6044-6045 . denotes .
T2999 6046-6049 NNP denotes BCR
T3000 6050-6053 CC denotes and
T3001 6054-6061 JJ denotes phorbol
T3002 6062-6067 NN denotes ester
T3003 6068-6075 NNS denotes signals
T3004 6076-6080 VBD denotes were
T3005 6081-6085 RB denotes also
T3006 6086-6090 JJ denotes able
T3007 6091-6093 TO denotes to
T3008 6094-6102 VB denotes increase
T3009 6103-6108 NNP denotes HSP27
T3010 6109-6124 NN denotes phosphorylation
T3011 6125-6127 IN denotes in
T3012 6128-6132 CD denotes PKD1
T3013 6133-6135 CC denotes or
T3014 6136-6140 CD denotes PKD3
T3015 6141-6147 JJ denotes single
T3016 6148-6156 NN denotes knockout
T3017 6157-6161 NNP denotes DT40
T3018 6162-6163 NNP denotes B
T3019 6164-6169 NNS denotes cells
T3020 6170-6171 -LRB- denotes (
T3021 6171-6175 NNP denotes Fig.
T3022 6176-6178 NNP denotes 1B
T3023 6178-6179 -RRB- denotes )
T3024 6179-6180 . denotes .
T3025 6181-6188 RB denotes However
T3026 6188-6189 , denotes ,
T3027 6190-6193 NNP denotes BCR
T3028 6193-6194 : denotes -
T3029 6195-6198 CC denotes and
T3030 6199-6206 VB denotes phorbol
T3031 6207-6220 JJ denotes ester-induced
T3032 6221-6236 NN denotes phosphorylation
T3033 6237-6239 IN denotes of
T3034 6240-6245 NNP denotes HSP27
T3035 6246-6248 IN denotes on
T3036 6249-6252 NNP denotes S82
T3037 6253-6256 VBD denotes was
T3038 6257-6266 VBN denotes abolished
T3039 6267-6269 IN denotes in
T3040 6270-6271 NNP denotes B
T3041 6272-6277 NNS denotes cells
T3042 6278-6282 WDT denotes that
T3043 6283-6289 VBD denotes lacked
T3044 6290-6294 DT denotes both
T3045 6295-6299 NNP denotes PKD1
T3046 6300-6303 CC denotes and
T3047 6304-6308 NNP denotes PKD3
T3048 6309-6310 -LRB- denotes (
T3049 6310-6314 NNP denotes Fig.
T3050 6315-6317 NNP denotes 1C
T3051 6317-6318 -RRB- denotes )
T3052 6318-6319 . denotes .
T3053 6320-6333 RB denotes Significantly
T3054 6333-6334 , denotes ,
T3055 6335-6354 JJ denotes doxycycline-induced
T3056 6355-6365 NN denotes expression
T3057 6366-6368 IN denotes of
T3058 6369-6372 DT denotes the
T3059 6373-6382 NN denotes Flag-PKD3
T3060 6383-6392 NN denotes transgene
T3061 6393-6395 IN denotes in
T3062 6396-6399 DT denotes the
T3063 6400-6406 JJ denotes double
T3064 6407-6415 NN denotes knockout
T3065 6416-6421 NNS denotes cells
T3066 6422-6425 VBD denotes was
T3067 6426-6436 JJ denotes sufficient
T3068 6437-6439 TO denotes to
T3069 6440-6447 VB denotes restore
T3070 6448-6454 JJ denotes normal
T3071 6455-6465 NN denotes regulation
T3072 6466-6468 IN denotes of
T3073 6469-6474 NNP denotes HSP27
T3074 6475-6490 NN denotes phosphorylation
T3075 6491-6492 -LRB- denotes (
T3076 6492-6496 NNP denotes Fig.
T3077 6497-6499 NNP denotes 1C
T3078 6499-6500 -RRB- denotes )
T3079 6500-6501 . denotes .
T3080 6502-6504 IN denotes In
T3081 6505-6513 NN denotes contrast
T3082 6513-6514 , denotes ,
T3083 6515-6525 NN denotes expression
T3084 6526-6528 IN denotes of
T3085 6529-6530 DT denotes a
T3086 6531-6547 JJ denotes kinase-deficient
T3087 6548-6552 NN denotes PKD3
T3088 6553-6559 JJ denotes mutant
T3089 6560-6567 NN denotes protein
T3090 6568-6570 IN denotes in
T3091 6571-6574 DT denotes the
T3092 6575-6581 JJ denotes double
T3093 6582-6590 NN denotes knockout
T3094 6591-6596 NNS denotes cells
T3095 6597-6600 VBD denotes was
T3096 6601-6604 RB denotes not
T3097 6605-6609 JJ denotes able
T3098 6610-6612 TO denotes to
T3099 6613-6620 VB denotes restore
T3100 6621-6624 NNP denotes BCR
T3101 6624-6625 : denotes -
T3102 6626-6628 CC denotes or
T3103 6629-6636 VB denotes phorbol
T3104 6637-6650 JJ denotes ester-induced
T3105 6651-6656 NNP denotes HSP27
T3106 6657-6672 NN denotes phosphorylation
T3107 6673-6674 -LRB- denotes (
T3108 6674-6678 NNP denotes Fig.
T3109 6679-6681 NNP denotes 1D
T3110 6681-6682 -RRB- denotes )
T3111 6682-6683 . denotes .
T3112 6684-6689 RB denotes Hence
T3113 6689-6690 , denotes ,
T3114 6691-6695 NNP denotes PKD3
T3115 6696-6698 RB denotes as
T3116 6699-6703 RB denotes well
T3117 6704-6706 IN denotes as
T3118 6707-6711 NNP denotes PKD1
T3119 6712-6715 MD denotes can
T3120 6716-6724 VB denotes regulate
T3121 6725-6730 NNP denotes HSP27
T3122 6731-6746 NN denotes phosphorylation
T3123 6747-6750 CC denotes and
T3124 6751-6753 IN denotes in
T3125 6754-6758 NNP denotes DT40
T3126 6759-6760 NNP denotes B
T3127 6761-6766 NNS denotes cells
T3128 6767-6771 PRP denotes they
T3129 6772-6775 VBP denotes are
T3130 6776-6788 RB denotes functionally
T3131 6789-6798 JJ denotes redundant
T3132 6799-6801 IN denotes as
T3133 6802-6807 NNP denotes HSP27
T3134 6808-6815 NNS denotes kinases
T3135 6815-6816 . denotes .
T4217 0-6823 CD denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2
T4218 6823-6831 NNP denotes Cellular
T4219 6832-6845 NN denotes proliferation
T4220 6846-6849 CC denotes and
T4221 6850-6858 NN denotes survival
T4222 6859-6861 IN denotes in
T4223 6862-6866 NNP denotes DT40
T4224 6867-6868 NNP denotes B
T4225 6869-6874 NNS denotes cells
T4226 6875-6882 VBG denotes lacking
T4227 6883-6893 NN denotes expression
T4228 6894-6896 IN denotes of
T4229 6897-6900 NNP denotes PKD
T4230 6901-6907 NN denotes family
T4231 6908-6915 VBZ denotes kinases
T4232 6916-6919 NNP denotes PKD
T4233 6920-6927 NNS denotes enzymes
T4234 6928-6932 VBP denotes have
T4235 6933-6943 RB denotes previously
T4236 6944-6948 VBN denotes been
T4237 6949-6955 VBN denotes linked
T4238 6956-6958 TO denotes to
T4239 6959-6962 DT denotes the
T4240 6963-6973 NN denotes regulation
T4241 6974-6976 IN denotes of
T4242 6977-6981 NN denotes cell
T4243 6982-6995 NN denotes proliferation
T4244 6996-6999 CC denotes and
T4245 7000-7008 NN denotes survival
T4246 7009-7010 -LRB- denotes (
T4247 7010-7018 VBN denotes reviewed
T4276 7165-7167 IN denotes in
T4277 7168-7171 DT denotes the
T4278 7172-7180 NN denotes presence
T4279 7181-7182 -LRB- denotes (
T4280 7182-7188 CD denotes PKD1/3
T4281 7188-7189 CD denotes −
T4282 7189-7190 NN denotes /
T4283 7190-7191 NN denotes −
T4284 7191-7192 : denotes :
T4285 7193-7202 NN denotes Flag-PKD3
T4286 7202-7203 NN denotes +
T4287 7203-7205 NN denotes ve
T4288 7205-7206 -RRB- denotes )
T4289 7207-7209 CC denotes or
T4290 7210-7217 NN denotes absence
T4291 7218-7219 -LRB- denotes (
T4292 7219-7225 CD denotes PKD1/3
T4293 7225-7226 CD denotes −
T4294 7226-7227 NN denotes /
T4295 7227-7228 NN denotes −
T4296 7228-7229 -RRB- denotes )
T4297 7230-7232 IN denotes of
T4298 7233-7244 NN denotes doxycycline
T4299 7245-7248 CC denotes and
T4300 7249-7258 VBD denotes monitored
T4301 7259-7270 JJ denotes exponential
T4302 7271-7277 NN denotes growth
T4303 7277-7278 . denotes .
T4304 7279-7281 IN denotes As
T4305 7282-7287 VBN denotes shown
T4306 7288-7290 IN denotes in
T4307 7291-7295 NNP denotes Fig.
T4308 7296-7298 NNP denotes 2A
T4309 7298-7299 , denotes ,
T4310 7300-7306 NNP denotes PKD1/3
T4311 7306-7307 VBD denotes −
T4312 7307-7308 CD denotes /
T4313 7308-7309 NN denotes −
T4314 7310-7315 NNS denotes cells
T4315 7316-7328 VBD denotes proliferated
T4316 7329-7342 RB denotes exponentially
T4317 7343-7346 CC denotes and
T4318 7347-7360 NN denotes re-expression
T4319 7361-7363 IN denotes of
T4320 7364-7373 NN denotes Flag-PKD3
T4321 7374-7376 IN denotes in
T4322 7377-7382 DT denotes these
T4323 7383-7388 NNS denotes cells
T4324 7389-7392 VBD denotes had
T4325 7393-7395 DT denotes no
T4326 7396-7402 NN denotes impact
T4327 7403-7405 IN denotes on
T4328 7406-7409 DT denotes the
T4329 7410-7414 NN denotes rate
T4330 7415-7417 IN denotes of
T4331 7418-7431 NN denotes proliferation
T4332 7431-7432 . denotes .
T4333 7433-7444 RB denotes Furthermore
T4334 7444-7445 , denotes ,
T4335 7446-7449 DT denotes the
T4336 7450-7459 NN denotes viability
T4337 7460-7462 IN denotes of
T4338 7463-7469 CD denotes PKD1/3
T4339 7469-7470 CD denotes −
T4340 7470-7471 NN denotes /
T4341 7471-7472 NN denotes −
T4342 7473-7474 NNP denotes B
T4343 7475-7480 NNS denotes cells
T4344 7481-7487 IN denotes during
T4345 7488-7495 JJ denotes routine
T4346 7496-7505 NN denotes culturing
T4347 7506-7509 VBD denotes was
T4348 7510-7513 RB denotes not
T4349 7514-7527 RB denotes significantly
T4350 7528-7537 JJ denotes different
T4351 7538-7542 IN denotes from
T4352 7543-7547 DT denotes that
T4353 7548-7550 IN denotes of
T4354 7551-7560 JJ denotes wild-type
T4355 7561-7562 NNP denotes B
T4356 7563-7568 NNS denotes cells
T4357 7569-7570 -LRB- denotes (
T4358 7570-7574 NNS denotes data
T4359 7575-7578 RB denotes not
T4360 7579-7584 VBN denotes shown
T4361 7584-7585 -RRB- denotes )
T4362 7585-7586 . denotes .
T4363 7587-7589 PRP denotes It
T4364 7590-7593 VBD denotes was
T4365 7594-7599 VBN denotes noted
T4366 7600-7604 IN denotes that
T4367 7605-7608 DT denotes the
T4368 7609-7619 NN denotes population
T4369 7620-7628 VBG denotes doubling
T4370 7629-7633 NN denotes time
T4371 7634-7636 IN denotes of
T4372 7637-7643 CD denotes PKD1/3
T4373 7643-7644 CD denotes −
T4374 7644-7645 NN denotes /
T4375 7645-7646 NN denotes −
T4376 7647-7652 NNS denotes cells
T4377 7653-7656 VBD denotes was
T4378 7657-7665 RB denotes slightly
T4379 7666-7672 JJR denotes slower
T4380 7673-7677 IN denotes than
T4381 7678-7682 DT denotes that
T4382 7683-7685 IN denotes of
T4383 7686-7690 JJ denotes wild
T4384 7691-7695 NN denotes type
T4385 7696-7700 CD denotes DT40
T4386 7701-7706 NNS denotes cells
T4387 7707-7708 -LRB- denotes (
T4388 7708-7712 CD denotes 12.7
T4389 7713-7714 CD denotes ±
T4390 7715-7718 CD denotes 2.8
T4391 7719-7720 NN denotes h
T4392 7721-7727 IN denotes versus
T4393 7728-7732 CD denotes 10.2
T4394 7733-7734 CD denotes ±
T4395 7735-7738 CD denotes 0.4
T4396 7739-7740 NN denotes h
T4397 7740-7741 -RRB- denotes )
T4398 7742-7745 CC denotes but
T4399 7746-7749 DT denotes the
T4400 7750-7757 NN denotes failure
T4401 7758-7760 IN denotes of
T4402 7761-7765 CD denotes PKD3
T4403 7766-7779 NN denotes re-expression
T4404 7780-7782 TO denotes to
T4405 7783-7789 VB denotes modify
T4406 7790-7793 DT denotes the
T4407 7794-7807 NN denotes proliferation
T4408 7808-7812 NN denotes rate
T4409 7813-7815 IN denotes of
T4410 7816-7822 CD denotes PKD1/3
T4411 7822-7823 CD denotes −
T4412 7823-7824 NN denotes /
T4413 7824-7825 NN denotes −
T4414 7826-7831 NNS denotes cells
T4415 7832-7840 VBZ denotes suggests
T4416 7841-7845 IN denotes that
T4417 7846-7851 DT denotes these
T4418 7852-7857 JJ denotes small
T4419 7858-7869 NNS denotes differences
T4420 7870-7874 VBD denotes were
T4421 7875-7879 RBS denotes most
T4422 7880-7886 JJ denotes likely
T4423 7887-7890 DT denotes the
T4424 7891-7897 NN denotes result
T4425 7898-7900 IN denotes of
T4426 7901-7907 JJ denotes clonal
T4427 7908-7917 NN denotes variation
T4428 7918-7921 CC denotes and
T4429 7922-7926 VBD denotes were
T4430 7927-7930 RB denotes not
T4431 7931-7937 VBN denotes caused
T4432 7938-7950 RB denotes specifically
T4433 7951-7953 IN denotes by
T4434 7954-7958 NN denotes loss
T4435 7959-7961 IN denotes of
T4436 7962-7965 NNP denotes PKD
T4437 7966-7973 VBZ denotes enzymes
T4438 7973-7974 . denotes .
T4439 7975-7979 RB denotes Thus
T4440 7979-7980 , denotes ,
T4441 7981-7984 NNP denotes PKD
T4442 7985-7991 NN denotes family
T4443 7992-7999 NNS denotes enzymes
T4444 8000-8003 VBP denotes are
T4445 8004-8007 RB denotes not
T4446 8008-8017 JJ denotes essential
T4447 8018-8021 IN denotes for
T4448 8022-8032 VBG denotes regulating
T4449 8033-8038 JJ denotes basal
T4450 8039-8047 NN denotes survival
T4451 8048-8051 CC denotes and
T4452 8052-8065 NN denotes proliferation
T4453 8066-8068 IN denotes of
T4454 8069-8073 NNP denotes DT40
T4455 8074-8075 NNP denotes B
T4456 8076-8081 NNS denotes cells
T4457 8081-8082 . denotes .
T4458 8083-8086 NNP denotes PKD
T4459 8087-8094 VBZ denotes enzymes
T4460 8094-8095 , denotes ,
T4461 8096-8108 RB denotes specifically
T4462 8109-8113 CD denotes PKD1
T4463 8114-8117 CC denotes and
T4464 8118-8122 NNP denotes PKD2
T4465 8122-8123 , denotes ,
T4466 8124-8128 VBP denotes have
T4467 8129-8139 RB denotes previously
T4468 8140-8144 VBN denotes been
T4469 8145-8151 VBN denotes linked
T4470 8152-8154 TO denotes to
T4471 8155-8156 DT denotes a
T4472 8157-8167 JJ denotes protective
T4473 8168-8172 NN denotes role
T4474 8173-8180 IN denotes against
T4475 8181-8190 JJ denotes oxidative
T4476 8191-8205 JJ denotes stress-induced
T4477 8206-8212 NN denotes injury
T4478 8213-8215 IN denotes in
T4479 8216-8219 NNP denotes 3T3
T4480 8220-8230 NN denotes fibroblast
T4481 8230-8231 , denotes ,
T4482 8232-8236 NNP denotes HeLa
T4483 8237-8240 CC denotes and
T4484 8241-8251 JJ denotes epithelial
T4485 8252-8256 NN denotes cell
T4486 8257-8262 NNS denotes lines
T4487 8263-8264 NNP denotes [
T4488 8264-8269 CD denotes 17,30
T4489 8270-8272 CD denotes 32
T4490 8272-8273 NN denotes ]
T4491 8273-8274 . denotes .
T4492 8275-8277 PRP denotes We
T4493 8278-8287 RB denotes therefore
T4494 8288-8297 VBD denotes addressed
T4495 8298-8301 DT denotes the
T4496 8302-8306 NN denotes role
T4497 8307-8309 IN denotes of
T4498 8310-8313 NNP denotes PKD
T4499 8314-8320 NN denotes family
T4500 8321-8328 NNS denotes kinases
T4501 8329-8331 IN denotes in
T4502 8332-8342 VBG denotes regulating
T4503 8343-8344 NNP denotes B
T4504 8345-8349 NN denotes cell
T4505 8350-8358 NN denotes survival
T4506 8359-8361 IN denotes in
T4507 8362-8370 NN denotes response
T4508 8371-8373 TO denotes to
T4509 8374-8383 JJ denotes oxidative
T4510 8384-8390 NN denotes stress
T4511 8391-8394 CC denotes and
T4512 8395-8400 JJ denotes other
T4513 8401-8407 NN denotes stress
T4514 8408-8415 NNS denotes stimuli
T4515 8415-8416 . denotes .
T4516 8417-8419 IN denotes As
T4517 8420-8425 VBN denotes shown
T4518 8426-8428 IN denotes in
T4519 8429-8433 NNP denotes Fig.
T4520 8434-8436 NNP denotes 2B
T4521 8436-8437 , denotes ,
T4522 8438-8442 NN denotes loss
T4523 8443-8445 IN denotes of
T4524 8446-8452 CD denotes PKD1/3
T4525 8453-8463 NN denotes expression
T4526 8464-8467 VBD denotes had
T4527 8468-8470 DT denotes no
T4528 8471-8482 JJ denotes significant
T4529 8483-8489 NN denotes impact
T4530 8490-8492 IN denotes on
T4531 8493-8496 DT denotes the
T4532 8497-8505 NN denotes survival
T4533 8506-8508 IN denotes of
T4534 8509-8513 NNP denotes DT40
T4535 8514-8515 NNP denotes B
T4536 8516-8521 NNS denotes cells
T4537 8522-8524 IN denotes in
T4538 8525-8533 NN denotes response
T4539 8534-8536 TO denotes to
T4540 8537-8550 JJ denotes mitochondrial
T4541 8551-8557 NN denotes stress
T4542 8558-8565 NNS denotes stimuli
T4543 8566-8567 -LRB- denotes (
T4544 8567-8571 CD denotes H2O2
T4545 8572-8574 CC denotes or
T4546 8575-8580 NN denotes serum
T4547 8581-8592 NN denotes deprivation
T4548 8592-8593 -RRB- denotes )
T4549 8593-8594 : denotes ;
T4550 8595-8598 NNP denotes DNA
T4551 8599-8607 VBG denotes damaging
T4552 8608-8614 NNS denotes agents
T4553 8615-8616 -LRB- denotes (
T4554 8616-8625 NN denotes etoposide
T4555 8626-8628 CC denotes or
T4556 8629-8640 NN denotes doxorubicin
T4557 8640-8641 -RRB- denotes )
T4558 8641-8642 : denotes ;
T4559 8643-8645 NNP denotes ER
T4560 8646-8653 NN denotes pathway
T4561 8654-8660 NN denotes stress
T4562 8661-8664 JJ denotes due
T4563 8665-8667 TO denotes to
T4564 8668-8675 NN denotes calcium
T4565 8676-8684 NN denotes overload
T4566 8685-8686 -LRB- denotes (
T4567 8686-8698 NN denotes thapsigargin
T4568 8698-8699 -RRB- denotes )
T4569 8700-8702 CC denotes or
T4570 8703-8712 VBG denotes following
T4571 8713-8722 JJ denotes prolonged
T4572 8723-8732 NN denotes treatment
T4573 8733-8737 IN denotes with
T4574 8738-8745 NN denotes phorbol
T4575 8746-8752 NNS denotes esters
T4576 8753-8755 CC denotes or
T4577 8756-8768 NNP denotes Trichostatin
T4578 8769-8770 NNP denotes A
T4579 8770-8771 , denotes ,
T4580 8772-8774 DT denotes an
T4581 8775-8784 NN denotes inhibitor
T4582 8785-8787 IN denotes of
T4583 8788-8793 NN denotes class
T4584 8794-8798 NNP denotes I/II
T4585 8799-8804 NNP denotes HDACs
T4586 8804-8805 . denotes .
T4587 8806-8810 RB denotes Thus
T4588 8810-8811 , denotes ,
T4589 8812-8815 NNP denotes PKD
T4590 8816-8823 NNS denotes kinases
T4591 8824-8826 VBP denotes do
T4592 8827-8830 RB denotes not
T4593 8831-8835 VB denotes play
T4594 8836-8838 DT denotes an
T4595 8839-8848 JJ denotes essential
T4596 8849-8853 NN denotes role
T4597 8854-8856 IN denotes in
T4598 8857-8867 VBG denotes regulating
T4599 8868-8869 NNP denotes B
T4600 8870-8874 NN denotes cell
T4601 8875-8883 NN denotes survival
T4602 8884-8886 IN denotes in
T4603 8887-8895 NN denotes response
T4604 8896-8898 TO denotes to
T4605 8899-8900 DT denotes a
T4606 8901-8906 NN denotes range
T4607 8907-8909 IN denotes of
T4608 8910-8919 JJ denotes different
T4609 8920-8926 NN denotes stress
T4610 8927-8934 NNS denotes stimuli
T4611 8934-8935 . denotes .
T5768 0-8942 CD denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3
T5769 8942-8949 NN denotes Antigen
T5770 8950-8958 NN denotes receptor
T5771 8959-8968 VBN denotes regulated
T5772 8969-8979 JJ denotes signalling
T5773 8980-8988 NNS denotes pathways
T5774 8989-8991 IN denotes in
T5775 8992-9000 NNP denotes PKD-null
T5776 9001-9005 NNP denotes DT40
T5777 9006-9007 NNP denotes B
T5778 9008-9013 NNS denotes cells
T5779 9014-9016 TO denotes To
T5780 9017-9024 RBR denotes further
T5781 9025-9032 VB denotes explore
T5782 9033-9036 DT denotes the
T5783 9037-9049 NN denotes contribution
T5784 9050-9052 IN denotes of
T5785 9053-9056 NNP denotes PKD
T5786 9057-9064 VBZ denotes kinases
T5787 9065-9067 TO denotes to
T5788 9068-9072 NNP denotes DT40
T5789 9073-9074 NNP denotes B
T5790 9075-9079 NN denotes cell
T5791 9080-9087 NN denotes biology
T5792 9088-9090 PRP denotes we
T5793 9091-9103 VBD denotes investigated
T5794 9104-9111 IN denotes whether
T5795 9112-9120 JJ denotes specific
T5796 9121-9134 JJ denotes BCR-regulated
T5797 9135-9145 JJ denotes signalling
T5798 9146-9152 NNS denotes events
T5799 9153-9157 VBD denotes were
T5800 9158-9167 JJ denotes defective
T5801 9168-9170 IN denotes in
T5802 9171-9174 DT denotes the
T5803 9175-9183 NNP denotes PKD-null
T5804 9184-9185 NNP denotes B
T5805 9186-9191 NNS denotes cells
T5806 9191-9192 . denotes .
T5807 9193-9200 JJ denotes Initial
T5808 9201-9212 NNS denotes experiments
T5809 9213-9221 VBD denotes revealed
T5810 9222-9226 IN denotes that
T5811 9227-9234 NN denotes surface
T5812 9235-9245 NN denotes expression
T5813 9246-9248 IN denotes of
T5814 9249-9252 DT denotes the
T5815 9253-9256 NNP denotes BCR
T5816 9257-9260 VBD denotes was
T5817 9261-9268 VBN denotes reduced
T5818 9269-9271 IN denotes in
T5819 9272-9278 CD denotes PKD1/3
T5820 9278-9279 CD denotes −
T5821 9279-9280 NN denotes /
T5822 9280-9281 NN denotes −
T5823 9282-9283 -LRB- denotes (
T5824 9283-9286 CC denotes and
T5825 9287-9289 IN denotes in
T5826 9290-9296 CD denotes PKD1/3
T5827 9296-9297 CD denotes −
T5828 9297-9298 NN denotes /
T5829 9298-9299 NN denotes −
T5830 9299-9300 : denotes :
T5831 9300-9309 NN denotes Flag-PKD3
T5832 9309-9310 NN denotes +
T5833 9310-9312 NN denotes ve
T5834 9312-9313 -RRB- denotes )
T5835 9314-9319 NNS denotes cells
T5836 9320-9328 VBN denotes compared
T5837 9329-9331 TO denotes to
T5838 9332-9341 JJ denotes wild-type
T5839 9342-9346 NNP denotes DT40
T5840 9347-9348 NNP denotes B
T5841 9349-9354 NNS denotes cells
T5842 9355-9356 -LRB- denotes (
T5843 9356-9360 NNP denotes Fig.
T5844 9361-9363 NNP denotes 3A
T5845 9364-9367 CC denotes and
T5846 9368-9372 NNS denotes data
T5847 9373-9376 RB denotes not
T5848 9377-9382 VBN denotes shown
T5849 9382-9383 -RRB- denotes )
T5850 9383-9384 . denotes .
T5851 9385-9397 RB denotes Nevertheless
T5852 9397-9398 , denotes ,
T5853 9399-9415 JJ denotes BCR-crosslinking
T5854 9416-9418 IN denotes of
T5855 9419-9425 CD denotes PKD1/3
T5856 9425-9426 CD denotes −
T5857 9426-9427 NN denotes /
T5858 9427-9428 NN denotes −
T5859 9429-9434 NNS denotes cells
T5860 9435-9438 VBD denotes was
T5861 9439-9449 JJ denotes sufficient
T5862 9450-9452 TO denotes to
T5863 9453-9459 VB denotes induce
T5864 9460-9463 DT denotes the
T5865 9464-9474 NN denotes activation
T5866 9475-9477 IN denotes of
T5867 9478-9479 DT denotes a
T5868 9480-9486 NN denotes number
T5869 9487-9489 IN denotes of
T5870 9490-9500 VBG denotes signalling
T5871 9501-9509 NNS denotes cascades
T5872 9509-9510 , denotes ,
T5873 9511-9518 JJ denotes similar
T5874 9519-9521 TO denotes to
T5875 9522-9526 DT denotes that
T5876 9527-9535 VBN denotes observed
T5877 9536-9538 IN denotes in
T5878 9539-9548 JJ denotes wild-type
T5879 9549-9554 NNS denotes cells
T5880 9555-9556 -LRB- denotes (
T5881 9556-9560 NNP denotes Fig.
T5882 9561-9563 NNP denotes 3B
T5883 9563-9564 -RRB- denotes )
T5884 9564-9565 . denotes .
T5885 9566-9571 RB denotes Hence
T5886 9571-9572 , denotes ,
T5887 9573-9584 JJ denotes BCR-induced
T5888 9585-9595 NN denotes activation
T5889 9596-9598 IN denotes of
T5890 9599-9602 DT denotes the
T5891 9603-9606 NNP denotes Akt
T5892 9606-9607 , denotes ,
T5893 9608-9616 NNP denotes mTOR/p70
T5894 9617-9619 NNP denotes S6
T5895 9620-9626 NN denotes kinase
T5896 9627-9628 -LRB- denotes (
T5897 9628-9630 IN denotes as
T5898 9631-9636 VBN denotes shown
T5899 9637-9639 IN denotes by
T5900 9640-9642 CD denotes S6
T5901 9643-9652 JJ denotes ribosomal
T5902 9653-9660 NN denotes protein
T5903 9661-9676 NN denotes phosphorylation
T5904 9676-9677 -RRB- denotes )
T5905 9678-9681 CC denotes and
T5906 9682-9686 NNP denotes MAPK
T5907 9687-9697 VBG denotes signalling
T5908 9698-9706 NNS denotes pathways
T5909 9707-9710 VBD denotes was
T5910 9711-9718 RB denotes clearly
T5911 9719-9729 JJ denotes detectable
T5912 9730-9732 IN denotes in
T5913 9733-9744 NNP denotes PKD1/3-null
T5914 9745-9746 NNP denotes B
T5915 9747-9752 NNS denotes cells
T5916 9753-9754 -LRB- denotes (
T5917 9754-9758 NNP denotes Fig.
T5918 9759-9761 NNP denotes 3B
T5919 9761-9762 -RRB- denotes )
T5920 9762-9763 . denotes .
T5921 9764-9775 RB denotes Furthermore
T5922 9775-9776 , denotes ,
T5923 9777-9785 JJ denotes enhanced
T5924 9786-9794 NN denotes tyrosine
T5925 9795-9810 NN denotes phosphorylation
T5926 9811-9813 IN denotes of
T5927 9814-9822 JJ denotes multiple
T5928 9823-9831 JJ denotes cellular
T5929 9832-9840 NNS denotes proteins
T5930 9841-9843 RB denotes as
T5931 9844-9848 RB denotes well
T5932 9849-9851 IN denotes as
T5933 9852-9854 DT denotes an
T5934 9855-9863 NN denotes increase
T5935 9864-9866 IN denotes in
T5936 9867-9880 JJ denotes intracellular
T5937 9881-9888 NN denotes calcium
T5938 9889-9895 NNS denotes levels
T5939 9896-9899 VBD denotes was
T5940 9900-9904 RB denotes also
T5941 9905-9913 VBN denotes observed
T5942 9914-9923 VBG denotes following
T5943 9924-9927 NNP denotes BCR
T5944 9928-9939 NN denotes stimulation
T5945 9940-9942 IN denotes of
T5946 9943-9954 NNP denotes PKD1/3-null
T5947 9955-9956 NNP denotes B
T5948 9957-9962 NNS denotes cells
T5949 9963-9964 -LRB- denotes (
T5950 9964-9968 NNS denotes data
T5951 9969-9972 RB denotes not
T5952 9973-9978 VBN denotes shown
T5953 9978-9979 -RRB- denotes )
T5954 9979-9980 . denotes .
T5955 9981-9983 PRP denotes We
T5956 9984-9987 VBD denotes did
T5957 9988-9995 VB denotes observe
T5958 9996-10000 IN denotes that
T5959 10001-10004 DT denotes the
T5960 10005-10013 NN denotes strength
T5961 10014-10016 IN denotes of
T5962 10017-10020 NNP denotes BCR
T5963 10021-10022 -LRB- denotes (
T5964 10022-10025 CC denotes but
T5965 10026-10029 RB denotes not
T5966 10030-10037 VB denotes phorbol
T5967 10038-10043 NN denotes ester
T5968 10043-10044 -RRB- denotes )
T5969 10044-10045 : denotes -
T5970 10045-10052 VBD denotes induced
T5971 10053-10063 NN denotes regulation
T5972 10064-10066 IN denotes of
T5973 10067-10070 DT denotes the
T5974 10071-10080 JJ denotes Erk1-RSK1
T5975 10081-10091 NN denotes signalling
T5976 10092-10099 NN denotes pathway
T5977 10100-10103 VBD denotes was
T5978 10104-10111 VBN denotes reduced
T5979 10112-10114 IN denotes in
T5980 10115-10121 CD denotes PKD1/3
T5981 10121-10122 CD denotes −
T5982 10122-10123 NN denotes /
T5983 10123-10124 NN denotes −
T5984 10125-10126 NNP denotes B
T5985 10127-10132 NNS denotes cells
T5986 10133-10141 VBN denotes compared
T5987 10142-10144 TO denotes to
T5988 10145-10154 JJ denotes wild-type
T5989 10155-10156 NNP denotes B
T5990 10157-10162 NNS denotes cells
T5991 10163-10164 -LRB- denotes (
T5992 10164-10168 NNP denotes Fig.
T5993 10169-10171 NNP denotes 3B
T5994 10171-10172 -RRB- denotes )
T5995 10172-10173 . denotes .
T5996 10174-10177 CD denotes One
T5997 10178-10192 NN denotes interpretation
T5998 10193-10195 IN denotes of
T5999 10196-10200 DT denotes this
T6000 10201-10205 NN denotes data
T6001 10206-10208 VBZ denotes is
T6002 10209-10213 IN denotes that
T6003 10214-10217 NNP denotes PKD
T6004 10218-10225 VBZ denotes enzymes
T6005 10226-10229 MD denotes may
T6006 10230-10238 VB denotes modulate
T6007 10239-10242 NNP denotes Erk
T6008 10243-10253 NN denotes activation
T6009 10253-10254 . denotes .
T6010 10255-10261 RB denotes Indeed
T6011 10261-10262 , denotes ,
T6012 10263-10266 NNP denotes PKD
T6013 10267-10274 NNS denotes enzymes
T6014 10275-10279 VBP denotes have
T6015 10280-10290 RB denotes previously
T6016 10291-10295 VBN denotes been
T6017 10296-10302 VBN denotes linked
T6018 10303-10305 TO denotes to
T6019 10306-10309 DT denotes the
T6020 10310-10316 NN denotes growth
T6021 10317-10333 JJ denotes factor-regulated
T6022 10334-10337 NNP denotes Erk
T6023 10338-10348 NN denotes signalling
T6024 10349-10351 IN denotes in
T6025 10352-10362 NN denotes fibroblast
T6026 10363-10366 CC denotes and
T6027 10367-10378 JJ denotes endothelial
T6028 10379-10383 NN denotes cell
T6029 10384-10389 NNS denotes lines
T6030 10390-10391 NNP denotes [
T6031 10391-10393 CD denotes 33
T6032 10394-10396 CD denotes 35
T6033 10396-10397 NN denotes ]
T6034 10397-10398 . denotes .
T6035 10399-10406 RB denotes However
T6036 10406-10407 , denotes ,
T6037 10408-10419 JJ denotes BCR-induced
T6038 10420-10423 NNP denotes Erk
T6039 10424-10439 NN denotes phosphorylation
T6040 10440-10443 VBD denotes was
T6041 10444-10448 RB denotes also
T6042 10449-10456 VBN denotes reduced
T6043 10457-10459 IN denotes in
T6044 10460-10466 CD denotes PKD1/3
T6045 10466-10467 CD denotes −
T6046 10467-10468 NN denotes /
T6047 10468-10469 SYM denotes −
T6048 10469-10470 : denotes -
T6049 10470-10479 NN denotes Flag-PKD3
T6050 10479-10480 NN denotes +
T6051 10481-10482 NNP denotes B
T6052 10483-10488 NNS denotes cells
T6053 10489-10490 -LRB- denotes (
T6054 10490-10494 NNS denotes data
T6055 10495-10498 RB denotes not
T6056 10499-10504 VBN denotes shown
T6057 10504-10505 -RRB- denotes )
T6058 10506-10516 VBG denotes suggesting
T6059 10517-10521 IN denotes that
T6060 10522-10529 VBN denotes reduced
T6061 10530-10533 NNP denotes BCR
T6062 10534-10540 NNS denotes levels
T6063 10541-10543 IN denotes on
T6064 10544-10547 DT denotes the
T6065 10548-10555 NN denotes surface
T6066 10556-10558 IN denotes of
T6067 10559-10565 CD denotes PKD1/3
T6068 10565-10566 CD denotes −
T6069 10566-10567 NN denotes /
T6070 10567-10568 NN denotes −
T6071 10569-10570 -LRB- denotes (
T6072 10570-10573 CC denotes and
T6073 10574-10580 NNP denotes PKD1/3
T6074 10580-10581 VBD denotes −
T6075 10581-10582 CD denotes /
T6076 10582-10583 SYM denotes −
T6077 10583-10584 : denotes -
T6078 10584-10593 NN denotes Flag-PKD3
T6079 10593-10594 NN denotes +
T6080 10594-10595 -RRB- denotes )
T6081 10596-10597 NNP denotes B
T6082 10598-10603 NNS denotes cells
T6083 10604-10607 MD denotes may
T6084 10608-10614 PRP denotes itself
T6085 10615-10621 VB denotes impact
T6086 10622-10624 IN denotes on
T6087 10625-10628 DT denotes the
T6088 10629-10637 NN denotes strength
T6089 10638-10640 IN denotes of
T6090 10641-10651 NN denotes activation
T6091 10652-10654 IN denotes of
T6092 10655-10659 DT denotes this
T6093 10660-10668 JJ denotes specific
T6094 10669-10682 JJ denotes intracellular
T6095 10683-10693 NN denotes signalling
T6096 10694-10701 NN denotes pathway
T6097 10701-10702 . denotes .
T6098 10703-10705 TO denotes To
T6099 10706-10712 VB denotes search
T6100 10713-10716 IN denotes for
T6101 10717-10722 JJ denotes other
T6102 10723-10732 JJ denotes potential
T6103 10733-10736 NNP denotes PKD
T6104 10737-10744 NNS denotes targets
T6105 10745-10749 WDT denotes that
T6106 10750-10753 MD denotes may
T6107 10754-10758 VB denotes show
T6108 10759-10768 JJ denotes defective
T6109 10769-10779 NN denotes regulation
T6110 10780-10782 IN denotes in
T6111 10783-10789 NNP denotes PKD1/3
T6112 10789-10790 NNP denotes −
T6113 10790-10791 NNP denotes /
T6114 10791-10792 NNP denotes −
T6115 10793-10797 NNP denotes DT40
T6116 10798-10799 NNP denotes B
T6117 10800-10805 NNS denotes cells
T6118 10805-10806 , denotes ,
T6119 10807-10809 PRP denotes we
T6120 10810-10814 VBD denotes used
T6121 10815-10816 DT denotes a
T6122 10817-10820 NNP denotes PKD
T6123 10821-10830 VB denotes substrate
T6124 10831-10847 JJ denotes phospho-antibody
T6125 10848-10852 IN denotes that
T6126 10853-10863 VBZ denotes recognises
T6127 10864-10873 NN denotes consensus
T6128 10874-10889 NN denotes phosphorylation
T6129 10890-10899 NNS denotes sequences
T6130 10900-10908 VBN denotes targeted
T6131 10909-10911 IN denotes by
T6132 10912-10915 NNP denotes PKD
T6133 10916-10923 VBZ denotes enzymes
T6134 10924-10925 -LRB- denotes (
T6135 10925-10934 NNP denotes LxRxxpS/T
T6136 10934-10935 -RRB- denotes )
T6137 10936-10937 NNP denotes [
T6138 10937-10939 CD denotes 36
T6139 10939-10940 NNP denotes ]
T6140 10940-10941 . denotes .
T6141 10942-10944 IN denotes As
T6142 10945-10950 VBN denotes shown
T6143 10951-10953 IN denotes in
T6144 10954-10958 NNP denotes Fig.
T6145 10959-10961 NNP denotes 3C
T6146 10961-10962 , denotes ,
T6147 10963-10970 NN denotes phorbol
T6148 10971-10976 NN denotes ester
T6149 10976-10977 : denotes -
T6150 10978-10981 CC denotes and
T6151 10982-10993 JJ denotes BCR-induced
T6152 10994-11009 NN denotes phosphorylation
T6153 11010-11012 IN denotes of
T6154 11013-11021 JJ denotes cellular
T6155 11022-11032 NNS denotes substrates
T6156 11033-11041 VBN denotes detected
T6157 11042-11044 IN denotes by
T6158 11045-11049 DT denotes this
T6159 11050-11066 NN denotes phospho-antibody
T6160 11067-11070 VBD denotes was
T6161 11071-11078 JJ denotes similar
T6162 11079-11081 IN denotes in
T6163 11082-11091 JJ denotes wild-type
T6164 11092-11095 CC denotes and
T6165 11096-11102 JJ denotes PKD1/3
T6166 11102-11103 NN denotes −
T6167 11103-11104 NN denotes /
T6168 11104-11105 NN denotes −
T6169 11106-11111 NNS denotes cells
T6170 11112-11115 CC denotes and
T6171 11116-11118 VBZ denotes is
T6172 11119-11128 RB denotes therefore
T6173 11129-11140 JJ denotes independent
T6174 11141-11143 IN denotes of
T6175 11144-11147 NNP denotes PKD
T6176 11148-11155 VBZ denotes enzymes
T6177 11155-11156 . denotes .
T6178 11157-11164 RB denotes However
T6179 11164-11165 , denotes ,
T6180 11166-11178 NN denotes pretreatment
T6181 11179-11181 IN denotes of
T6182 11182-11186 DT denotes both
T6183 11187-11196 JJ denotes wild-type
T6184 11197-11200 CC denotes and
T6185 11201-11207 JJ denotes PKD1/3
T6186 11207-11208 NN denotes −
T6187 11208-11209 NN denotes /
T6188 11209-11210 NN denotes −
T6189 11211-11215 NNP denotes DT40
T6190 11216-11217 NNP denotes B
T6191 11218-11223 NNS denotes cells
T6192 11224-11228 IN denotes with
T6193 11229-11238 NNP denotes GF109203X
T6194 11238-11239 , denotes ,
T6195 11240-11241 DT denotes a
T6196 11242-11260 NN denotes bisindoylmaleimide
T6197 11261-11271 NN denotes derivative
T6198 11272-11276 WDT denotes that
T6199 11277-11285 VBZ denotes inhibits
T6200 11286-11290 NNS denotes PKCs
T6201 11291-11300 VBD denotes prevented
T6202 11301-11304 DT denotes the
T6203 11305-11314 NN denotes induction
T6204 11315-11317 IN denotes of
T6205 11318-11326 NNS denotes proteins
T6206 11327-11331 WDT denotes that
T6207 11332-11339 VBP denotes contain
T6208 11340-11354 VBN denotes phosphorylated
T6209 11355-11363 NNP denotes LxRxxS/T
T6210 11364-11370 NNS denotes motifs
T6211 11370-11371 . denotes .
T6212 11372-11376 RB denotes Thus
T6213 11377-11381 NN denotes loss
T6214 11382-11384 IN denotes of
T6215 11385-11391 NNP denotes PKD1/3
T6216 11392-11399 VBZ denotes enzymes
T6217 11400-11404 VBZ denotes does
T6218 11405-11408 RB denotes not
T6219 11409-11417 RB denotes globally
T6220 11418-11425 VB denotes disrupt
T6221 11426-11429 DT denotes the
T6222 11430-11445 NN denotes phosphorylation
T6223 11446-11448 IN denotes of
T6224 11449-11457 JJ denotes cellular
T6225 11458-11466 NNS denotes proteins
T6226 11467-11471 WDT denotes that
T6227 11472-11479 VBP denotes contain
T6228 11480-11489 NNP denotes LxRxxpS/T
T6229 11490-11496 NNS denotes motifs
T6230 11496-11497 . denotes .
T6231 11498-11502 DT denotes This
T6232 11503-11509 NN denotes result
T6233 11510-11512 VBZ denotes is
T6234 11513-11520 RB denotes perhaps
T6235 11521-11524 RB denotes not
T6236 11525-11535 JJ denotes surprising
T6237 11536-11538 IN denotes as
T6238 11539-11547 NNP denotes LxRxxS/T
T6239 11548-11554 NNS denotes motifs
T6240 11555-11559 RB denotes also
T6241 11560-11563 VBP denotes act
T6242 11564-11566 RB denotes as
T6243 11567-11571 JJ denotes good
T6244 11572-11582 NNS denotes substrates
T6245 11583-11586 IN denotes for
T6246 11587-11592 JJ denotes other
T6247 11593-11609 JJ denotes serine/threonine
T6248 11610-11617 NNS denotes kinases
T6249 11618-11622 JJ denotes such
T6250 11623-11625 IN denotes as
T6251 11626-11634 NNP denotes MAPKAPK2
T6252 11634-11635 . denotes .
T6253 11636-11643 RB denotes However
T6254 11644-11649 DT denotes these
T6255 11650-11661 NNS denotes experiments
T6256 11662-11664 VBP denotes do
T6257 11665-11672 VB denotes provide
T6258 11673-11680 JJ denotes further
T6259 11681-11689 NN denotes evidence
T6260 11690-11694 IN denotes that
T6261 11695-11710 JJ denotes phosphospecific
T6262 11711-11719 NN denotes antisera
T6263 11720-11723 VBP denotes are
T6264 11724-11727 RB denotes not
T6265 11728-11740 RB denotes sufficiently
T6266 11741-11750 JJ denotes selective
T6267 11751-11753 TO denotes to
T6268 11754-11756 VB denotes be
T6269 11757-11767 VBN denotes designated
T6270 11768-11774 NN denotes kinase
T6271 11775-11783 JJ denotes specific
T6272 11784-11793 JJ denotes substrate
T6273 11794-11802 NN denotes antisera
T6274 11802-11803 . denotes .
T6275 11804-11815 JJ denotes BCR-induced
T6276 11816-11826 JJ denotes signalling
T6277 11827-11835 NNS denotes pathways
T6278 11836-11845 VBP denotes culminate
T6279 11846-11848 IN denotes in
T6280 11849-11852 DT denotes the
T6281 11853-11863 NN denotes activation
T6282 11864-11866 IN denotes of
T6283 11867-11871 NN denotes gene
T6284 11872-11885 NN denotes transcription
T6285 11886-11892 NNS denotes events
T6286 11893-11897 WDT denotes that
T6287 11898-11905 VBP denotes control
T6288 11906-11907 NNP denotes B
T6289 11908-11912 NN denotes cell
T6290 11913-11921 NN denotes survival
T6291 11921-11922 , denotes ,
T6292 11923-11936 NN denotes proliferation
T6293 11937-11940 CC denotes and
T6294 11941-11949 NN denotes function
T6295 11949-11950 . denotes .
T6296 11951-11953 IN denotes In
T6297 11954-11958 DT denotes this
T6298 11959-11966 NN denotes context
T6299 11966-11967 , denotes ,
T6300 11968-11970 PRP denotes it
T6301 11971-11974 VBZ denotes has
T6302 11975-11979 VBN denotes been
T6303 11980-11988 VBN denotes proposed
T6304 11989-11993 IN denotes that
T6305 11994-11997 NNP denotes PKD
T6306 11998-12004 NN denotes family
T6307 12005-12012 NNS denotes members
T6308 12013-12020 NN denotes control
T6309 12021-12023 IN denotes of
T6310 12024-12028 NN denotes gene
T6311 12029-12042 NN denotes transcription
T6312 12043-12050 IN denotes through
T6313 12051-12061 NN denotes activation
T6314 12062-12064 IN denotes of
T6315 12065-12068 DT denotes the
T6316 12069-12073 NNP denotes NFκB
T6317 12074-12087 NN denotes transcription
T6318 12088-12094 NN denotes factor
T6319 12094-12095 . denotes .
T6320 12096-12100 RB denotes Thus
T6321 12100-12101 , denotes ,
T6322 12102-12114 JJ denotes PKD-mediated
T6323 12115-12125 NN denotes activation
T6324 12126-12128 IN denotes of
T6325 12129-12133 NNP denotes NFκB
T6326 12134-12140 VBZ denotes occurs
T6327 12141-12151 JJ denotes downstream
T6328 12152-12154 IN denotes of
T6329 12155-12156 DT denotes a
T6330 12157-12164 NN denotes variety
T6331 12165-12167 IN denotes of
T6332 12168-12177 JJ denotes different
T6333 12178-12185 NNS denotes signals
T6334 12185-12186 , denotes ,
T6335 12187-12196 VBG denotes including
T6336 12197-12211 JJ denotes mROS/oxidative
T6337 12212-12218 NN denotes stress
T6338 12218-12219 , denotes ,
T6339 12220-12236 JJ denotes lysophosphatidic
T6340 12237-12241 NN denotes acid
T6341 12242-12245 CC denotes and
T6342 12246-12249 DT denotes the
T6343 12250-12257 NNP denotes Bcr-Abl
T6344 12258-12266 NN denotes oncogene
T6345 12267-12268 NNP denotes [
T6346 12268-12282 CD denotes 17,21,23,30,37
T6347 12282-12283 NNP denotes ]
T6348 12283-12284 . denotes .
T6349 12285-12296 RB denotes Furthermore
T6350 12296-12297 , denotes ,
T6351 12298-12308 NN denotes expression
T6352 12309-12311 IN denotes of
T6353 12312-12314 DT denotes an
T6354 12315-12324 VBN denotes activated
T6355 12325-12329 NNP denotes PKD1
T6356 12330-12336 JJ denotes mutant
T6357 12337-12345 VBZ denotes enhances
T6358 12346-12359 JJ denotes HPK1-mediated
T6359 12360-12364 NNP denotes NFκB
T6360 12365-12375 NN denotes activation
T6361 12376-12377 NNP denotes [
T6362 12377-12379 CD denotes 38
T6363 12379-12380 NNP denotes ]
T6364 12380-12381 . denotes .
T6365 12382-12384 IN denotes In
T6366 12385-12386 NNP denotes B
T6367 12387-12392 NNS denotes cells
T6368 12392-12393 , denotes ,
T6369 12394-12398 NNP denotes NFκB
T6370 12399-12401 VBZ denotes is
T6371 12402-12407 VBN denotes known
T6372 12408-12410 TO denotes to
T6373 12411-12413 VB denotes be
T6374 12414-12423 VBN denotes regulated
T6375 12424-12427 IN denotes via
T6376 12428-12431 NNP denotes DAG
T6377 12432-12435 CC denotes and
T6378 12436-12440 NNP denotes PKCβ
T6379 12441-12442 NNP denotes [
T6380 12442-12447 CD denotes 39,40
T6381 12447-12448 NNP denotes ]
T6382 12449-12452 CC denotes but
T6383 12453-12460 IN denotes whether
T6384 12461-12465 NNS denotes PKDs
T6385 12466-12469 VBP denotes are
T6386 12470-12473 JJ denotes key
T6387 12474-12488 NNS denotes intermediaries
T6388 12489-12492 IN denotes for
T6389 12493-12497 NNP denotes NFκB
T6390 12498-12508 NN denotes regulation
T6391 12509-12512 VBZ denotes has
T6392 12513-12516 RB denotes not
T6393 12517-12521 VBN denotes been
T6394 12522-12530 VBN denotes explored
T6395 12530-12531 . denotes .
T6396 12532-12535 DT denotes The
T6397 12536-12540 NNS denotes data
T6398 12541-12542 -LRB- denotes (
T6399 12542-12546 NNP denotes Fig.
T6400 12547-12549 NNP denotes 4A
T6401 12549-12550 -RRB- denotes )
T6402 12551-12555 VBP denotes show
T6403 12556-12560 IN denotes that
T6404 12561-12565 NNP denotes NFκB
T6405 12566-12581 JJ denotes transcriptional
T6406 12582-12590 NN denotes activity
T6407 12591-12594 VBD denotes was
T6408 12595-12603 RB denotes strongly
T6409 12604-12611 VBN denotes induced
T6410 12612-12614 IN denotes in
T6411 12615-12619 DT denotes both
T6412 12620-12629 JJ denotes wild-type
T6413 12630-12633 CC denotes and
T6414 12634-12640 JJ denotes PKD1/3
T6415 12640-12641 NN denotes −
T6416 12641-12642 NN denotes /
T6417 12642-12643 NN denotes −
T6418 12644-12648 NNP denotes DT40
T6419 12649-12650 NNP denotes B
T6420 12651-12656 NNS denotes cells
T6421 12657-12659 IN denotes in
T6422 12660-12668 NN denotes response
T6423 12669-12671 TO denotes to
T6424 12672-12678 DT denotes either
T6425 12679-12686 NN denotes phorbol
T6426 12687-12692 NN denotes ester
T6427 12693-12695 CC denotes or
T6428 12696-12699 NNP denotes BCR
T6429 12700-12711 NN denotes stimulation
T6430 12711-12712 . denotes .
T6431 12713-12715 IN denotes In
T6432 12716-12724 NN denotes contrast
T6433 12724-12725 , denotes ,
T6434 12726-12729 NNP denotes BCR
T6435 12730-12733 CC denotes and
T6436 12734-12741 JJ denotes phorbol
T6437 12742-12755 JJ denotes ester-induced
T6438 12756-12760 NNP denotes NFκB
T6439 12761-12776 JJ denotes transcriptional
T6440 12777-12785 NN denotes activity
T6441 12786-12789 VBD denotes was
T6442 12790-12799 VBN denotes abolished
T6443 12800-12802 IN denotes in
T6444 12803-12807 NNP denotes PKCβ
T6445 12807-12808 NNP denotes −
T6446 12808-12809 NNP denotes /
T6447 12809-12810 NNP denotes −
T6448 12811-12815 NNP denotes DT40
T6449 12816-12817 NNP denotes B
T6450 12818-12823 NNS denotes cells
T6451 12824-12825 -LRB- denotes (
T6452 12825-12829 NNP denotes Fig.
T6453 12830-12832 NNP denotes 4A
T6454 12832-12833 -RRB- denotes )
T6455 12833-12834 , denotes ,
T6456 12835-12843 IN denotes although
T6457 12844-12850 JJ denotes strong
T6458 12851-12861 NN denotes activation
T6459 12862-12864 IN denotes of
T6460 12865-12868 NNP denotes PKD
T6461 12869-12876 NNS denotes kinases
T6462 12877-12878 -LRB- denotes (
T6463 12878-12880 IN denotes as
T6464 12881-12889 VBN denotes assessed
T6465 12890-12892 IN denotes by
T6466 12893-12912 NN denotes autophosphorylation
T6467 12913-12915 IN denotes of
T6468 12916-12920 NNP denotes PKD1
T6469 12921-12923 IN denotes at
T6470 12924-12928 NNP denotes S916
T6471 12928-12929 -RRB- denotes )
T6472 12930-12933 VBD denotes was
T6473 12934-12942 VBN denotes observed
T6474 12943-12945 IN denotes in
T6475 12946-12949 DT denotes the
T6476 12950-12954 NNP denotes PKCβ
T6477 12954-12955 NNP denotes −
T6478 12955-12956 VBD denotes /
T6479 12956-12957 CD denotes −
T6480 12958-12963 NNS denotes cells
T6481 12964-12965 -LRB- denotes (
T6482 12965-12969 NNP denotes Fig.
T6483 12970-12972 NNP denotes 4B
T6484 12972-12973 -RRB- denotes )
T6485 12973-12974 . denotes .
T6486 12975-12979 RB denotes Thus
T6487 12979-12980 , denotes ,
T6488 12981-12984 NNP denotes PKD
T6489 12985-12992 NNS denotes kinases
T6490 12993-12996 VBP denotes are
T6491 12997-13004 DT denotes neither
T6492 13005-13014 JJ denotes essential
T6493 13015-13018 CC denotes nor
T6494 13019-13029 JJ denotes sufficient
T6495 13030-13032 TO denotes to
T6496 13033-13040 VB denotes mediate
T6497 13041-13052 JJ denotes BCR-induced
T6498 13053-13057 NN denotes NFκB
T6499 13058-13068 NN denotes activation
T6500 13069-13071 IN denotes in
T6501 13072-13076 NNP denotes DT40
T6502 13077-13078 NNP denotes B
T6503 13079-13084 NNS denotes cells
T6504 13085-13088 CC denotes and
T6505 13089-13094 RB denotes hence
T6506 13095-13097 VBP denotes do
T6507 13098-13101 RB denotes not
T6508 13102-13113 VB denotes participate
T6509 13114-13116 IN denotes in
T6510 13117-13124 NNP denotes DAG/PKC
T6511 13125-13133 VBD denotes mediated
T6512 13134-13141 NN denotes control
T6513 13142-13144 IN denotes of
T6514 13145-13149 NNP denotes NFκB
T6515 13149-13150 . denotes .
T7377 237-13173 JJ denotes 40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway. To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera. BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor. Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38]. In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored. The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB. 4 Discussion Protein
T7378 13174-13180 NN denotes kinase
T7379 13181-13182 NNP denotes D
T7380 13183-13189 NN denotes serine
T7381 13190-13197 NNS denotes kinases
T7382 13198-13202 VBP denotes have
T7383 13203-13207 VBN denotes been
T7384 13208-13216 VBN denotes proposed
T7385 13217-13219 TO denotes to
T7386 13220-13228 VB denotes regulate
T7387 13229-13236 JJ denotes diverse
T7388 13237-13245 JJ denotes cellular
T7389 13246-13255 NNS denotes functions
T7390 13256-13265 VBG denotes including
T7391 13266-13269 DT denotes the
T7392 13270-13285 NN denotes phosphorylation
T7393 13286-13289 CC denotes and
T7394 13290-13297 JJ denotes nuclear
T7395 13298-13310 NN denotes localisation
T7396 13311-13313 IN denotes of
T7397 13314-13319 NN denotes class
T7398 13320-13322 NNP denotes II
T7399 13323-13328 NNP denotes HDACs
T7400 13329-13332 CC denotes and
T7401 13333-13336 DT denotes the
T7402 13337-13352 NN denotes phosphorylation
T7403 13353-13355 IN denotes of
T7404 13356-13361 NNP denotes HSP27
T7405 13361-13362 . denotes .
T7406 13363-13365 PRP denotes It
T7407 13366-13369 VBZ denotes has
T7408 13370-13374 RB denotes also
T7409 13375-13379 VBN denotes been
T7410 13380-13389 VBN denotes suggested
T7411 13390-13394 IN denotes that
T7412 13395-13399 NNP denotes PKDs
T7413 13400-13403 NN denotes act
T7414 13404-13406 IN denotes as
T7415 13407-13420 JJ denotes mitochondrial
T7416 13421-13428 NNS denotes sensors
T7417 13429-13432 IN denotes for
T7418 13433-13442 JJ denotes oxidative
T7419 13443-13449 NN denotes stress
T7420 13450-13453 CC denotes and
T7421 13454-13458 VB denotes play
T7422 13459-13460 DT denotes a
T7423 13461-13465 NN denotes role
T7424 13466-13468 IN denotes in
T7425 13469-13479 VBG denotes regulating
T7426 13480-13484 NNP denotes NFκB
T7427 13485-13498 NN denotes transcription
T7428 13499-13506 NNS denotes factors
T7429 13507-13508 NNP denotes [
T7430 13508-13510 CD denotes 41
T7431 13510-13511 NNP denotes ]
T7432 13511-13512 . denotes .
T7433 13513-13517 JJS denotes Most
T7434 13518-13520 IN denotes of
T7435 13521-13524 DT denotes the
T7436 13525-13529 NNS denotes data
T7437 13530-13535 IN denotes about
T7438 13536-13539 DT denotes the
T7439 13540-13548 NN denotes function
T7440 13549-13551 IN denotes of
T7441 13552-13556 NNP denotes PKDs
T7442 13557-13560 VBZ denotes has
T7443 13561-13565 VBN denotes come
T7444 13566-13570 IN denotes from
T7445 13571-13582 NNS denotes experiments
T7446 13583-13587 WDT denotes that
T7447 13588-13599 RB denotes ectopically
T7448 13600-13607 VBP denotes express
T7449 13608-13614 JJ denotes active
T7450 13615-13617 CC denotes or
T7451 13618-13628 JJ denotes inhibitory
T7452 13629-13632 NNP denotes PKD
T7453 13633-13640 NNS denotes mutants
T7454 13641-13643 CC denotes or
T7455 13644-13648 DT denotes that
T7456 13649-13652 NN denotes use
T7457 13653-13657 NNS denotes RNAi
T7458 13658-13660 TO denotes to
T7459 13661-13667 VB denotes reduce
T7460 13668-13671 NNP denotes PKD
T7461 13672-13682 NN denotes expression
T7462 13682-13683 . denotes .
T7463 13684-13686 PRP denotes We
T7464 13687-13691 VBP denotes have
T7465 13692-13696 VBN denotes used
T7466 13697-13701 NN denotes gene
T7467 13702-13711 VBG denotes targeting
T7468 13712-13714 TO denotes to
T7469 13715-13727 RB denotes specifically
T7470 13728-13734 VB denotes delete
T7471 13735-13738 NNP denotes PKD
T7472 13739-13746 NNS denotes alleles
T7473 13747-13749 IN denotes in
T7474 13750-13754 NNP denotes DT40
T7475 13755-13762 NN denotes chicken
T7476 13763-13764 NNP denotes B
T7477 13765-13770 NNS denotes cells
T7478 13771-13774 CC denotes and
T7479 13775-13778 MD denotes can
T7480 13779-13783 RB denotes thus
T7481 13784-13787 VB denotes use
T7482 13788-13796 NNP denotes PKD-null
T7483 13797-13801 NNP denotes DT40
T7484 13802-13807 NNS denotes cells
T7485 13808-13810 TO denotes to
T7486 13811-13817 VB denotes assess
T7487 13818-13821 DT denotes the
T7488 13822-13830 JJ denotes relative
T7489 13831-13843 NN denotes contribution
T7490 13844-13846 IN denotes of
T7491 13847-13857 JJ denotes individual
T7492 13858-13861 NNP denotes PKD
T7493 13862-13870 NNS denotes isoforms
T7494 13871-13873 IN denotes in
T7495 13874-13879 NN denotes class
T7496 13880-13882 NNP denotes II
T7497 13883-13887 NNP denotes HDAC
T7498 13888-13895 NN denotes control
T7499 13896-13902 IN denotes versus
T7500 13903-13912 JJ denotes oxidative
T7501 13913-13919 NN denotes stress
T7502 13920-13929 NNS denotes responses
T7503 13930-13933 CC denotes and
T7504 13934-13938 NNP denotes NFκB
T7505 13939-13949 NN denotes regulation
T7506 13950-13952 IN denotes in
T7507 13953-13964 NNS denotes lymphocytes
T7508 13964-13965 . denotes .
T7509 13966-13968 PRP denotes We
T7510 13969-13973 VBP denotes have
T7511 13974-13984 RB denotes previously
T7512 13985-13989 VBN denotes used
T7513 13990-13995 DT denotes these
T7514 13996-14004 NNP denotes PKD-null
T7515 14005-14009 NNP denotes DT40
T7516 14010-14015 NNS denotes cells
T7517 14016-14018 TO denotes to
T7518 14019-14025 VB denotes define
T7519 14026-14028 DT denotes an
T7520 14029-14038 JJ denotes essential
T7521 14039-14043 NN denotes role
T7522 14044-14047 IN denotes for
T7523 14048-14052 NNS denotes PKDs
T7524 14053-14055 IN denotes in
T7525 14056-14066 NN denotes regulation
T7526 14067-14069 IN denotes of
T7527 14070-14075 NN denotes class
T7528 14076-14078 NNP denotes II
T7529 14079-14084 NNP denotes HDACs
T7530 14084-14085 , denotes ,
T7531 14086-14089 DT denotes the
T7532 14090-14097 JJ denotes present
T7533 14098-14104 NN denotes report
T7534 14105-14108 RB denotes now
T7535 14109-14118 VBZ denotes describes
T7536 14119-14121 DT denotes an
T7537 14122-14135 JJ denotes indispensable
T7538 14136-14140 NN denotes role
T7539 14141-14144 IN denotes for
T7540 14145-14149 NNS denotes PKDs
T7541 14150-14152 IN denotes in
T7542 14153-14163 VBG denotes regulating
T7543 14164-14167 DT denotes the
T7544 14168-14183 NN denotes phosphorylation
T7545 14184-14186 IN denotes of
T7546 14187-14192 NNP denotes HSP27
T7547 14193-14195 IN denotes on
T7548 14196-14202 NN denotes serine
T7549 14203-14205 CD denotes 82
T7550 14205-14206 , denotes ,
T7551 14207-14208 DT denotes a
T7552 14209-14213 NN denotes site
T7553 14214-14224 RB denotes previously
T7554 14225-14235 VBN denotes identified
T7555 14236-14238 IN denotes as
T7556 14239-14240 DT denotes a
T7557 14241-14247 NN denotes target
T7558 14248-14251 IN denotes for
T7559 14252-14255 DT denotes the
T7560 14256-14268 NN denotes p38-MAPKAPK2
T7561 14269-14279 VBG denotes signalling
T7562 14280-14287 NN denotes cascade
T7563 14288-14289 NNP denotes [
T7564 14289-14291 CD denotes 42
T7565 14291-14292 NNP denotes ]
T7566 14292-14293 . denotes .
T7567 14294-14301 RB denotes However
T7568 14301-14302 , denotes ,
T7569 14303-14310 NNS denotes studies
T7570 14311-14313 IN denotes of
T7571 14314-14322 NNP denotes PKD-null
T7572 14323-14327 NNP denotes DT40
T7573 14328-14333 NNS denotes cells
T7574 14334-14340 VBP denotes reveal
T7575 14341-14345 IN denotes that
T7576 14346-14349 NNP denotes PKD
T7577 14350-14356 NN denotes family
T7578 14357-14364 NNS denotes kinases
T7579 14365-14368 VBP denotes are
T7580 14369-14372 RB denotes not
T7581 14373-14382 JJ denotes essential
T7582 14383-14386 IN denotes for
T7583 14387-14396 JJ denotes oxidative
T7584 14397-14403 NN denotes stress
T7585 14404-14412 NN denotes survival
T7586 14413-14422 NNS denotes responses
T7587 14423-14426 CC denotes nor
T7588 14427-14430 VBP denotes are
T7589 14431-14435 PRP denotes they
T7590 14436-14444 VBN denotes required
T7591 14445-14448 IN denotes for
T7592 14449-14459 NN denotes activation
T7593 14460-14462 IN denotes of
T7594 14463-14467 NNP denotes NFκB
T7595 14468-14481 NN denotes transcription
T7596 14482-14489 NNS denotes factors
T7597 14489-14490 . denotes .
T7598 14491-14496 DT denotes These
T7599 14497-14503 JJ denotes latter
T7600 14504-14512 NNS denotes findings
T7601 14513-14516 VBP denotes are
T7602 14517-14519 IN denotes in
T7603 14520-14528 JJ denotes striking
T7604 14529-14537 NN denotes contrast
T7605 14538-14540 TO denotes to
T7606 14541-14549 JJ denotes previous
T7607 14550-14562 NNS denotes observations
T7608 14563-14565 IN denotes in
T7609 14566-14570 NNP denotes HeLa
T7610 14571-14574 CC denotes and
T7611 14575-14585 JJ denotes epithelial
T7612 14586-14590 NN denotes cell
T7613 14591-14596 NNS denotes lines
T7614 14597-14602 WRB denotes where
T7615 14603-14622 NNP denotes overexpression/RNAi
T7616 14623-14633 NNS denotes approaches
T7617 14634-14638 VBP denotes have
T7618 14639-14649 VBN denotes implicated
T7619 14650-14656 NNP denotes PKD1/2
T7620 14657-14659 IN denotes in
T7621 14660-14663 DT denotes the
T7622 14664-14671 NN denotes control
T7623 14672-14674 IN denotes of
T7624 14675-14688 NN denotes proliferation
T7625 14688-14689 , denotes ,
T7626 14690-14698 NN denotes survival
T7627 14699-14702 CC denotes and
T7628 14703-14707 NNP denotes NFκB
T7629 14708-14718 NN denotes activation
T7630 14719-14720 NNP denotes [
T7631 14720-14725 CD denotes 20,23
T7632 14725-14726 NNP denotes ]
T7633 14726-14727 . denotes .
T7634 14728-14733 RB denotes Hence
T7635 14733-14734 , denotes ,
T7636 14735-14738 DT denotes the
T7637 14739-14746 JJ denotes present
T7638 14747-14753 NN denotes report
T7639 14754-14759 VBZ denotes shows
T7640 14760-14764 IN denotes that
T7641 14765-14768 DT denotes the
T7642 14769-14777 VBN denotes proposed
T7643 14778-14783 NNS denotes roles
T7644 14784-14787 IN denotes for
T7645 14788-14792 NNS denotes PKDs
T7646 14793-14795 IN denotes as
T7647 14796-14799 JJ denotes key
T7648 14800-14807 NNS denotes sensors
T7649 14808-14812 WDT denotes that
T7650 14813-14821 VBP denotes modulate
T7651 14822-14830 NN denotes survival
T7652 14831-14839 NNS denotes pathways
T7653 14840-14842 IN denotes in
T7654 14843-14851 NN denotes response
T7655 14852-14854 TO denotes to
T7656 14855-14864 JJ denotes oxidative
T7657 14865-14871 NN denotes stress
T7658 14872-14875 CC denotes and
T4248 7019-7021 IN denotes in
T4249 7022-7023 NNP denotes [
T4250 7023-7025 CD denotes 20
T4251 7025-7026 NNP denotes ]
T4252 7026-7027 -RRB- denotes )
T4253 7027-7028 . denotes .
T4254 7029-7031 TO denotes To
T4255 7032-7043 VB denotes investigate
T4256 7044-7047 DT denotes the
T4257 7048-7054 NN denotes effect
T4258 7055-7059 WDT denotes that
T4259 7060-7064 NN denotes loss
T4260 7065-7067 IN denotes of
T4261 7068-7071 NNP denotes PKD
T4262 7072-7079 NNS denotes kinases
T4263 7080-7083 VBD denotes had
T4264 7084-7086 IN denotes on
T4265 7087-7088 NNP denotes B
T4266 7089-7093 NN denotes cell
T4267 7094-7102 NN denotes survival
T4268 7103-7109 CC denotes and/or
T4269 7110-7123 NN denotes proliferation
T4270 7124-7126 PRP denotes we
T4271 7127-7135 VBD denotes cultured
T4272 7136-7145 JJ denotes wild-type
T4273 7146-7149 CC denotes and
T4274 7150-7158 JJ denotes PKD-null
T4275 7159-7164 NNS denotes cells
T7659 14876-14884 VB denotes regulate
T7660 14885-14889 NN denotes cell
T7661 14890-14898 NN denotes survival
T7662 14899-14902 CC denotes and
T7663 14903-14916 NN denotes proliferation
T7664 14917-14920 VBP denotes are
T7665 14921-14924 RB denotes not
T7666 14925-14935 JJ denotes ubiquitous
T7667 14936-14939 CC denotes and
T7668 14940-14943 MD denotes may
T7669 14944-14946 VB denotes be
T7670 14947-14957 VBN denotes restricted
T7671 14958-14960 TO denotes to
T7672 14961-14968 JJ denotes certain
T7673 14969-14973 NN denotes cell
T7674 14974-14982 NNS denotes lineages
T7675 14982-14983 . denotes .
T7676 14984-14989 VBN denotes Taken
T7677 14990-14998 RB denotes together
T7678 14998-14999 , denotes ,
T7679 15000-15005 DT denotes these
T7680 15006-15010 NNS denotes data
T7681 15011-15019 VBP denotes indicate
T7682 15020-15024 IN denotes that
T7683 15025-15029 NN denotes loss
T7684 15030-15032 IN denotes of
T7685 15033-15043 NN denotes expression
T7686 15044-15046 IN denotes of
T7687 15047-15050 NNP denotes PKD
T7688 15051-15057 NN denotes family
T7689 15058-15065 NNS denotes members
T7690 15066-15070 VBZ denotes does
T7691 15071-15074 RB denotes not
T7692 15075-15083 RB denotes globally
T7693 15084-15090 NN denotes impact
T7694 15091-15093 IN denotes on
T7695 15094-15099 JJ denotes early
T7696 15100-15113 JJ denotes BCR-regulated
T7697 15114-15124 NN denotes signalling
T7698 15125-15133 NNS denotes pathways
T7699 15133-15134 . denotes .
R204 T241 T231 dobj enzymes,investigate
R205 T242 T243 nsubj we,generated
R206 T243 T241 relcl generated,enzymes
R207 T244 T249 det a,line
R208 T245 T249 compound PKD-null,line
R209 T246 T247 compound DT40,B-lymphocyte
R210 T247 T249 compound B-lymphocyte,line
R211 T248 T249 compound cell,line
R212 T249 T243 dobj line,generated
R213 T250 T216 punct .,are
R214 T251 T254 advmod Previously,shown
R215 T252 T254 nsubj we,shown
R216 T253 T254 aux have,shown
R217 T254 T254 ROOT shown,shown
R218 T255 T257 mark that,have
R219 T256 T257 nsubj PKDs,have
R220 T257 T254 ccomp have,shown
R221 T258 T260 det an,role
R222 T259 T260 amod essential,role
R223 T260 T257 dobj role,have
R224 T261 T260 prep in,role
R225 T262 T261 pcomp regulating,in
R226 T263 T266 compound class,deacetylases
R227 T264 T266 compound II,deacetylases
R228 T265 T266 compound histone,deacetylases
R229 T266 T262 dobj deacetylases,regulating
R230 T267 T262 prep in,regulating
R231 T268 T271 compound DT40,Matthews
R232 T269 T271 compound B-cells,Matthews
R233 T270 T271 compound [,Matthews
R234 T271 T267 pobj Matthews,in
R235 T272 T271 punct ",",Matthews
R236 T273 T271 conj S.A.,Matthews
R237 T274 T271 punct ",",Matthews
R238 T275 T271 conj Liu,Matthews
R239 T276 T275 punct ",",Liu
R240 T277 T275 conj P.,Liu
R241 T278 T277 punct ",",P.
R242 T279 T277 conj Spitaler,P.
R243 T280 T279 punct ",",Spitaler
R244 T281 T279 conj M.,Spitaler
R245 T282 T281 punct ",",M.
R246 T283 T281 conj Olson,M.
R247 T284 T283 punct ",",Olson
R248 T285 T283 conj E.N.,Olson
R249 T286 T285 punct ",",E.N.
R250 T287 T285 conj McKinsey,E.N.
R251 T288 T287 punct ",",McKinsey
R252 T289 T287 conj T.A.,McKinsey
R253 T290 T287 punct ",",McKinsey
R254 T291 T287 conj Cantrell,McKinsey
R255 T292 T291 punct ",",Cantrell
R256 T293 T291 conj D.A.,Cantrell
R257 T294 T293 cc and,D.A.
R258 T295 T293 conj Scharenberg,D.A.
R259 T296 T295 punct ",",Scharenberg
R260 T297 T295 conj A.M.,Scharenberg
R261 T298 T297 punct (,A.M.
R262 T299 T297 appos 2006,A.M.
R263 T300 T297 punct ),A.M.
R264 T301 T302 amod Essential,role
R265 T302 T260 appos role,role
R266 T303 T302 prep for,role
R267 T304 T303 pobj protein,for
R268 T305 T308 compound kinase,kinases
R269 T306 T307 compound D,family
R270 T307 T308 compound family,kinases
R271 T308 T302 conj kinases,role
R272 T309 T308 prep in,kinases
R273 T310 T311 det the,regulation
R274 T311 T309 pobj regulation,in
R275 T312 T311 prep of,regulation
R276 T313 T316 compound class,deacetylases
R277 T314 T316 compound II,deacetylases
R278 T315 T316 compound histone,deacetylases
R279 T316 T312 pobj deacetylases,of
R280 T317 T316 prep in,deacetylases
R281 T318 T319 compound B,lymphocytes
R282 T319 T317 pobj lymphocytes,in
R283 T320 T254 punct .,shown
R284 T321 T321 ROOT Mol,Mol
R285 T322 T321 punct .,Mol
R286 T323 T324 compound Cell,Biol
R287 T324 T324 ROOT Biol,Biol
R288 T325 T326 punct .,26
R289 T326 T324 nummod 26,Biol
R290 T327 T324 punct ",",Biol
R291 T328 T324 appos 1569,Biol
R292 T329 T324 appos 1577,Biol
R293 T330 T330 ROOT ],]
R294 T331 T324 punct .,Biol
R295 T332 T334 nsubj We,show
R296 T333 T334 advmod now,show
R297 T334 T334 ROOT show,show
R298 T335 T339 mark that,required
R299 T336 T339 nsubjpass PKDs,required
R300 T337 T339 auxpass are,required
R301 T338 T339 advmod also,required
R302 T339 T334 ccomp required,show
R303 T340 T341 aux to,regulate
R304 T341 T339 xcomp regulate,required
R305 T342 T343 compound HSP27,phosphorylation
R306 T343 T341 dobj phosphorylation,regulate
R307 T344 T343 prep in,phosphorylation
R308 T345 T346 compound DT40,B-cells
R309 T346 T344 pobj B-cells,in
R310 T347 T334 punct .,show
R311 T348 T364 advmod However,regulate
R312 T349 T364 punct ",",regulate
R313 T350 T364 prep in,regulate
R314 T351 T350 pobj contrast,in
R315 T352 T351 prep to,contrast
R316 T353 T354 amod previous,observations
R317 T354 T352 pobj observations,to
R318 T355 T354 prep in,observations
R319 T356 T358 amod other,types
R320 T357 T358 compound cell,types
R321 T358 T355 pobj types,in
R322 T359 T364 punct ",",regulate
R323 T360 T364 nsubj PKD,regulate
R324 T361 T364 nsubj enzymes,regulate
R325 T362 T364 aux do,regulate
R326 T363 T364 neg not,regulate
R327 T364 T364 ROOT regulate,regulate
R328 T365 T367 amod basic,processes
R329 T366 T367 amod cellular,processes
R330 T367 T364 dobj processes,regulate
R331 T368 T369 amod such,as
R332 T369 T367 prep as,processes
R333 T370 T369 pobj proliferation,as
R334 T371 T370 cc or,proliferation
R335 T372 T370 conj survival,proliferation
R336 T373 T370 conj responses,proliferation
R337 T374 T373 punct ",",responses
R338 T375 T364 cc nor,regulate
R339 T376 T378 nmod NFκB,activity
R340 T377 T378 amod transcriptional,activity
R341 T378 T364 conj activity,regulate
R342 T379 T378 amod downstream,activity
R343 T380 T379 prep of,downstream
R344 T381 T385 det the,receptor
R345 T382 T384 compound B,antigen
R346 T383 T384 compound cell,antigen
R347 T384 T385 compound antigen,receptor
R348 T385 T380 pobj receptor,of
R349 T386 T364 punct .,regulate
R350 T387 T390 advmod Thus,have
R351 T388 T390 punct ",",have
R352 T389 T390 nsubj PKDs,have
R353 T390 T390 ROOT have,have
R354 T391 T393 det a,role
R355 T392 T393 amod selective,role
R356 T393 T390 dobj role,have
R357 T394 T393 prep in,role
R358 T395 T396 compound DT40,B-cell
R359 T396 T397 compound B-cell,biology
R360 T397 T394 pobj biology,in
R361 T398 T390 punct .,have
R894 T1008 T1011 amod "1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The",D
R895 T1009 T1011 nmod protein,D
R896 T1010 T1011 compound kinase,D
R897 T1011 T1011 ROOT D,D
R898 T1012 T1011 punct (,D
R899 T1013 T1011 appos PKD,D
R900 T1014 T1011 punct ),D
R901 T1015 T1017 amod serine/threonine,family
R902 T1016 T1017 compound kinase,family
R903 T1017 T1018 nsubj family,has
R904 T1018 T1018 ROOT has,has
R905 T1019 T1020 nummod three,members
R906 T1020 T1018 dobj members,has
R907 T1021 T1018 punct :,has
R908 T1022 T1022 ROOT PKD1,PKD1
R909 T1023 T1022 punct ",",PKD1
R910 T1024 T1022 conj PKD2,PKD1
R911 T1025 T1024 cc and,PKD2
R912 T1026 T1024 conj PKD3,PKD2
R913 T1027 T1018 punct .,has
R914 T1028 T1030 amod Most,types
R915 T1029 T1030 compound cell,types
R916 T1030 T1031 nsubj types,express
R917 T1031 T1031 ROOT express,express
R918 T1032 T1033 advmod at,least
R919 T1033 T1034 advmod least,two
R920 T1034 T1036 nummod two,isoforms
R921 T1035 T1036 compound PKD,isoforms
R922 T1036 T1031 dobj isoforms,express
R923 T1037 T1031 cc but,express
R924 T1038 T1039 compound PKD,enzymes
R925 T1039 T1040 nsubj enzymes,are
R926 T1040 T1043 auxpass are,expressed
R927 T1041 T1042 advmod especially,highly
R928 T1042 T1043 advmod highly,expressed
R929 T1043 T1031 conj expressed,express
R930 T1044 T1043 prep in,expressed
R931 T1045 T1046 amod haematopoietic,cells
R932 T1046 T1044 pobj cells,in
R933 T1047 T1043 punct ",",expressed
R934 T1048 T1051 advmod where,activated
R935 T1049 T1051 nsubjpass they,activated
R936 T1050 T1051 auxpass are,activated
R937 T1051 T1043 advcl activated,expressed
R938 T1052 T1051 prep in,activated
R939 T1053 T1052 pobj response,in
R940 T1054 T1053 prep to,response
R941 T1055 T1056 compound antigen,receptors
R942 T1056 T1057 compound receptors,stimulation
R943 T1057 T1054 pobj stimulation,to
R944 T1058 T1060 compound [,]
R945 T1059 T1060 compound "2,3",]
R946 T1060 T1057 appos ],stimulation
R947 T1061 T1043 punct .,expressed
R948 T1062 T1065 det A,pathway
R949 T1063 T1065 amod conserved,pathway
R950 T1064 T1065 amod signalling,pathway
R951 T1065 T1071 nsubj pathway,involves
R952 T1066 T1065 acl linking,pathway
R953 T1067 T1068 compound antigen,receptors
R954 T1068 T1066 dobj receptors,linking
R955 T1069 T1066 prep to,linking
R956 T1070 T1069 pobj PKDs,to
R957 T1071 T1071 ROOT involves,involves
R958 T1072 T1073 det the,activation
R959 T1073 T1071 dobj activation,involves
R960 T1074 T1073 prep of,activation
R961 T1075 T1074 pobj PLCγ,of
R962 T1076 T1073 cc and,activation
R963 T1077 T1079 det the,production
R964 T1078 T1079 amod subsequent,production
R965 T1079 T1073 conj production,activation
R966 T1080 T1079 prep of,production
R967 T1081 T1080 pobj diacylglycerol,of
R968 T1082 T1083 punct (,DAG
R969 T1083 T1081 appos DAG,diacylglycerol
R970 T1084 T1081 punct ),diacylglycerol
R971 T1085 T1086 nsubj which,stimulates
R972 T1086 T1079 relcl stimulates,production
R973 T1087 T1090 amod classical,protein
R974 T1088 T1087 cc and/or,classical
R975 T1089 T1087 conj novel,classical
R976 T1090 T1092 compound protein,Cs
R977 T1091 T1092 compound kinase,Cs
R978 T1092 T1086 dobj Cs,stimulates
R979 T1093 T1092 punct (,Cs
R980 T1094 T1092 appos PKC,Cs
R981 T1095 T1092 punct ),Cs
R982 T1096 T1109 nsubj that,kinases
R983 T1097 T1096 aux phosphorylate,that
R984 T1098 T1102 nummod two,residues
R985 T1099 T1102 amod key,residues
R986 T1100 T1102 amod regulatory,residues
R987 T1101 T1102 compound serine,residues
R988 T1102 T1097 dobj residues,phosphorylate
R989 T1103 T1102 prep in,residues
R990 T1104 T1106 det the,loop
R991 T1105 T1106 compound activation,loop
R992 T1106 T1103 pobj loop,in
R993 T1107 T1106 prep of,loop
R994 T1108 T1107 pobj PKD,of
R995 T1109 T1079 relcl kinases,production
R996 T1110 T1109 dobj [,kinases
R997 T1111 T1110 nummod 3,[
R998 T1112 T1113 nummod 6,]
R999 T1113 T1079 appos ],production
R1000 T1114 T1071 punct .,involves
R1001 T1115 T1118 det The,region
R1002 T1116 T1118 amod N-terminal,region
R1003 T1117 T1118 amod regulatory,region
R1004 T1118 T1121 nsubj region,enzymes
R1005 T1119 T1118 prep of,region
R1006 T1120 T1119 pobj PKD,of
R1007 T1121 T1121 ROOT enzymes,enzymes
R1008 T1122 T1121 conj contains,enzymes
R1009 T1123 T1124 det a,DAG
R1010 T1124 T1126 nmod DAG,domain
R1011 T1125 T1126 amod binding,domain
R1012 T1126 T1122 dobj domain,contains
R1013 T1127 T1126 cc and,domain
R1014 T1128 T1129 amod direct,binding
R1015 T1129 T1126 conj binding,domain
R1016 T1130 T1129 prep of,binding
R1017 T1131 T1130 pobj DAG,of
R1018 T1132 T1133 advmod also,contributes
R1019 T1133 T1121 conj contributes,enzymes
R1020 T1134 T1133 prep to,contributes
R1021 T1135 T1139 nummod PKD1,]
R1022 T1136 T1137 compound activation,[
R1023 T1137 T1139 nmod [,]
R1024 T1138 T1137 nummod 7,[
R1025 T1139 T1134 pobj ],to
R1026 T1140 T1142 advmod as,as
R1027 T1141 T1142 advmod well,as
R1028 T1142 T1133 prep as,contributes
R1029 T1143 T1142 pcomp regulating,as
R1030 T1144 T1146 det the,location
R1031 T1145 T1146 amod spatial,location
R1032 T1146 T1143 dobj location,regulating
R1033 T1147 T1146 prep of,location
R1034 T1148 T1149 compound PKD,enzymes
R1035 T1149 T1147 pobj enzymes,of
R1036 T1150 T1143 prep within,regulating
R1037 T1151 T1150 pobj cells,within
R1038 T1152 T1153 nmod [,8
R1039 T1153 T1143 npadvmod 8,regulating
R1040 T1154 T1155 nummod 12,]
R1041 T1155 T1153 appos ],8
R1042 T1156 T1121 punct .,enzymes
R1043 T1157 T1158 compound PKD,enzymes
R1044 T1158 T1161 nsubjpass enzymes,proposed
R1045 T1159 T1161 aux have,proposed
R1062 T1176 T1177 amod anti-apoptotic,signals
R1063 T1177 T1171 appos signals,[
R1064 T1178 T1180 compound [,]
R1065 T1179 T1180 compound "17,18",]
R1066 T1180 T1177 dobj ],signals
R1067 T1181 T1180 cc and,]
R1068 T1182 T1183 amod thymocyte,development
R1069 T1183 T1186 nmod development,]
R1070 T1184 T1186 nmod [,]
R1071 T1185 T1186 nummod 19,]
R1072 T1186 T1180 conj ],]
R1073 T1187 T1177 punct .,signals
R1074 T1188 T1199 nsubj Expression,modify
R1075 T1189 T1188 prep of,Expression
R1076 T1190 T1192 amod mutant,inactive
R1077 T1191 T1192 advmod catalytically,inactive
R1078 T1192 T1196 amod inactive,PKDs
R1079 T1193 T1192 cc and,inactive
R1080 T1194 T1195 advmod constitutively,activated
R1081 T1195 T1192 conj activated,inactive
R1082 T1196 T1188 appos PKDs,Expression
R1083 T1197 T1199 aux can,modify
R1084 T1198 T1199 advmod also,modify
R1085 T1199 T1199 ROOT modify,modify
R1086 T1200 T1201 compound Golgi,function
R1087 T1201 T1199 dobj function,modify
R1088 T1202 T1201 punct ",",function
R1089 T1203 T1204 compound cell,adhesion
R1090 T1204 T1201 conj adhesion,function
R1091 T1205 T1204 cc and,adhesion
R1092 T1206 T1207 compound cell,motility
R1093 T1207 T1204 conj motility,adhesion
R1094 T1208 T1209 punct (,reviewed
R1095 T1209 T1199 advcl reviewed,modify
R1096 T1210 T1209 prep in,reviewed
R1097 T1211 T1210 pobj [,in
R1098 T1212 T1213 nummod 20,]
R1099 T1213 T1211 appos ],[
R1100 T1214 T1211 punct ),[
R1101 T1215 T1199 punct .,modify
R1102 T1216 T1223 prep In,linked
R1103 T1217 T1216 amod particular,In
R1104 T1218 T1223 punct ",",linked
R1105 T1219 T1223 nsubjpass PKDs,linked
R1106 T1220 T1223 aux have,linked
R1107 T1221 T1223 auxpass been,linked
R1108 T1222 T1223 advmod widely,linked
R1109 T1223 T1223 ROOT linked,linked
R1110 T1224 T1223 prep to,linked
R1111 T1225 T1226 det the,activation
R1112 T1226 T1224 pobj activation,to
R1113 T1227 T1226 prep of,activation
R1114 T1228 T1231 det the,factor
R1115 T1229 T1231 compound NFκB,factor
R1116 T1230 T1231 compound transcription,factor
R1117 T1231 T1227 pobj factor,of
R1118 T1232 T1224 cc and,to
R1119 T1233 T1223 prep in,linked
R1120 T1234 T1233 pcomp regulating,in
R1121 T1235 T1236 compound cell,survival
R1122 T1236 T1234 dobj survival,regulating
R1123 T1237 T1234 prep during,regulating
R1124 T1238 T1239 amod oxidative,stress
R1125 T1239 T1241 compound stress,"17,21"
R1126 T1240 T1241 compound [,"17,21"
R1127 T1241 T1237 pobj "17,21",during
R1175 T1289 T1289 ROOT enzymes,enzymes
R1176 T1290 T1291 mark that,repress
R1196 T1310 T1309 prep of,role
R1197 T1311 T1310 pobj PKDs,of
R1198 T1312 T1314 nsubj we,generated
R1199 T1313 T1314 aux have,generated
R1200 T1314 T1314 ROOT generated,generated
R1201 T1315 T1318 compound DT40,lines
R1202 T1316 T1318 compound B,lines
R1203 T1317 T1318 compound cell,lines
R1204 T1318 T1314 dobj lines,generated
R1205 T1319 T1320 nsubj that,lack
R1206 T1320 T1318 relcl lack,lines
R1207 T1321 T1320 dobj expression,lack
R1208 T1322 T1321 prep of,expression
R1209 T1323 T1326 nummod one,members
R1210 T1324 T1323 cc or,one
R1211 T1325 T1323 conj more,one
R1212 T1326 T1322 pobj members,of
R1213 T1327 T1326 prep of,members
R1214 T1328 T1330 det the,family
R1215 T1329 T1330 compound PKD,family
R1216 T1330 T1327 pobj family,of
R1217 T1331 T1333 nmod [,]
R1218 T1332 T1333 nummod 1,]
R1219 T1333 T1326 appos ],members
R1220 T1334 T1314 punct ",",generated
R1221 T1335 T1314 advcl allowing,generated
R1222 T1336 T1338 nsubj us,investigate
R1223 T1337 T1338 aux to,investigate
R1224 T1338 T1335 ccomp investigate,allowing
R1225 T1339 T1340 det the,function
R1226 T1340 T1338 dobj function,investigate
R1227 T1341 T1340 punct (,function
R1228 T1342 T1340 appos s,function
R1229 T1343 T1340 punct ),function
R1230 T1344 T1340 prep of,function
R1231 T1345 T1344 pobj PKD,of
R1232 T1346 T1344 pobj isoforms,of
R1233 T1347 T1346 acl following,isoforms
R1234 T1348 T1350 compound B,antigen
R1235 T1349 T1350 compound cell,antigen
R1236 T1350 T1351 compound antigen,receptor
R1237 T1351 T1347 dobj receptor,following
R1238 T1352 T1353 punct (,BCR
R1239 T1353 T1351 appos BCR,receptor
R1240 T1354 T1353 punct ),BCR
R1241 T1355 T1351 conj stimulation,receptor
R1242 T1356 T1347 punct ",",following
R1243 T1357 T1359 advmod as,addressing
R1244 T1358 T1359 advmod well,addressing
R1245 T1359 T1338 advcl addressing,investigate
R1246 T1360 T1361 det the,issue
R1247 T1361 T1359 dobj issue,addressing
R1248 T1362 T1361 prep of,issue
R1249 T1363 T1364 amod functional,redundancy
R1250 T1364 T1362 pobj redundancy,of
R1251 T1365 T1364 prep between,redundancy
R1252 T1366 T1370 det the,members
R1253 T1367 T1370 amod different,members
R1254 T1368 T1370 compound PKD,members
R1255 T1369 T1370 compound family,members
R1256 T1370 T1365 pobj members,between
R1257 T1371 T1314 punct .,generated
R1258 T1372 T1373 amod Previous,studies
R1259 T1373 T1375 nsubj studies,shown
R1261 T1374 T1375 aux have,shown
R1264 T1375 T1375 ROOT shown,shown
R1267 T1376 T1378 mark that,are
R1275 T1377 T1378 nsubj PKDs,are
R1279 T1378 T1375 ccomp are,shown
R1284 T1379 T1378 acomp indispensable,are
R1288 T1380 T1378 prep for,are
R1292 T1381 T1382 compound HDAC,regulation
R1296 T1382 T1380 pobj regulation,for
R1298 T1383 T1382 prep in,regulation
R1299 T1477 T1474 prep in,ascribed
R1300 T1478 T1480 amod other,systems
R1301 T1479 T1480 amod cellular,systems
R1302 T1480 T1477 pobj systems,in
R1303 T1384 T1385 compound B,cells
R1304 T1481 T1455 punct .,reveal
R1305 T1385 T1383 pobj cells,in
R1306 T1386 T1388 nmod [,]
R1307 T1387 T1388 nummod 1,]
R1308 T1388 T1382 appos ],regulation
R1309 T1389 T1375 punct .,shown
R1310 T1390 T1392 npadvmod Herein,show
R1311 T1391 T1392 nsubj we,show
R1312 T1392 T1392 ROOT show,show
R1313 T1393 T1395 mark that,are
R1314 T1394 T1395 nsubj PKDs,are
R1315 T1395 T1392 ccomp are,show
R1316 T1396 T1395 advmod also,are
R1317 T1397 T1395 acomp indispensable,are
R1318 T1398 T1395 prep for,are
R1319 T1399 T1400 compound HSP27,phosphorylation
R1320 T1400 T1398 pobj phosphorylation,for
R1321 T1401 T1400 prep in,phosphorylation
R1322 T1402 T1403 compound B,cells
R1323 T1403 T1401 pobj cells,in
R1324 T1404 T1392 punct .,show
R1325 T1405 T1411 advmod However,are
R1326 T1406 T1411 punct ",",are
R1327 T1407 T1409 compound PKD-null,B
R1328 T1408 T1409 compound DT40,B
R1329 T1409 T1410 compound B,cells
R1331 T1410 T1411 nsubj cells,are
R1335 T1411 T1411 ROOT are,are
R1336 T1412 T1411 acomp viable,are
R1338 T1413 T1411 cc and,are
R1340 T1414 T1411 conj proliferate,are
R1341 T1415 T1414 advmod normally,proliferate
R1342 T1416 T1411 punct .,are
R1343 T1417 T1430 advmod Moreover,affect
R1344 T1418 T1430 punct ",",affect
R1345 T1419 T1430 nsubj loss,affect
R1347 T1420 T1419 prep of,loss
R1351 T1421 T1424 det the,pool
R1352 T1422 T1424 amod entire,pool
R1355 T1423 T1424 amod cellular,pool
R1356 T1424 T1420 pobj pool,of
R1357 T1425 T1424 prep of,pool
R1358 T1426 T1425 pobj PKD,of
R1359 T1427 T1430 aux does,affect
R1360 T1428 T1430 neg not,affect
R1361 T1429 T1430 advmod critically,affect
R1362 T1430 T1430 ROOT affect,affect
R1365 T1431 T1433 amod oxidative,responses
R1367 T1432 T1433 compound stress,responses
R1369 T1433 T1430 dobj responses,affect
R1371 T1434 T1433 prep in,responses
R1373 T1435 T1436 compound B,cells
R1374 T1436 T1434 pobj cells,in
R1376 T1437 T1430 cc nor,affect
R1378 T1438 T1441 aux do,play
R1379 T1439 T1440 compound PKD,kinases
R1380 T1440 T1441 nsubj kinases,play
R1381 T1441 T1430 conj play,affect
R1382 T1442 T1444 det an,role
R1383 T1443 T1444 amod essential,role
R1384 T1444 T1441 dobj role,play
R1385 T1445 T1444 prep in,role
R1387 T1446 T1445 pcomp regulating,in
R1391 T1447 T1449 nmod NFκB,activity
R1519 T1759 T1761 nummod −,−
R1520 T1760 T1761 compound /,−
R1521 T1761 T1757 pobj −,of
R1522 T1762 T1765 punct ",",/
R1523 T1763 T1764 compound PKD3,−
R1524 T1764 T1765 nsubj −,/
R1525 T1765 T1765 ROOT /,/
R1526 T1766 T1776 nummod −,lines
R1527 T1767 T1766 cc and,−
R1528 T1768 T1776 nummod PKD1/3,lines
R1529 T1769 T1776 nummod −,lines
R1530 T1770 T1772 nmod /,knockout
R1531 T1771 T1772 compound −,knockout
R1532 T1772 T1776 compound knockout,lines
R1533 T1773 T1774 compound DT40,B
R1534 T1774 T1775 compound B,cell
R1535 T1775 T1776 compound cell,lines
R1536 T1776 T1779 nsubjpass lines,described
R1537 T1777 T1779 aux have,described
R1538 T1778 T1779 auxpass been,described
R1539 T1779 T1765 ccomp described,/
R1540 T1780 T1783 advmod previously,]
R1541 T1781 T1783 nmod [,]
R1542 T1782 T1781 nummod 1,[
R1543 T1783 T1779 dobj ],described
R1544 T1784 T1765 punct .,/
R1545 T1785 T1787 nsubjpass Cells,lysed
R1546 T1786 T1787 auxpass were,lysed
R1547 T1787 T1787 ROOT lysed,lysed
R1548 T1788 T1787 cc and,lysed
R1549 T1789 T1790 compound protein,extracts
R1550 T1790 T1792 nsubjpass extracts,analysed
R1551 T1791 T1792 auxpass were,analysed
R1552 T1792 T1787 conj analysed,lysed
R1553 T1793 T1792 prep in,analysed
R1554 T1794 T1796 amod Western,experiments
R1555 T1795 T1796 amod blotting,experiments
R1556 T1796 T1793 pobj experiments,in
R1557 T1797 T1799 mark as,described
R1558 T1798 T1799 advmod previously,described
R1559 T1799 T1792 advcl described,analysed
R1560 T1800 T1802 nmod [,]
R1561 T1801 T1802 nummod 1,]
R1562 T1802 T1799 dobj ],described
R1563 T1803 T1792 punct .,analysed
R1564 T1804 T1807 nmod Chloramphenicol,assays
R1565 T1805 T1807 nmod acetyl,assays
R1566 T1806 T1807 compound transferase,assays
R1567 T1807 T1810 nsubjpass assays,described
R1568 T1808 T1810 aux have,described
R1569 T1809 T1810 auxpass been,described
R1570 T1810 T1810 ROOT described,described
R1571 T1811 T1814 advmod previously,]
R1572 T1812 T1814 nmod [,]
R1573 T1813 T1814 nummod 29,]
R1574 T1814 T1810 dobj ],described
R1575 T1815 T1810 punct .,described
R1663 T1939 T1940 nummod "Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 ",sIgM
R1664 T1940 T1944 amod sIgM,cells
R1665 T1941 T1944 amod staining,cells
R1666 T1942 T1943 compound DT40,B
R1667 T1943 T1944 compound B,cells
R1668 T1944 T1954 nsubjpass cells,resuspended
R1669 T1945 T1944 punct (,cells
R1670 T1946 T1949 nummod 2,cells
R1671 T1947 T1949 nummod ×,cells
R1677 T1953 T1954 auxpass were,resuspended
R1678 T1954 T1954 ROOT resuspended,resuspended
R1679 T1955 T1954 prep in,resuspended
R1680 T1956 T1958 nummod 200,buffer
R1681 T1957 T1958 compound μl,buffer
R1682 T1958 T1955 pobj buffer,in
R1683 T1959 T1958 punct (,buffer
R1684 T1960 T1962 compound RPMI,media
R1685 T1961 T1962 compound 1640,media
R1686 T1962 T1958 appos media,buffer
R1687 T1963 T1962 punct ",",media
R1688 T1964 T1965 nummod 1,%
R1689 T1965 T1968 nmod %,serum
R1690 T1966 T1968 amod foetal,serum
R1691 T1967 T1968 compound calf,serum
R1692 T1968 T1962 appos serum,media
R1693 T1969 T1962 punct ),media
R1694 T1970 T1958 acl containing,buffer
R1695 T1971 T1975 amod anti-chicken,conjugated
R1696 T1972 T1974 nummod M1,antibody
R1697 T1973 T1974 amod monoclonal,antibody
R1698 T1974 T1975 compound antibody,conjugated
R1699 T1975 T1954 conj conjugated,resuspended
R1700 T1976 T1975 prep to,conjugated
R1701 T1977 T1976 pobj FITC,to
R1702 T1978 T1975 prep for,conjugated
R1703 T1979 T1980 nummod 20,min
R1704 T1980 T1978 pobj min,for
R1705 T1981 T1980 prep on,min
R1706 T1982 T1981 pobj ice,on
R1707 T1983 T1954 punct .,resuspended
R1708 T1984 T1985 det The,cells
R1709 T1985 T1987 nsubjpass cells,washed
R1710 T1986 T1987 auxpass were,washed
R1711 T1987 T1987 ROOT washed,washed
R1712 T1988 T1987 advmod twice,washed
R1713 T1989 T1988 cc and,twice
R1714 T1990 T1991 amod fluorescent,intensity
R1715 T1991 T1993 nsubjpass intensity,analysed
R1716 T1992 T1993 auxpass was,analysed
R1717 T1993 T1987 conj analysed,washed
R1718 T1994 T1993 agent by,analysed
R1719 T1995 T1996 compound flow,cytometry
R1720 T1996 T1994 pobj cytometry,by
R1721 T1997 T1993 punct .,analysed
R1722 T1998 T1999 det All,results
R1723 T1999 T2001 nsubj results,are
R1724 T2000 T1999 acl shown,results
R1725 T2001 T2001 ROOT are,are
R1726 T2002 T2001 attr representative,are
R1727 T2003 T2002 prep of,representative
R1728 T2004 T2001 prep at,are
R1729 T2005 T2007 quantmod two,four
R1730 T2006 T2007 quantmod to,four
R1731 T2007 T2009 nummod four,experiments
R1732 T2008 T2009 amod independent,experiments
R1733 T2009 T2004 pobj experiments,at
R1734 T2010 T2012 mark unless,indicated
R1735 T2011 T2012 advmod otherwise,indicated
R1736 T2012 T2001 advcl indicated,are
R1737 T2013 T2001 punct .,are
R2279 T2730 T2731 nummod "Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 ",Loss
R2280 T2731 T2744 nsubj Loss,kinases
R2281 T2732 T2731 prep of,Loss
R2282 T2733 T2734 compound HSP27,phosphorylation
R2283 T2734 T2732 pobj phosphorylation,of
R2284 T2735 T2731 prep in,Loss
R2285 T2736 T2737 compound DT40,B
R2286 T2737 T2738 compound B,cells
R2287 T2738 T2735 pobj cells,in
R2288 T2739 T2744 advcl lacking,kinases
R2289 T2740 T2739 dobj expression,lacking
R2290 T2741 T2740 prep of,expression
R2291 T2742 T2743 compound PKD,family
R2292 T2743 T2741 pobj family,of
R2293 T2744 T2744 ROOT kinases,kinases
R2294 T2745 T2746 compound DT40,B
R2295 T2746 T2747 compound B,cells
R2296 T2747 T2748 nsubj cells,express
R2297 T2748 T2744 ccomp express,kinases
R2298 T2749 T2751 nummod two,isoforms
R2299 T2750 T2751 compound PKD,isoforms
R2300 T2751 T2748 dobj isoforms,express
R2301 T2752 T2751 punct ",",isoforms
R2302 T2753 T2751 conj PKD1,isoforms
R2303 T2754 T2753 cc and,PKD1
R2304 T2755 T2753 conj PKD3,PKD1
R2305 T2756 T2755 punct ",",PKD3
R2306 T2757 T2748 cc and,express
R2307 T2758 T2760 mark as,described
R2308 T2759 T2760 advmod previously,described
R2309 T2760 T2764 advcl described,generated
R2310 T2761 T2764 nsubj we,generated
R2311 T2762 T2764 aux have,generated
R2312 T2763 T2764 advmod recently,generated
R2313 T2764 T2748 conj generated,express
R2314 T2765 T2768 compound DT40,lines
R2315 T2766 T2768 compound B,lines
R2316 T2767 T2768 compound cell,lines
R2317 T2768 T2764 dobj lines,generated
R2318 T2769 T2770 nsubj that,lack
R2319 T2770 T2768 relcl lack,lines
R2320 T2771 T2770 dobj expression,lack
R2321 T2772 T2771 prep of,expression
R2322 T2773 T2774 det either,PKD1
R2323 T2774 T2772 pobj PKD1,of
R2324 T2775 T2774 cc or,PKD1
R2325 T2776 T2774 conj PKD3,PKD1
R2326 T2777 T2776 cc or,PKD3
R2327 T2778 T2779 det both,enzymes
R2328 T2779 T2772 pobj enzymes,of
R2329 T2780 T2782 nmod [,]
R2330 T2781 T2782 nummod 1,]
R2331 T2782 T2764 npadvmod ],generated
R2332 T2783 T2744 punct .,kinases
R2333 T2784 T2800 prep In,/
R2334 T2785 T2784 pcomp generating,In
R2335 T2786 T2790 det the,lines
R2336 T2787 T2789 amod double,cell
R2337 T2788 T2789 compound knockout,cell
R2338 T2789 T2790 compound cell,lines
R2339 T2790 T2785 dobj lines,generating
R2340 T2791 T2792 nsubj we,targeted
R2341 T2792 T2790 relcl targeted,lines
R2342 T2793 T2795 det the,loci
R2343 T2794 T2795 compound PKD1,loci
R2344 T2795 T2792 dobj loci,targeted
R2345 T2796 T2792 prep in,targeted
R2346 T2797 T2799 det a,−
R2347 T2798 T2799 compound PKD3,−
R2348 T2799 T2796 pobj −,in
R2349 T2800 T2800 ROOT /,/
R2350 T2801 T2803 nummod −,line
R2351 T2802 T2803 compound cell,line
R2352 T2803 T2800 dobj line,/
R2353 T2804 T2805 nsubj that,expressed
R2354 T2805 T2803 relcl expressed,line
R2355 T2806 T2808 det a,transgene
R2356 T2807 T2808 amod Flag-PKD3,transgene
R2357 T2808 T2805 dobj transgene,expressed
R2358 T2809 T2808 prep under,transgene
R2359 T2810 T2811 det the,control
R2360 T2811 T2809 pobj control,under
R2361 T2812 T2811 prep of,control
R2362 T2813 T2815 det a,promoter
R2363 T2814 T2815 amod doxycycline-inducible,promoter
R2364 T2815 T2812 pobj promoter,of
R2365 T2816 T2800 punct .,/
R2366 T2817 T2832 advmod Hence,is
R2367 T2818 T2832 punct ",",is
R2368 T2819 T2826 nmod in,expression
R2369 T2820 T2821 det the,presence
R2370 T2821 T2819 pobj presence,in
R2371 T2822 T2821 prep of,presence
R2372 T2823 T2822 pobj doxycycline,of
R2373 T2824 T2821 punct ",",presence
R2374 T2825 T2826 compound Flag-PKD3,expression
R2375 T2826 T2832 nsubj expression,is
R2376 T2827 T2826 prep in,expression
R2377 T2828 T2831 nummod PKD1/3,cells
R2378 T2829 T2831 amod double,cells
R2379 T2830 T2831 compound knockout,cells
R2380 T2831 T2827 pobj cells,in
R2381 T2832 T2832 ROOT is,is
R2382 T2833 T2832 acomp comparable,is
R2383 T2834 T2833 prep to,comparable
R2384 T2835 T2837 amod endogenous,present
R2385 T2836 T2837 amod PKD3,present
R2386 T2837 T2834 pobj present,to
R2387 T2838 T2837 prep in,present
R2388 T2839 T2841 amod wild-type,cells
R2389 T2840 T2841 compound DT40,cells
R2390 T2841 T2838 pobj cells,in
R2391 T2842 T2837 cc and,present
R2392 T2843 T2837 conj removal,present
R2393 T2844 T2843 prep of,removal
R2394 T2845 T2844 pobj doxycycline,of
R2395 T2846 T2843 prep from,removal
R2396 T2847 T2849 det the,media
R2397 T2848 T2849 compound culture,media
R2398 T2849 T2846 pobj media,from
R2399 T2850 T2832 prep for,is
R2400 T2851 T2852 nummod 5,days
R2401 T2852 T2853 compound days,results
R2402 T2853 T2850 pobj results,for
R2403 T2854 T2853 prep in,results
R2404 T2855 T2859 det a,phenotype
R2405 T2856 T2857 advmod completely,null
R2406 T2857 T2859 amod null,phenotype
R2407 T2858 T2859 compound PKD,phenotype
R2408 T2859 T2854 pobj phenotype,in
R2409 T2860 T2862 punct (,1A
R2410 T2861 T2862 compound Fig.,1A
R2411 T2862 T2859 appos 1A,phenotype
R2412 T2863 T2862 punct ),1A
R2413 T2864 T2832 punct .,is
R2414 T2865 T2869 advmod Previously,demonstrated
R2415 T2866 T2869 punct ",",demonstrated
R2416 T2867 T2869 nsubj we,demonstrated
R2417 T2868 T2869 aux have,demonstrated
R2418 T2869 T2869 ROOT demonstrated,demonstrated
R2419 T2870 T2886 mark that,is
R2420 T2871 T2886 nsubj phosphorylation,is
R2421 T2872 T2871 cc and,phosphorylation
R2422 T2873 T2874 amod nuclear,exclusion
R2423 T2874 T2871 conj exclusion,phosphorylation
R2424 T2875 T2874 prep of,exclusion
R2425 T2876 T2879 nmod class,deacetylases
R2426 T2877 T2879 nmod II,deacetylases
R2427 T2878 T2879 compound histone,deacetylases
R2428 T2879 T2875 pobj deacetylases,of
R2429 T2880 T2879 punct (,deacetylases
R2430 T2881 T2879 appos HDACs,deacetylases
R2431 T2882 T2879 punct ),deacetylases
R2432 T2883 T2871 prep during,phosphorylation
R2433 T2884 T2885 compound BCR,engagement
R2434 T2885 T2883 pobj engagement,during
R2435 T2886 T2869 ccomp is,demonstrated
R2436 T2887 T2886 acomp defective,is
R2437 T2888 T2886 prep in,is
R2438 T2889 T2894 nummod PKD1/3,cells
R2439 T2890 T2894 nummod −,cells
R2440 T2891 T2894 nmod /,cells
R2441 T2892 T2894 compound −,cells
R2442 T2893 T2894 compound B,cells
R2443 T2894 T2888 pobj cells,in
R2444 T2895 T2886 cc and,is
R2445 T2896 T2897 aux can,restored
R2446 T2897 T2886 conj restored,is
R2451 T2902 T2904 amod single,isoform
R2488 T2939 T2936 prep on,phosphorylation
R2489 T2940 T2939 pobj serine,on
R2490 T2941 T2940 nummod 82,serine
R2491 T2942 T2936 punct ",",phosphorylation
R2492 T2943 T2947 det the,sequence
R2493 T2944 T2947 amod proposed,sequence
R2494 T2945 T2946 compound PKD1,substrate
R2495 T2946 T2947 compound substrate,sequence
R2496 T2947 T2936 appos sequence,phosphorylation
R2497 T2948 T2929 punct .,assessed
R2498 T2949 T2951 nsubj We,investigated
R2499 T2950 T2951 advmod initially,investigated
R2500 T2951 T2951 ROOT investigated,investigated
R2501 T2952 T2953 det the,regulation
R2502 T2953 T2951 dobj regulation,investigated
R2503 T2954 T2953 prep of,regulation
R2504 T2955 T2956 compound HSP27,phosphorylation
R2505 T2956 T2954 pobj phosphorylation,of
R2506 T2957 T2956 prep in,phosphorylation
R2507 T2958 T2959 amod single,knockout
R2508 T2959 T2962 compound knockout,cells
R2509 T2960 T2961 compound DT40,B
R2510 T2961 T2962 compound B,cells
R2511 T2962 T2957 pobj cells,in
R2512 T2963 T2951 advcl lacking,investigated
R2513 T2964 T2965 preconj either,PKD1
R2514 T2965 T2963 dobj PKD1,lacking
R2515 T2966 T2965 cc or,PKD1
R2516 T2967 T2965 conj PKD3,PKD1
R2517 T2968 T2951 punct .,investigated
R2518 T2969 T2970 mark As,shown
R2519 T2970 T2985 advcl shown,increased
R2520 T2971 T2970 prep in,shown
R2521 T2972 T2973 compound Fig.,1B
R2522 T2973 T2971 pobj 1B,in
R2523 T2974 T2973 punct ",",1B
R2524 T2975 T2973 appos activation,1B
R2525 T2976 T2975 prep of,activation
R2526 T2977 T2978 det the,BCR
R2527 T2978 T2976 pobj BCR,of
R2528 T2979 T2975 cc or,activation
R2529 T2980 T2975 conj treatment,activation
R2530 T2981 T2975 prep with,activation
R2531 T2982 T2984 det the,PdBu
R2532 T2983 T2984 compound DAG-mimetic,PdBu
R2533 T2984 T2981 pobj PdBu,with
R2534 T2985 T2985 ROOT increased,increased
R2535 T2986 T2987 det the,levels
R2536 T2987 T2985 dobj levels,increased
R2537 T2988 T2987 prep of,levels
R2538 T2989 T2990 compound HSP27,phosphorylation
R2539 T2990 T2988 pobj phosphorylation,of
R2540 T2991 T2987 prep at,levels
R2541 T2992 T2991 pobj S82,at
R2542 T2993 T2992 prep in,S82
R2543 T2994 T2997 amod wild-type,cells
R2544 T2995 T2996 compound DT40,B
R2545 T2996 T2997 compound B,cells
R2546 T2997 T2993 pobj cells,in
R2547 T2998 T2985 punct .,increased
R2548 T2999 T3004 nsubj BCR,were
R2549 T3000 T2999 cc and,BCR
R2550 T3001 T3003 amod phorbol,signals
R2551 T3002 T3003 compound ester,signals
R2552 T3003 T2999 conj signals,BCR
R2553 T3004 T3004 ROOT were,were
R2554 T3005 T3004 advmod also,were
R2555 T3006 T3004 acomp able,were
R2556 T3007 T3008 aux to,increase
R2557 T3008 T3006 xcomp increase,able
R2558 T3009 T3010 compound HSP27,phosphorylation
R2559 T3010 T3008 dobj phosphorylation,increase
R2560 T3011 T3008 prep in,increase
R2561 T3012 T3016 nummod PKD1,knockout
R2562 T3013 T3012 cc or,PKD1
R2563 T3014 T3012 conj PKD3,PKD1
R2564 T3015 T3016 amod single,knockout
R2565 T3016 T3019 compound knockout,cells
R2566 T3017 T3018 compound DT40,B
R2567 T3018 T3019 compound B,cells
R2568 T3019 T3011 pobj cells,in
R2569 T3020 T3004 punct (,were
R2570 T3021 T3022 compound Fig.,1B
R2571 T3022 T3004 npadvmod 1B,were
R2572 T3023 T3004 punct ),were
R2573 T3024 T3004 punct .,were
R2574 T3025 T3038 advmod However,abolished
R2575 T3026 T3038 punct ",",abolished
R2576 T3027 T3038 nsubjpass BCR,abolished
R2577 T3028 T3027 punct -,BCR
R2578 T3029 T3027 cc and,BCR
R2579 T3030 T3032 nmod phorbol,phosphorylation
R2580 T3031 T3032 amod ester-induced,phosphorylation
R2581 T3032 T3027 conj phosphorylation,BCR
R2582 T3033 T3032 prep of,phosphorylation
R2583 T3034 T3033 pobj HSP27,of
R2584 T3035 T3032 prep on,phosphorylation
R2585 T3036 T3035 pobj S82,on
R2586 T3037 T3038 auxpass was,abolished
R2587 T3038 T3038 ROOT abolished,abolished
R2588 T3039 T3038 prep in,abolished
R2589 T3040 T3041 compound B,cells
R2590 T3041 T3039 pobj cells,in
R2591 T3042 T3043 nsubj that,lacked
R2592 T3043 T3041 relcl lacked,cells
R2593 T3044 T3045 preconj both,PKD1
R2594 T3045 T3043 dobj PKD1,lacked
R2595 T3046 T3045 cc and,PKD1
R2596 T3047 T3045 conj PKD3,PKD1
R2597 T3048 T3047 punct (,PKD3
R2598 T3049 T3050 compound Fig.,1C
R2599 T3050 T3047 appos 1C,PKD3
R2600 T3051 T3047 punct ),PKD3
R2601 T3052 T3038 punct .,abolished
R2602 T3053 T3066 advmod Significantly,was
R2603 T3054 T3056 punct ",",expression
R2604 T3055 T3056 amod doxycycline-induced,expression
R2605 T3056 T3066 nsubj expression,was
R2606 T3057 T3056 prep of,expression
R2607 T3058 T3060 det the,transgene
R2608 T3059 T3060 compound Flag-PKD3,transgene
R2609 T3060 T3057 pobj transgene,of
R2610 T3061 T3060 prep in,transgene
R2611 T3062 T3065 det the,cells
R2612 T3063 T3065 amod double,cells
R2613 T3064 T3065 compound knockout,cells
R2614 T3065 T3061 pobj cells,in
R2615 T3066 T3066 ROOT was,was
R2616 T3067 T3066 acomp sufficient,was
R2617 T3068 T3069 aux to,restore
R2618 T3069 T3067 xcomp restore,sufficient
R2619 T3070 T3071 amod normal,regulation
R2620 T3071 T3069 dobj regulation,restore
R2621 T3072 T3071 prep of,regulation
R2622 T3073 T3074 compound HSP27,phosphorylation
R2623 T3074 T3072 pobj phosphorylation,of
R2624 T3075 T3077 punct (,1C
R2625 T3076 T3077 compound Fig.,1C
R2626 T3077 T3074 appos 1C,phosphorylation
R2627 T3078 T3077 punct ),1C
R2628 T3079 T3066 punct .,was
R2629 T3080 T3095 prep In,was
R2630 T3081 T3080 pobj contrast,In
R2631 T3082 T3095 punct ",",was
R2632 T3083 T3095 nsubj expression,was
R2633 T3084 T3083 prep of,expression
R2634 T3085 T3089 det a,protein
R2635 T3086 T3089 amod kinase-deficient,protein
R2636 T3087 T3089 amod PKD3,protein
R2637 T3088 T3089 amod mutant,protein
R2638 T3089 T3084 pobj protein,of
R2639 T3090 T3089 prep in,protein
R2640 T3091 T3094 det the,cells
R2641 T3092 T3094 amod double,cells
R2642 T3093 T3094 compound knockout,cells
R2643 T3094 T3090 pobj cells,in
R2644 T3095 T3095 ROOT was,was
R2645 T3096 T3095 neg not,was
R2646 T3097 T3095 acomp able,was
R2647 T3098 T3099 aux to,restore
R2648 T3099 T3097 xcomp restore,able
R2649 T3100 T3099 dobj BCR,restore
R2650 T3101 T3100 punct -,BCR
R2651 T3102 T3099 cc or,restore
R2652 T3103 T3099 conj phorbol,restore
R2653 T3104 T3106 amod ester-induced,phosphorylation
R2654 T3105 T3106 compound HSP27,phosphorylation
R2655 T3106 T3103 dobj phosphorylation,phorbol
R2656 T3107 T3106 punct (,phosphorylation
R2657 T3108 T3109 compound Fig.,1D
R2658 T3109 T3106 appos 1D,phosphorylation
R2659 T3110 T3106 punct ),phosphorylation
R2660 T3111 T3095 punct .,was
R2661 T3112 T3120 advmod Hence,regulate
R2662 T3113 T3120 punct ",",regulate
R2663 T3114 T3120 nsubj PKD3,regulate
R2664 T3115 T3117 advmod as,as
R2665 T3116 T3117 advmod well,as
R2666 T3117 T3114 cc as,PKD3
R2667 T3118 T3114 conj PKD1,PKD3
R2668 T3119 T3120 aux can,regulate
R2669 T3120 T3120 ROOT regulate,regulate
R2670 T3121 T3122 compound HSP27,phosphorylation
R2671 T3122 T3120 dobj phosphorylation,regulate
R2672 T3123 T3120 cc and,regulate
R2673 T3124 T3120 prep in,regulate
R2674 T3125 T3126 compound DT40,B
R2675 T3126 T3127 compound B,cells
R2676 T3127 T3124 pobj cells,in
R2677 T3128 T3129 nsubj they,are
R2678 T3129 T3120 conj are,regulate
R2679 T3130 T3131 advmod functionally,redundant
R2680 T3131 T3129 acomp redundant,are
R2681 T3132 T3131 prep as,redundant
R2682 T3133 T3134 compound HSP27,kinases
R2683 T3134 T3132 pobj kinases,as
R2684 T3135 T3129 punct .,are
R3416 T4217 T4219 nummod "Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 ",proliferation
R3417 T4218 T4219 compound Cellular,proliferation
R3418 T4219 T4231 nsubj proliferation,kinases
R3419 T4220 T4219 cc and,proliferation
R3420 T4221 T4219 conj survival,proliferation
R3421 T4222 T4219 prep in,proliferation
R3422 T4223 T4224 compound DT40,B
R3423 T4224 T4225 compound B,cells
R3424 T4225 T4222 pobj cells,in
R3425 T4226 T4231 advcl lacking,kinases
R3426 T4227 T4226 dobj expression,lacking
R3427 T4228 T4227 prep of,expression
R3428 T4229 T4230 compound PKD,family
R3429 T4230 T4228 pobj family,of
R3430 T4231 T4231 ROOT kinases,kinases
R3431 T4232 T4233 compound PKD,enzymes
R3432 T4233 T4237 nsubjpass enzymes,linked
R3433 T4234 T4237 aux have,linked
R3434 T4235 T4237 advmod previously,linked
R3435 T4236 T4237 auxpass been,linked
R3436 T4237 T4231 ccomp linked,kinases
R3437 T4238 T4237 prep to,linked
R3438 T4239 T4240 det the,regulation
R3439 T4240 T4238 pobj regulation,to
R3440 T4241 T4240 prep of,regulation
R3441 T4242 T4243 compound cell,proliferation
R3442 T4243 T4241 pobj proliferation,of
R3443 T4244 T4243 cc and,proliferation
R3444 T4245 T4243 conj survival,proliferation
R3445 T4246 T4243 punct (,proliferation
R3446 T4247 T4240 acl reviewed,regulation
R3447 T4248 T4247 prep in,reviewed
R3448 T4249 T4248 pobj [,in
R3449 T4250 T4251 nummod 20,]
R3450 T4251 T4249 appos ],[
R3451 T4252 T4249 punct ),[
R3452 T4253 T4231 punct .,kinases
R3453 T4254 T4255 aux To,investigate
R3454 T4255 T4271 advcl investigate,cultured
R3455 T4256 T4257 det the,effect
R3456 T4257 T4255 dobj effect,investigate
R3457 T4258 T4263 dobj that,had
R3458 T4259 T4263 nsubj loss,had
R3459 T4260 T4259 prep of,loss
R3460 T4261 T4262 compound PKD,kinases
R3461 T4262 T4260 pobj kinases,of
R3462 T4263 T4257 relcl had,effect
R3463 T4264 T4263 prep on,had
R3464 T4265 T4267 compound B,survival
R3465 T4266 T4267 compound cell,survival
R3466 T4267 T4264 pobj survival,on
R3467 T4268 T4263 cc and/or,had
R3468 T4269 T4271 dobj proliferation,cultured
R3469 T4270 T4271 nsubj we,cultured
R3470 T4271 T4271 ROOT cultured,cultured
R3471 T4272 T4275 amod wild-type,cells
R3472 T4273 T4272 cc and,wild-type
R3473 T4274 T4272 conj PKD-null,wild-type
R3474 T4275 T4271 dobj cells,cultured
R3475 T4276 T4271 prep in,cultured
R3476 T4277 T4278 det the,presence
R3477 T4278 T4276 pobj presence,in
R3478 T4279 T4278 punct (,presence
R3479 T4280 T4300 nsubj PKD1/3,monitored
R3480 T4281 T4283 nummod −,−
R3481 T4282 T4283 compound /,−
R3482 T4283 T4300 dep −,monitored
R3483 T4284 T4283 punct :,−
R3484 T4285 T4287 nmod Flag-PKD3,ve
R3485 T4286 T4287 compound +,ve
R3486 T4287 T4283 appos ve,−
R3487 T4288 T4287 punct ),ve
R3488 T4289 T4287 cc or,ve
R3489 T4290 T4287 conj absence,ve
R3490 T4291 T4290 punct (,absence
R3491 T4292 T4295 nummod PKD1/3,−
R3492 T4293 T4295 nummod −,−
R3493 T4294 T4295 compound /,−
R3494 T4295 T4290 appos −,absence
R3495 T4296 T4295 punct ),−
R3496 T4297 T4290 prep of,absence
R3497 T4298 T4297 pobj doxycycline,of
R3498 T4299 T4290 cc and,absence
R3499 T4300 T4271 advcl monitored,cultured
R3500 T4301 T4302 amod exponential,growth
R3501 T4302 T4300 dobj growth,monitored
R3502 T4303 T4271 punct .,cultured
R3503 T4304 T4305 mark As,shown
R3504 T4305 T4311 advcl shown,−
R3505 T4306 T4305 prep in,shown
R3506 T4307 T4308 compound Fig.,2A
R3507 T4308 T4306 pobj 2A,in
R3508 T4309 T4311 punct ",",−
R3509 T4310 T4311 nsubj PKD1/3,−
R3510 T4311 T4311 ROOT −,−
R3511 T4312 T4314 nummod /,cells
R3512 T4313 T4314 compound −,cells
R3513 T4314 T4315 nsubj cells,proliferated
R3514 T4315 T4311 ccomp proliferated,−
R3515 T4316 T4315 advmod exponentially,proliferated
R3516 T4317 T4315 cc and,proliferated
R3517 T4318 T4324 nsubj re-expression,had
R3518 T4319 T4318 prep of,re-expression
R3519 T4320 T4319 pobj Flag-PKD3,of
R3520 T4321 T4318 prep in,re-expression
R3521 T4322 T4323 det these,cells
R3522 T4323 T4321 pobj cells,in
R3523 T4324 T4315 conj had,proliferated
R3524 T4325 T4326 det no,impact
R3525 T4326 T4324 dobj impact,had
R3526 T4327 T4326 prep on,impact
R3527 T4328 T4329 det the,rate
R3528 T4329 T4327 pobj rate,on
R3529 T4330 T4329 prep of,rate
R3530 T4331 T4330 pobj proliferation,of
R3531 T4332 T4311 punct .,−
R3532 T4333 T4347 advmod Furthermore,was
R3533 T4334 T4347 punct ",",was
R3534 T4335 T4336 det the,viability
R3535 T4336 T4347 nsubj viability,was
R3536 T4337 T4336 prep of,viability
R3537 T4338 T4343 nummod PKD1/3,cells
R3538 T4339 T4343 nummod −,cells
R3539 T4340 T4343 compound /,cells
R3540 T4341 T4343 compound −,cells
R3541 T4342 T4343 compound B,cells
R3542 T4343 T4337 pobj cells,of
R3543 T4344 T4336 prep during,viability
R3544 T4345 T4346 amod routine,culturing
R3545 T4346 T4344 pobj culturing,during
R3546 T4347 T4347 ROOT was,was
R3547 T4348 T4347 neg not,was
R3548 T4349 T4350 advmod significantly,different
R3549 T4350 T4347 acomp different,was
R3550 T4351 T4350 prep from,different
R3551 T4352 T4351 pobj that,from
R3552 T4353 T4352 prep of,that
R3553 T4354 T4356 amod wild-type,cells
R3554 T4355 T4356 compound B,cells
R3555 T4356 T4353 pobj cells,of
R3556 T4357 T4360 punct (,shown
R3557 T4358 T4360 nsubjpass data,shown
R3558 T4359 T4360 neg not,shown
R3559 T4360 T4347 conj shown,was
R3560 T4361 T4360 punct ),shown
R3561 T4362 T4347 punct .,was
R3562 T4363 T4365 nsubjpass It,noted
R3563 T4364 T4365 auxpass was,noted
R3564 T4365 T4365 ROOT noted,noted
R3565 T4366 T4377 mark that,was
R3566 T4367 T4370 det the,time
R3567 T4368 T4369 compound population,doubling
R3568 T4369 T4370 amod doubling,time
R3569 T4370 T4377 nsubj time,was
R3570 T4371 T4370 prep of,time
R3571 T4372 T4376 nummod PKD1/3,cells
R3572 T4373 T4376 nummod −,cells
R3573 T4374 T4376 compound /,cells
R3574 T4375 T4376 compound −,cells
R3575 T4376 T4371 pobj cells,of
R3576 T4377 T4365 ccomp was,noted
R3577 T4378 T4379 advmod slightly,slower
R3578 T4379 T4377 acomp slower,was
R3579 T4380 T4379 prep than,slower
R3580 T4381 T4380 pobj that,than
R3581 T4382 T4381 prep of,that
R3582 T4383 T4384 amod wild,type
R3583 T4384 T4382 pobj type,of
R3584 T4385 T4386 nummod DT40,cells
R3585 T4386 T4384 appos cells,type
R3586 T4387 T4386 punct (,cells
R3587 T4388 T4391 nummod 12.7,h
R3588 T4389 T4391 nmod ±,h
R3589 T4390 T4391 nummod 2.8,h
R3590 T4391 T4386 appos h,cells
R3591 T4392 T4391 prep versus,h
R3592 T4393 T4396 nummod 10.2,h
R3593 T4394 T4396 nmod ±,h
R3594 T4395 T4396 nummod 0.4,h
R3595 T4396 T4392 pobj h,versus
R3596 T4397 T4386 punct ),cells
R3597 T4398 T4365 cc but,noted
R3598 T4399 T4400 det the,failure
R3599 T4400 T4415 nsubj failure,suggests
R3600 T4401 T4400 prep of,failure
R3601 T4402 T4403 nummod PKD3,re-expression
R3602 T4403 T4401 pobj re-expression,of
R3603 T4404 T4405 aux to,modify
R3604 T4405 T4400 acl modify,failure
R3605 T4406 T4408 det the,rate
R3606 T4407 T4408 compound proliferation,rate
R3607 T4408 T4405 dobj rate,modify
R3608 T4409 T4408 prep of,rate
R3609 T4410 T4414 nummod PKD1/3,cells
R3610 T4411 T4414 nummod −,cells
R3611 T4412 T4414 compound /,cells
R3612 T4413 T4414 compound −,cells
R3613 T4414 T4409 pobj cells,of
R3614 T4415 T4365 conj suggests,noted
R3615 T4416 T4420 mark that,were
R3616 T4417 T4419 det these,differences
R3617 T4418 T4419 amod small,differences
R3618 T4419 T4420 nsubj differences,were
R3619 T4420 T4415 ccomp were,suggests
R3620 T4421 T4422 advmod most,likely
R3621 T4422 T4420 acomp likely,were
R3622 T4423 T4424 det the,result
R3623 T4424 T4420 attr result,were
R3624 T4425 T4424 prep of,result
R3625 T4426 T4427 amod clonal,variation
R3626 T4427 T4425 pobj variation,of
R3627 T4428 T4420 cc and,were
R3628 T4429 T4431 auxpass were,caused
R3629 T4430 T4431 neg not,caused
R3630 T4431 T4420 conj caused,were
R3631 T4432 T4431 advmod specifically,caused
R3632 T4433 T4431 agent by,caused
R3633 T4434 T4433 pobj loss,by
R3634 T4435 T4434 prep of,loss
R3635 T4436 T4437 compound PKD,enzymes
R3636 T4437 T4435 pobj enzymes,of
R3637 T4438 T4415 punct .,suggests
R3638 T4439 T4444 advmod Thus,are
R3639 T4440 T4444 punct ",",are
R3640 T4441 T4443 compound PKD,enzymes
R3641 T4442 T4443 compound family,enzymes
R3642 T4443 T4444 nsubj enzymes,are
R3643 T4444 T4444 ROOT are,are
R3644 T4445 T4444 neg not,are
R3645 T4446 T4444 acomp essential,are
R3646 T4447 T4446 prep for,essential
R3647 T4448 T4447 pcomp regulating,for
R3648 T4449 T4450 amod basal,survival
R3649 T4450 T4448 dobj survival,regulating
R3650 T4451 T4450 cc and,survival
R3658 T4459 T4469 nsubj enzymes,linked
R3659 T4460 T4459 punct ",",enzymes
R3660 T4461 T4462 advmod specifically,PKD1
R3661 T4462 T4459 appos PKD1,enzymes
R3662 T4463 T4462 cc and,PKD1
R3663 T4464 T4462 conj PKD2,PKD1
R3664 T4465 T4469 punct ",",linked
R3665 T4466 T4469 aux have,linked
R3666 T4467 T4469 advmod previously,linked
R3667 T4468 T4469 auxpass been,linked
R3668 T4469 T4469 ROOT linked,linked
R3669 T4470 T4469 prep to,linked
R3670 T4471 T4473 det a,role
R3671 T4472 T4473 amod protective,role
R3672 T4473 T4470 pobj role,to
R3673 T4474 T4473 prep against,role
R3674 T4475 T4477 amod oxidative,injury
R3675 T4476 T4477 amod stress-induced,injury
R3676 T4477 T4486 nmod injury,lines
R3677 T4478 T4477 prep in,injury
R3678 T4479 T4480 compound 3T3,fibroblast
R3679 T4480 T4478 pobj fibroblast,in
R3680 T4481 T4480 punct ",",fibroblast
R3681 T4482 T4480 conj HeLa,fibroblast
R3682 T4483 T4482 cc and,HeLa
R3683 T4484 T4486 amod epithelial,lines
R3684 T4485 T4486 compound cell,lines
R3685 T4486 T4474 pobj lines,against
R3686 T4487 T4488 compound [,"17,30"
R3687 T4488 T4486 appos "17,30",lines
R3688 T4489 T4490 nummod 32,]
R3689 T4490 T4486 appos ],lines
R3690 T4491 T4469 punct .,linked
R3691 T4492 T4494 nsubj We,addressed
R3692 T4493 T4494 advmod therefore,addressed
R3693 T4494 T4494 ROOT addressed,addressed
R3694 T4495 T4496 det the,role
R3695 T4496 T4494 dobj role,addressed
R3696 T4497 T4496 prep of,role
R3697 T4498 T4499 compound PKD,family
R3698 T4499 T4500 compound family,kinases
R3699 T4500 T4497 pobj kinases,of
R3700 T4501 T4496 prep in,role
R3701 T4502 T4501 pcomp regulating,in
R3702 T4503 T4504 compound B,cell
R3703 T4504 T4505 compound cell,survival
R3704 T4505 T4502 dobj survival,regulating
R3705 T4506 T4502 prep in,regulating
R3706 T4507 T4506 pobj response,in
R3707 T4508 T4507 prep to,response
R3708 T4509 T4510 amod oxidative,stress
R3709 T4510 T4508 pobj stress,to
R3710 T4511 T4510 cc and,stress
R3711 T4512 T4514 amod other,stimuli
R3712 T4513 T4514 compound stress,stimuli
R3713 T4514 T4510 conj stimuli,stress
R3714 T4515 T4494 punct .,addressed
R3715 T4516 T4517 mark As,shown
R3716 T4517 T4526 advcl shown,had
R3717 T4518 T4517 prep in,shown
R3718 T4519 T4520 compound Fig.,2B
R3719 T4520 T4518 pobj 2B,in
R3720 T4521 T4520 punct ",",2B
R3721 T4522 T4520 appos loss,2B
R3722 T4523 T4522 prep of,loss
R3723 T4524 T4525 nummod PKD1/3,expression
R3724 T4525 T4523 pobj expression,of
R3725 T4526 T4526 ROOT had,had
R3726 T4527 T4529 det no,impact
R3727 T4528 T4529 amod significant,impact
R3728 T4529 T4526 dobj impact,had
R3729 T4530 T4529 prep on,impact
R3730 T4531 T4532 det the,survival
R3731 T4532 T4530 pobj survival,on
R3732 T4533 T4532 prep of,survival
R3733 T4534 T4535 compound DT40,B
R3734 T4535 T4536 compound B,cells
R3735 T4536 T4533 pobj cells,of
R3736 T4537 T4526 prep in,had
R3737 T4538 T4537 pobj response,in
R3738 T4539 T4538 prep to,response
R3739 T4540 T4542 amod mitochondrial,stimuli
R3740 T4541 T4542 compound stress,stimuli
R3741 T4542 T4539 pobj stimuli,to
R3742 T4543 T4542 punct (,stimuli
R3743 T4544 T4542 appos H2O2,stimuli
R3744 T4545 T4544 cc or,H2O2
R3745 T4546 T4547 compound serum,deprivation
R3746 T4547 T4544 conj deprivation,H2O2
R3747 T4548 T4542 punct ),stimuli
R3748 T4549 T4542 punct ;,stimuli
R3749 T4550 T4552 amod DNA,agents
R3750 T4551 T4552 amod damaging,agents
R3751 T4552 T4542 conj agents,stimuli
R3752 T4553 T4552 punct (,agents
R3753 T4554 T4552 appos etoposide,agents
R3754 T4555 T4554 cc or,etoposide
R3755 T4556 T4554 conj doxorubicin,etoposide
R3756 T4557 T4552 punct ),agents
R3757 T4558 T4526 punct ;,had
R3758 T4559 T4561 compound ER,stress
R3759 T4560 T4561 compound pathway,stress
R3760 T4561 T4570 nsubj stress,following
R3761 T4562 T4561 amod due,stress
R3762 T4563 T4562 pcomp to,due
R3763 T4564 T4565 compound calcium,overload
R3764 T4565 T4562 pobj overload,due
R3765 T4566 T4567 punct (,thapsigargin
R3766 T4567 T4565 appos thapsigargin,overload
R3767 T4568 T4567 punct ),thapsigargin
R3768 T4569 T4565 cc or,overload
R3769 T4570 T4526 advcl following,had
R3770 T4571 T4572 amod prolonged,treatment
R3771 T4572 T4570 dobj treatment,following
R3772 T4573 T4572 prep with,treatment
R3773 T4574 T4575 compound phorbol,esters
R3774 T4575 T4573 pobj esters,with
R3775 T4576 T4575 cc or,esters
R3776 T4577 T4578 compound Trichostatin,A
R3777 T4578 T4575 conj A,esters
R3778 T4579 T4578 punct ",",A
R3779 T4580 T4581 det an,inhibitor
R3780 T4581 T4578 appos inhibitor,A
R3781 T4582 T4581 prep of,inhibitor
R3782 T4583 T4585 compound class,HDACs
R3783 T4584 T4585 compound I/II,HDACs
R3784 T4585 T4582 pobj HDACs,of
R3785 T4586 T4526 punct .,had
R3786 T4587 T4593 advmod Thus,play
R3787 T4588 T4593 punct ",",play
R3788 T4589 T4590 compound PKD,kinases
R3789 T4590 T4593 nsubj kinases,play
R3790 T4591 T4593 aux do,play
R3791 T4592 T4593 neg not,play
R3792 T4593 T4593 ROOT play,play
R3793 T4594 T4596 det an,role
R3794 T4595 T4596 amod essential,role
R3795 T4596 T4593 dobj role,play
R3796 T4597 T4593 prep in,play
R3797 T4598 T4597 pcomp regulating,in
R3798 T4599 T4600 compound B,cell
R3799 T4600 T4601 compound cell,survival
R3800 T4601 T4598 dobj survival,regulating
R3801 T4602 T4598 prep in,regulating
R3802 T4603 T4602 pobj response,in
R3803 T4604 T4603 prep to,response
R3804 T4605 T4606 det a,range
R3805 T4606 T4604 pobj range,to
R3806 T4607 T4606 prep of,range
R3807 T4608 T4610 amod different,stimuli
R3808 T4609 T4610 compound stress,stimuli
R3809 T4610 T4607 pobj stimuli,of
R3810 T4611 T4593 punct .,play
R4621 T5768 T5770 nummod "Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 ",receptor
R4622 T5769 T5770 compound Antigen,receptor
R4623 T5770 T5773 amod receptor,pathways
R4624 T5771 T5773 amod regulated,pathways
R4625 T5772 T5773 amod signalling,pathways
R4626 T5773 T5781 nsubj pathways,explore
R4627 T5774 T5773 prep in,pathways
R4628 T5775 T5777 compound PKD-null,B
R4629 T5776 T5777 compound DT40,B
R4630 T5777 T5778 compound B,cells
R4631 T5778 T5774 pobj cells,in
R4632 T5779 T5781 aux To,explore
R4633 T5780 T5781 advmod further,explore
R4634 T5781 T5781 ROOT explore,explore
R4635 T5782 T5783 det the,contribution
R4636 T5783 T5781 dobj contribution,explore
R4637 T5784 T5783 prep of,contribution
R4638 T5785 T5786 compound PKD,kinases
R4639 T5786 T5784 pobj kinases,of
R4640 T5787 T5786 prep to,kinases
R4641 T5788 T5791 compound DT40,biology
R4642 T5789 T5791 compound B,biology
R4643 T5790 T5791 compound cell,biology
R4644 T5791 T5787 pobj biology,to
R4645 T5792 T5793 nsubj we,investigated
R4646 T5793 T5783 relcl investigated,contribution
R4647 T5794 T5799 mark whether,were
R4648 T5795 T5798 amod specific,events
R4649 T5796 T5798 amod BCR-regulated,events
R4650 T5797 T5798 amod signalling,events
R4651 T5798 T5799 nsubj events,were
R4652 T5799 T5793 ccomp were,investigated
R4653 T5800 T5799 acomp defective,were
R4654 T5801 T5800 prep in,defective
R4655 T5802 T5805 det the,cells
R4656 T5803 T5804 compound PKD-null,B
R4657 T5804 T5805 compound B,cells
R4658 T5805 T5801 pobj cells,in
R4659 T5806 T5781 punct .,explore
R4660 T5807 T5808 amod Initial,experiments
R4661 T5808 T5809 nsubj experiments,revealed
R4662 T5809 T5809 ROOT revealed,revealed
R4663 T5810 T5817 mark that,reduced
R4664 T5811 T5812 compound surface,expression
R4665 T5812 T5817 nsubjpass expression,reduced
R4666 T5813 T5812 prep of,expression
R4667 T5814 T5815 det the,BCR
R4668 T5815 T5813 pobj BCR,of
R4669 T5816 T5817 auxpass was,reduced
R4670 T5817 T5809 ccomp reduced,revealed
R4671 T5818 T5817 prep in,reduced
R4672 T5819 T5822 nummod PKD1/3,−
R4673 T5820 T5822 nummod −,−
R4674 T5821 T5822 compound /,−
R4675 T5822 T5818 pobj −,in
R4676 T5823 T5822 punct (,−
R4677 T5824 T5818 cc and,in
R4678 T5825 T5817 prep in,reduced
R4679 T5826 T5835 nummod PKD1/3,cells
R4680 T5827 T5829 nummod −,−
R4681 T5828 T5829 compound /,−
R4682 T5829 T5835 nummod −,cells
R4683 T5830 T5829 punct :,−
R4684 T5831 T5829 appos Flag-PKD3,−
R4685 T5832 T5833 advmod +,ve
R4686 T5833 T5829 appos ve,−
R4687 T5834 T5829 punct ),−
R4688 T5835 T5825 pobj cells,in
R4689 T5836 T5817 prep compared,reduced
R4690 T5837 T5836 prep to,compared
R4691 T5838 T5841 amod wild-type,cells
R4692 T5839 T5840 compound DT40,B
R4693 T5840 T5841 compound B,cells
R4694 T5841 T5837 pobj cells,to
R4695 T5842 T5841 punct (,cells
R4696 T5843 T5844 compound Fig.,3A
R4697 T5844 T5841 appos 3A,cells
R4698 T5845 T5844 cc and,3A
R4699 T5846 T5848 nsubj data,shown
R4700 T5847 T5848 neg not,shown
R4701 T5848 T5841 relcl shown,cells
R4702 T5849 T5817 punct ),reduced
R4703 T5850 T5809 punct .,revealed
R4704 T5851 T5860 advmod Nevertheless,was
R4705 T5852 T5860 punct ",",was
R4706 T5853 T5860 nsubj BCR-crosslinking,was
R4707 T5854 T5853 prep of,BCR-crosslinking
R4708 T5855 T5859 nummod PKD1/3,cells
R4709 T5856 T5859 nummod −,cells
R4710 T5857 T5859 nmod /,cells
R4711 T5858 T5859 amod −,cells
R4712 T5859 T5854 pobj cells,of
R4713 T5860 T5860 ROOT was,was
R4714 T5861 T5860 acomp sufficient,was
R4715 T5862 T5863 aux to,induce
R4716 T5863 T5861 xcomp induce,sufficient
R4717 T5864 T5865 det the,activation
R4718 T5865 T5863 dobj activation,induce
R4719 T5866 T5865 prep of,activation
R4720 T5867 T5868 det a,number
R4721 T5868 T5866 pobj number,of
R4722 T5869 T5868 prep of,number
R4723 T5870 T5869 pcomp signalling,of
R4724 T5871 T5870 dobj cascades,signalling
R4725 T5872 T5871 punct ",",cascades
R4726 T5873 T5871 amod similar,cascades
R4727 T5874 T5873 prep to,similar
R4728 T5875 T5876 nsubj that,observed
R4729 T5876 T5871 relcl observed,cascades
R4730 T5877 T5876 prep in,observed
R4731 T5878 T5879 amod wild-type,cells
R4732 T5879 T5877 pobj cells,in
R4733 T5880 T5871 punct (,cascades
R4734 T5881 T5882 compound Fig.,3B
R4735 T5882 T5871 appos 3B,cascades
R4736 T5883 T5871 punct ),cascades
R4737 T5884 T5860 punct .,was
R4738 T5885 T5888 advmod Hence,activation
R4739 T5886 T5888 punct ",",activation
R4740 T5887 T5888 amod BCR-induced,activation
R4741 T5888 T5909 nsubj activation,was
R4742 T5889 T5888 prep of,activation
R4743 T5890 T5891 det the,Akt
R4744 T5891 T5889 pobj Akt,of
R4745 T5892 T5891 punct ",",Akt
R4746 T5893 T5895 compound mTOR/p70,kinase
R4747 T5894 T5895 compound S6,kinase
R4748 T5895 T5891 appos kinase,Akt
R4749 T5896 T5898 punct (,shown
R4750 T5897 T5898 mark as,shown
R4751 T5898 T5888 advcl shown,activation
R4752 T5899 T5898 agent by,shown
R4753 T5900 T5908 nummod S6,pathways
R4754 T5901 T5902 amod ribosomal,protein
R4755 T5902 T5908 nmod protein,pathways
R4756 T5903 T5902 appos phosphorylation,protein
R4757 T5904 T5902 punct ),protein
R4758 T5905 T5902 cc and,protein
R4759 T5906 T5902 conj MAPK,protein
R4760 T5907 T5908 amod signalling,pathways
R4761 T5908 T5899 pobj pathways,by
R4762 T5909 T5909 ROOT was,was
R4763 T5910 T5909 advmod clearly,was
R4764 T5911 T5909 acomp detectable,was
R4765 T5912 T5911 prep in,detectable
R4766 T5913 T5914 compound PKD1/3-null,B
R4767 T5914 T5915 compound B,cells
R4768 T5915 T5912 pobj cells,in
R4769 T5916 T5918 punct (,3B
R4770 T5917 T5918 compound Fig.,3B
R4771 T5918 T5911 appos 3B,detectable
R4772 T5919 T5918 punct ),3B
R4773 T5920 T5909 punct .,was
R4774 T5921 T5941 advmod Furthermore,observed
R4775 T5922 T5941 punct ",",observed
R4776 T5923 T5925 amod enhanced,phosphorylation
R4777 T5924 T5925 compound tyrosine,phosphorylation
R4778 T5925 T5941 nsubjpass phosphorylation,observed
R4779 T5926 T5925 prep of,phosphorylation
R4780 T5927 T5929 amod multiple,proteins
R4781 T5928 T5929 amod cellular,proteins
R4782 T5929 T5926 pobj proteins,of
R4783 T5930 T5932 advmod as,as
R4784 T5931 T5932 advmod well,as
R4785 T5932 T5925 cc as,phosphorylation
R4786 T5933 T5934 det an,increase
R4787 T5934 T5925 conj increase,phosphorylation
R4788 T5935 T5934 prep in,increase
R4789 T5936 T5938 amod intracellular,levels
R4790 T5937 T5938 compound calcium,levels
R4791 T5938 T5935 pobj levels,in
R4792 T5939 T5941 auxpass was,observed
R4793 T5940 T5941 advmod also,observed
R4794 T5941 T5941 ROOT observed,observed
R4795 T5942 T5941 prep following,observed
R4796 T5943 T5944 compound BCR,stimulation
R4797 T5944 T5942 pobj stimulation,following
R4798 T5945 T5944 prep of,stimulation
R4799 T5946 T5948 nmod PKD1/3-null,cells
R4800 T5947 T5948 compound B,cells
R4801 T5948 T5945 pobj cells,of
R4802 T5949 T5950 punct (,data
R4803 T5950 T5948 appos data,cells
R4804 T5951 T5952 neg not,shown
R4805 T5952 T5950 acl shown,data
R4806 T5953 T5950 punct ),data
R4807 T5954 T5941 punct .,observed
R4808 T5955 T5957 nsubj We,observe
R4809 T5956 T5957 aux did,observe
R4810 T5957 T5957 ROOT observe,observe
R4811 T5958 T5970 mark that,induced
R4812 T5959 T5960 det the,strength
R4813 T5960 T5970 nsubj strength,induced
R4814 T5961 T5960 prep of,strength
R4815 T5962 T5961 pobj BCR,of
R4816 T5963 T5960 punct (,strength
R4817 T5964 T5960 cc but,strength
R4818 T5965 T5966 neg not,phorbol
R4819 T5966 T5967 advmod phorbol,ester
R4820 T5967 T5960 conj ester,strength
R4821 T5968 T5960 punct ),strength
R4822 T5969 T5970 punct -,induced
R4823 T5970 T5957 ccomp induced,observe
R4824 T5971 T5970 dobj regulation,induced
R4825 T5972 T5971 prep of,regulation
R4826 T5973 T5976 det the,pathway
R4827 T5974 T5976 amod Erk1-RSK1,pathway
R4828 T5975 T5976 compound signalling,pathway
R4829 T5976 T5972 pobj pathway,of
R4830 T5977 T5978 auxpass was,reduced
R4831 T5978 T5970 conj reduced,induced
R4832 T5979 T5978 prep in,reduced
R4833 T5980 T5985 nummod PKD1/3,cells
R4834 T5981 T5985 nummod −,cells
R4835 T5982 T5985 compound /,cells
R4836 T5983 T5985 compound −,cells
R4837 T5984 T5985 compound B,cells
R4838 T5985 T5979 pobj cells,in
R4839 T5986 T5978 prep compared,reduced
R4840 T5987 T5986 prep to,compared
R4841 T5988 T5990 amod wild-type,cells
R4842 T5989 T5990 compound B,cells
R4843 T5990 T5987 pobj cells,to
R4844 T5991 T5993 punct (,3B
R4845 T5992 T5993 compound Fig.,3B
R4846 T5993 T5990 appos 3B,cells
R4847 T5994 T5990 punct ),cells
R4848 T5995 T5957 punct .,observe
R4849 T5996 T5997 nummod One,interpretation
R4854 T6001 T6001 ROOT is,is
R4855 T6002 T6006 mark that,modulate
R4856 T6003 T6004 compound PKD,enzymes
R4857 T6004 T6006 nsubj enzymes,modulate
R4858 T6005 T6006 aux may,modulate
R4859 T6006 T6001 ccomp modulate,is
R4860 T6007 T6008 compound Erk,activation
R4861 T6008 T6006 dobj activation,modulate
R4862 T6009 T6001 punct .,is
R4863 T6010 T6017 advmod Indeed,linked
R4864 T6011 T6017 punct ",",linked
R4865 T6012 T6013 compound PKD,enzymes
R4866 T6013 T6017 nsubj enzymes,linked
R4867 T6014 T6017 aux have,linked
R4868 T6015 T6017 advmod previously,linked
R4869 T6016 T6017 auxpass been,linked
R4870 T6017 T6017 ROOT linked,linked
R4871 T6018 T6017 prep to,linked
R4872 T6019 T6020 det the,growth
R4873 T6020 T6018 pobj growth,to
R4874 T6021 T6023 amod factor-regulated,signalling
R4875 T6022 T6023 compound Erk,signalling
R4876 T6023 T6017 dobj signalling,linked
R4877 T6024 T6023 prep in,signalling
R4878 T6025 T6029 nmod fibroblast,lines
R4879 T6026 T6025 cc and,fibroblast
R4880 T6027 T6029 amod endothelial,lines
R4881 T6028 T6029 compound cell,lines
R4882 T6029 T6024 pobj lines,in
R4883 T6030 T6023 dobj [,signalling
R4884 T6031 T6030 nummod 33,[
R4885 T6032 T6033 nummod 35,]
R4886 T6033 T6030 appos ],[
R4887 T6034 T6017 punct .,linked
R4888 T6035 T6042 advmod However,reduced
R4889 T6036 T6042 punct ",",reduced
R4890 T6037 T6039 amod BCR-induced,phosphorylation
R4891 T6038 T6039 compound Erk,phosphorylation
R4892 T6039 T6042 nsubjpass phosphorylation,reduced
R4893 T6040 T6042 auxpass was,reduced
R4894 T6041 T6042 advmod also,reduced
R4895 T6042 T6042 ROOT reduced,reduced
R4896 T6043 T6042 prep in,reduced
R4897 T6044 T6052 nummod PKD1/3,cells
R4898 T6045 T6046 nummod −,/
R4899 T6046 T6052 compound /,cells
R4900 T6047 T6049 punct −,Flag-PKD3
R4901 T6048 T6049 punct -,Flag-PKD3
R4902 T6049 T6052 compound Flag-PKD3,cells
R4903 T6050 T6052 compound +,cells
R4904 T6051 T6052 compound B,cells
R4905 T6052 T6043 pobj cells,in
R4906 T6053 T6052 punct (,cells
R4907 T6054 T6054 ROOT data,data
R4908 T6055 T6056 neg not,shown
R4909 T6056 T6054 acl shown,data
R4910 T6057 T6054 punct ),data
R4911 T6058 T6042 advcl suggesting,reduced
R4912 T6059 T6074 mark that,−
R4913 T6060 T6062 amod reduced,levels
R4914 T6061 T6062 compound BCR,levels
R4915 T6062 T6074 nsubj levels,−
R4916 T6063 T6062 prep on,levels
R4917 T6064 T6065 det the,surface
R4918 T6065 T6063 pobj surface,on
R4919 T6066 T6065 prep of,surface
R4920 T6067 T6070 nummod PKD1/3,−
R4921 T6068 T6070 nummod −,−
R4922 T6069 T6070 compound /,−
R4923 T6070 T6066 pobj −,of
R4924 T6071 T6070 punct (,−
R4925 T6072 T6070 cc and,−
R4926 T6073 T6074 nsubj PKD1/3,−
R4927 T6074 T6058 ccomp −,suggesting
R4928 T6075 T6079 nummod /,+
R4929 T6076 T6078 punct −,Flag-PKD3
R4930 T6077 T6078 punct -,Flag-PKD3
R4939 T6086 T6085 prep on,impact
R4940 T6087 T6088 det the,strength
R4941 T6088 T6086 pobj strength,on
R4942 T6089 T6088 prep of,strength
R4943 T6090 T6089 pobj activation,of
R4944 T6091 T6090 prep of,activation
R4945 T6092 T6096 det this,pathway
R4946 T6093 T6096 amod specific,pathway
R4947 T6094 T6096 amod intracellular,pathway
R4948 T6095 T6096 compound signalling,pathway
R4949 T6096 T6091 pobj pathway,of
R4950 T6097 T6085 punct .,impact
R4951 T6098 T6099 aux To,search
R4952 T6099 T6120 advcl search,used
R4953 T6100 T6099 prep for,search
R4954 T6101 T6104 amod other,targets
R4955 T6102 T6104 amod potential,targets
R4956 T6103 T6104 compound PKD,targets
R4957 T6104 T6100 pobj targets,for
R4958 T6105 T6107 nsubj that,show
R4959 T6106 T6107 aux may,show
R4960 T6107 T6104 relcl show,targets
R4961 T6108 T6109 amod defective,regulation
R4962 T6109 T6107 dobj regulation,show
R4963 T6110 T6109 prep in,regulation
R4964 T6111 T6116 compound PKD1/3,B
R4965 T6112 T6116 compound −,B
R4966 T6113 T6116 compound /,B
R4967 T6114 T6116 compound −,B
R4968 T6115 T6116 compound DT40,B
R4969 T6116 T6117 compound B,cells
R4970 T6117 T6110 pobj cells,in
R4971 T6118 T6120 punct ",",used
R4972 T6119 T6120 nsubj we,used
R4973 T6120 T6120 ROOT used,used
R4974 T6121 T6123 det a,substrate
R4975 T6122 T6123 compound PKD,substrate
R4976 T6123 T6120 dobj substrate,used
R4977 T6124 T6123 amod phospho-antibody,substrate
R4978 T6125 T6126 nsubj that,recognises
R4979 T6126 T6124 relcl recognises,phospho-antibody
R4980 T6127 T6129 compound consensus,sequences
R4981 T6128 T6129 compound phosphorylation,sequences
R4982 T6129 T6126 dobj sequences,recognises
R4983 T6130 T6129 acl targeted,sequences
R4984 T6131 T6130 agent by,targeted
R4985 T6132 T6133 compound PKD,enzymes
R4986 T6133 T6131 pobj enzymes,by
R4987 T6134 T6137 punct (,[
R4988 T6135 T6137 nmod LxRxxpS/T,[
R4989 T6136 T6137 punct ),[
R4990 T6137 T6133 appos [,enzymes
R4991 T6138 T6139 nummod 36,]
R4992 T6139 T6137 appos ],[
R4993 T6140 T6120 punct .,used
R4994 T6141 T6142 mark As,shown
R4995 T6142 T6160 advcl shown,was
R4996 T6143 T6142 prep in,shown
R4997 T6144 T6145 compound Fig.,3C
R4998 T6145 T6143 pobj 3C,in
R4999 T6146 T6145 punct ",",3C
R5000 T6147 T6160 nsubj phorbol,was
R5001 T6148 T6160 nsubj ester,was
R5002 T6149 T6148 punct -,ester
R5003 T6150 T6148 cc and,ester
R5004 T6151 T6152 amod BCR-induced,phosphorylation
R5005 T6152 T6148 conj phosphorylation,ester
R5006 T6153 T6152 prep of,phosphorylation
R5007 T6154 T6155 amod cellular,substrates
R5008 T6155 T6153 pobj substrates,of
R5009 T6156 T6155 acl detected,substrates
R5010 T6157 T6156 agent by,detected
R5011 T6158 T6159 det this,phospho-antibody
R5012 T6159 T6157 pobj phospho-antibody,by
R5013 T6160 T6160 ROOT was,was
R5014 T6161 T6160 acomp similar,was
R5015 T6162 T6161 prep in,similar
R5016 T6163 T6169 amod wild-type,cells
R5017 T6164 T6163 cc and,wild-type
R5018 T6165 T6163 conj PKD1/3,wild-type
R5019 T6166 T6169 compound −,cells
R5020 T6167 T6169 compound /,cells
R5021 T6168 T6169 compound −,cells
R5022 T6169 T6162 pobj cells,in
R5023 T6170 T6160 cc and,was
R5024 T6171 T6160 conj is,was
R5025 T6172 T6173 advmod therefore,independent
R5026 T6173 T6171 acomp independent,is
R5027 T6174 T6173 prep of,independent
R5028 T6175 T6176 compound PKD,enzymes
R5029 T6176 T6174 pobj enzymes,of
R5030 T6177 T6171 punct .,is
R5031 T6178 T6201 advmod However,prevented
R5032 T6179 T6201 punct ",",prevented
R5033 T6180 T6201 nsubj pretreatment,prevented
R5034 T6181 T6180 prep of,pretreatment
R5035 T6182 T6183 preconj both,wild-type
R5036 T6183 T6181 pobj wild-type,of
R5037 T6184 T6183 cc and,wild-type
R5038 T6185 T6183 conj PKD1/3,wild-type
R5039 T6186 T6188 compound −,−
R5040 T6187 T6188 compound /,−
R5041 T6188 T6191 compound −,cells
R5042 T6189 T6190 compound DT40,B
R5043 T6190 T6191 compound B,cells
R5044 T6191 T6183 appos cells,wild-type
R5045 T6192 T6180 prep with,pretreatment
R5046 T6193 T6192 pobj GF109203X,with
R5047 T6194 T6193 punct ",",GF109203X
R5048 T6195 T6197 det a,derivative
R5049 T6196 T6197 compound bisindoylmaleimide,derivative
R5050 T6197 T6193 appos derivative,GF109203X
R5051 T6198 T6199 nsubj that,inhibits
R5052 T6199 T6197 relcl inhibits,derivative
R5053 T6200 T6199 dobj PKCs,inhibits
R5054 T6201 T6201 ROOT prevented,prevented
R5055 T6202 T6203 det the,induction
R5056 T6203 T6201 dobj induction,prevented
R5057 T6204 T6203 prep of,induction
R5058 T6205 T6204 pobj proteins,of
R5059 T6206 T6207 nsubj that,contain
R5060 T6207 T6205 relcl contain,proteins
R5061 T6208 T6209 compound phosphorylated,LxRxxS/T
R5062 T6209 T6210 compound LxRxxS/T,motifs
R5063 T6210 T6207 dobj motifs,contain
R5064 T6211 T6201 punct .,prevented
R5065 T6212 T6216 advmod Thus,enzymes
R5066 T6213 T6216 nsubj loss,enzymes
R5067 T6214 T6213 prep of,loss
R5068 T6215 T6214 pobj PKD1/3,of
R5069 T6216 T6220 nsubj enzymes,disrupt
R5070 T6217 T6220 aux does,disrupt
R5071 T6218 T6220 neg not,disrupt
R5072 T6219 T6220 advmod globally,disrupt
R5073 T6220 T6220 ROOT disrupt,disrupt
R5074 T6221 T6222 det the,phosphorylation
R5075 T6222 T6220 dobj phosphorylation,disrupt
R5076 T6223 T6222 prep of,phosphorylation
R5077 T6224 T6225 amod cellular,proteins
R5078 T6225 T6223 pobj proteins,of
R5132 T6279 T6278 prep in,culminate
R5133 T6280 T6281 det the,activation
R5134 T6281 T6279 pobj activation,in
R5135 T6282 T6281 prep of,activation
R5136 T6283 T6284 compound gene,transcription
R5137 T6284 T6285 compound transcription,events
R5138 T6285 T6282 pobj events,of
R5139 T6286 T6287 nsubj that,control
R5140 T6287 T6285 relcl control,events
R5141 T6288 T6290 compound B,survival
R5142 T6289 T6290 compound cell,survival
R5143 T6290 T6287 dobj survival,control
R5144 T6291 T6290 punct ",",survival
R5145 T6292 T6290 conj proliferation,survival
R5146 T6293 T6292 cc and,proliferation
R5147 T6294 T6292 conj function,proliferation
R5148 T6295 T6278 punct .,culminate
R5149 T6296 T6303 prep In,proposed
R5150 T6297 T6298 det this,context
R5151 T6298 T6296 pobj context,In
R5152 T6299 T6303 punct ",",proposed
R5153 T6300 T6303 nsubjpass it,proposed
R5154 T6301 T6303 aux has,proposed
R5155 T6302 T6303 auxpass been,proposed
R5156 T6303 T6303 ROOT proposed,proposed
R5157 T6304 T6303 dobj that,proposed
R5158 T6305 T6307 compound PKD,members
R5159 T6306 T6307 compound family,members
R5160 T6307 T6304 nsubj members,that
R5161 T6308 T6307 dobj control,members
R5162 T6309 T6308 prep of,control
R5163 T6310 T6311 compound gene,transcription
R5164 T6311 T6309 pobj transcription,of
R5165 T6312 T6307 prep through,members
R5166 T6313 T6312 pobj activation,through
R5167 T6314 T6313 prep of,activation
R5168 T6315 T6318 det the,factor
R5169 T6316 T6318 compound NFκB,factor
R5170 T6317 T6318 compound transcription,factor
R5171 T6318 T6314 pobj factor,of
R5172 T6319 T6303 punct .,proposed
R5173 T6320 T6326 advmod Thus,occurs
R5174 T6321 T6326 punct ",",occurs
R5175 T6322 T6323 amod PKD-mediated,activation
R5176 T6323 T6326 nsubj activation,occurs
R5177 T6324 T6323 prep of,activation
R5178 T6325 T6324 pobj NFκB,of
R5179 T6326 T6326 ROOT occurs,occurs
R5180 T6327 T6326 advmod downstream,occurs
R5181 T6328 T6327 prep of,downstream
R5182 T6329 T6330 det a,variety
R5183 T6330 T6328 pobj variety,of
R5184 T6331 T6330 prep of,variety
R5185 T6332 T6333 amod different,signals
R5186 T6333 T6331 pobj signals,of
R5187 T6334 T6333 punct ",",signals
R5188 T6335 T6333 prep including,signals
R5189 T6336 T6337 amod mROS/oxidative,stress
R5190 T6337 T6335 pobj stress,including
R5191 T6338 T6337 punct ",",stress
R5192 T6339 T6340 amod lysophosphatidic,acid
R5193 T6340 T6337 conj acid,stress
R5194 T6341 T6340 cc and,acid
R5195 T6342 T6344 det the,oncogene
R5196 T6343 T6344 compound Bcr-Abl,oncogene
R5197 T6344 T6340 conj oncogene,acid
R5198 T6345 T6347 compound [,]
R5199 T6346 T6347 compound "17,21,23,30,37",]
R5200 T6347 T6344 appos ],oncogene
R5201 T6348 T6326 punct .,occurs
R5202 T6349 T6357 advmod Furthermore,enhances
R5203 T6350 T6351 punct ",",expression
R5204 T6351 T6357 nsubj expression,enhances
R5205 T6352 T6351 prep of,expression
R5206 T6353 T6356 det an,mutant
R5207 T6354 T6356 amod activated,mutant
R5208 T6355 T6356 compound PKD1,mutant
R5209 T6356 T6352 pobj mutant,of
R5210 T6357 T6357 ROOT enhances,enhances
R5211 T6358 T6360 amod HPK1-mediated,activation
R5212 T6359 T6360 compound NFκB,activation
R5213 T6360 T6357 dobj activation,enhances
R5214 T6361 T6363 nmod [,]
R5215 T6362 T6363 nummod 38,]
R5216 T6363 T6357 npadvmod ],enhances
R5217 T6364 T6357 punct .,enhances
R5218 T6365 T6371 prep In,known
R5219 T6366 T6367 compound B,cells
R5220 T6367 T6365 pobj cells,In
R5221 T6368 T6371 punct ",",known
R5222 T6369 T6371 nsubjpass NFκB,known
R5223 T6370 T6371 auxpass is,known
R5224 T6371 T6371 ROOT known,known
R5225 T6372 T6374 aux to,regulated
R5226 T6373 T6374 auxpass be,regulated
R5227 T6374 T6371 xcomp regulated,known
R5228 T6375 T6374 prep via,regulated
R5229 T6376 T6375 pobj DAG,via
R5230 T6377 T6376 cc and,DAG
R5231 T6378 T6379 compound PKCβ,[
R5232 T6379 T6376 conj [,DAG
R5233 T6380 T6381 compound "39,40",]
R5234 T6381 T6376 conj ],DAG
R5235 T6382 T6371 cc but,known
R5236 T6383 T6385 mark whether,are
R5237 T6384 T6385 nsubj PKDs,are
R5238 T6385 T6371 conj are,known
R5239 T6386 T6387 amod key,intermediaries
R5240 T6387 T6385 attr intermediaries,are
R5241 T6388 T6385 prep for,are
R5242 T6389 T6390 compound NFκB,regulation
R5243 T6406 T6409 nsubjpass activity,induced
R5244 T6390 T6388 pobj regulation,for
R5245 T6407 T6409 auxpass was,induced
R5246 T6408 T6409 advmod strongly,induced
R5247 T6391 T6394 aux has,explored
R5248 T6409 T6402 ccomp induced,show
R5249 T6410 T6409 prep in,induced
R5250 T6392 T6394 neg not,explored
R5251 T6411 T6412 preconj both,wild-type
R5252 T6393 T6394 auxpass been,explored
R5253 T6412 T6417 nmod wild-type,−
R5254 T6394 T6385 conj explored,are
R5255 T6413 T6412 cc and,wild-type
R5256 T6414 T6412 conj PKD1/3,wild-type
R5257 T6415 T6417 compound −,−
R5258 T6395 T6371 punct .,known
R5259 T6416 T6417 compound /,−
R5260 T6417 T6420 compound −,cells
R5261 T6418 T6419 compound DT40,B
R5262 T6396 T6397 det The,data
R5263 T6419 T6420 compound B,cells
R5264 T6420 T6410 pobj cells,in
R5265 T6421 T6409 prep in,induced
R5266 T6397 T6402 nsubj data,show
R5267 T6422 T6421 pobj response,in
R5268 T6423 T6422 prep to,response
R5269 T6398 T6397 punct (,data
R5270 T6424 T6426 preconj either,ester
R5271 T6425 T6426 compound phorbol,ester
R5272 T6399 T6400 compound Fig.,4A
R5273 T6426 T6423 pobj ester,to
R5274 T6427 T6426 cc or,ester
R5275 T6428 T6429 compound BCR,stimulation
R5276 T6429 T6426 conj stimulation,ester
R5277 T6430 T6402 punct .,show
R5278 T6431 T6442 prep In,abolished
R5279 T6432 T6431 pobj contrast,In
R5280 T6400 T6397 appos 4A,data
R5281 T6433 T6442 punct ",",abolished
R5282 T6434 T6440 nmod BCR,activity
R5283 T6401 T6397 punct ),data
R5284 T6435 T6434 cc and,BCR
R5285 T6436 T6440 amod phorbol,activity
R5286 T6437 T6440 amod ester-induced,activity
R5287 T6438 T6440 nmod NFκB,activity
R5288 T6439 T6440 amod transcriptional,activity
R5289 T6402 T6402 ROOT show,show
R5290 T6440 T6442 nsubjpass activity,abolished
R5291 T6441 T6442 auxpass was,abolished
R5292 T6403 T6409 mark that,induced
R5293 T6442 T6442 ROOT abolished,abolished
R5294 T6443 T6442 prep in,abolished
R5295 T6444 T6449 compound PKCβ,B
R5296 T6404 T6406 nmod NFκB,activity
R5297 T6445 T6449 compound −,B
R5298 T6446 T6449 compound /,B
R5299 T6447 T6449 compound −,B
R5300 T6405 T6406 amod transcriptional,activity
R5301 T6448 T6449 compound DT40,B
R5302 T6449 T6450 compound B,cells
R5304 T6450 T6443 pobj cells,in
R5305 T6451 T6442 punct (,abolished
R5870 T7383 T7384 auxpass been,proposed
R5871 T7384 T7384 ROOT proposed,proposed
R5872 T7385 T7386 aux to,regulate
R5873 T7386 T7384 xcomp regulate,proposed
R5874 T7387 T7389 amod diverse,functions
R5875 T7388 T7389 amod cellular,functions
R5876 T7389 T7386 dobj functions,regulate
R5877 T7390 T7389 prep including,functions
R5913 T7426 T7427 compound NFκB,transcription
R5914 T7427 T7428 compound transcription,factors
R5915 T7428 T7425 dobj factors,regulating
R5916 T7429 T7431 nmod [,]
R5917 T7430 T7431 nummod 41,]
R5918 T7431 T7428 appos ],factors
R5919 T7432 T7410 punct .,suggested
R5920 T7433 T7443 nsubj Most,come
R5921 T7434 T7433 prep of,Most
R5922 T7435 T7436 det the,data
R5923 T7436 T7434 pobj data,of
R5924 T7437 T7436 prep about,data
R5925 T7438 T7439 det the,function
R5926 T7439 T7437 pobj function,about
R5927 T7440 T7439 prep of,function
R5928 T7441 T7440 pobj PKDs,of
R5929 T7442 T7443 aux has,come
R5930 T7443 T7443 ROOT come,come
R5931 T7444 T7443 prep from,come
R5932 T7445 T7444 pobj experiments,from
R5933 T7446 T7448 nsubj that,express
R5934 T7447 T7448 advmod ectopically,express
R5935 T7448 T7445 relcl express,experiments
R5936 T7449 T7453 amod active,mutants
R5937 T7450 T7449 cc or,active
R5938 T7451 T7449 conj inhibitory,active
R5939 T7452 T7453 compound PKD,mutants
R5968 T7481 T7470 conj use,delete
R5969 T7482 T7483 compound PKD-null,DT40
R5970 T7483 T7484 compound DT40,cells
R5971 T7484 T7481 dobj cells,use
R5972 T7485 T7486 aux to,assess
R5973 T7486 T7481 xcomp assess,use
R5974 T7487 T7489 det the,contribution
R5975 T7488 T7489 amod relative,contribution
R5976 T7489 T7486 dobj contribution,assess
R5977 T7490 T7489 prep of,contribution
R5978 T7491 T7493 amod individual,isoforms
R5979 T7492 T7493 compound PKD,isoforms
R5980 T7493 T7490 pobj isoforms,of
R5981 T7494 T7493 prep in,isoforms
R5982 T7495 T7494 pobj class,in
R5983 T7496 T7497 compound II,HDAC
R5984 T7497 T7498 compound HDAC,control
R5985 T7498 T7489 conj control,contribution
R5986 T7499 T7498 prep versus,control
R5987 T7500 T7502 amod oxidative,responses
R5988 T7501 T7502 compound stress,responses
R5989 T7502 T7499 pobj responses,versus
R5990 T7503 T7502 cc and,responses
R5991 T7504 T7505 compound NFκB,regulation
R5992 T7505 T7502 conj regulation,responses
R5993 T7506 T7505 prep in,regulation
R5994 T7507 T7506 pobj lymphocytes,in
R5995 T7508 T7465 punct .,used
R5996 T7509 T7512 nsubj We,used
R5997 T7510 T7512 aux have,used
R5998 T7511 T7512 advmod previously,used
R5999 T7512 T7512 ROOT used,used
R6000 T7513 T7516 det these,cells
R6001 T7514 T7515 compound PKD-null,DT40
R6002 T7515 T7516 compound DT40,cells
R6003 T7516 T7512 dobj cells,used
R6004 T7517 T7518 aux to,define
R6005 T7518 T7512 xcomp define,used
R6006 T7519 T7521 det an,role
R6007 T7520 T7521 amod essential,role
R6008 T7521 T7518 dobj role,define
R6009 T7522 T7521 prep for,role
R6010 T7523 T7522 pobj PKDs,for
R6011 T7524 T7523 prep in,PKDs
R6012 T7525 T7524 pobj regulation,in
R6013 T7526 T7525 prep of,regulation
R6014 T7527 T7529 compound class,HDACs
R6015 T7528 T7529 compound II,HDACs
R6016 T7529 T7526 pobj HDACs,of
R6017 T7530 T7535 punct ",",describes
R6018 T7531 T7533 det the,report
R6019 T7532 T7533 amod present,report
R6020 T7533 T7535 nsubj report,describes
R6021 T7534 T7535 advmod now,describes
R6022 T7535 T7512 conj describes,used
R6023 T7536 T7538 det an,role
R6024 T7537 T7538 amod indispensable,role
R6025 T7538 T7535 dobj role,describes
R6026 T7539 T7538 prep for,role
R6027 T7540 T7539 pobj PKDs,for
R6028 T7541 T7540 prep in,PKDs
R6029 T7542 T7541 pcomp regulating,in
R6030 T7543 T7544 det the,phosphorylation
R6031 T7544 T7542 dobj phosphorylation,regulating
R6032 T7545 T7544 prep of,phosphorylation
R6033 T7546 T7545 pobj HSP27,of
R6034 T7547 T7544 prep on,phosphorylation
R6035 T7548 T7547 pobj serine,on
R6036 T7549 T7548 nummod 82,serine
R6037 T7550 T7548 punct ",",serine
R6038 T7551 T7552 det a,site
R6039 T7552 T7542 dobj site,regulating
R6040 T7553 T7554 advmod previously,identified
R6041 T7554 T7552 acl identified,site
R6042 T7555 T7554 prep as,identified
R6043 T7556 T7557 det a,target
R6044 T7557 T7555 pobj target,as
R6045 T7558 T7557 prep for,target
R6046 T7559 T7562 det the,cascade
R6047 T7560 T7561 advmod p38-MAPKAPK2,signalling
R6048 T7561 T7562 amod signalling,cascade
R6049 T7562 T7558 pobj cascade,for
R6050 T7563 T7565 nmod [,]
R6051 T7564 T7565 nummod 42,]
R6052 T7565 T7562 appos ],cascade
R6053 T7566 T7535 punct .,describes
R6054 T7567 T7574 advmod However,reveal
R6055 T7568 T7574 punct ",",reveal
R6056 T7569 T7574 nsubj studies,reveal
R6057 T7570 T7569 prep of,studies
R6058 T7571 T7572 compound PKD-null,DT40
R6059 T7572 T7573 compound DT40,cells
R6060 T7573 T7570 pobj cells,of
R6061 T7574 T7574 ROOT reveal,reveal
R6062 T7575 T7579 mark that,are
R6063 T7576 T7578 compound PKD,kinases
R6064 T7577 T7578 compound family,kinases
R6065 T7578 T7579 nsubj kinases,are
R6066 T7579 T7574 ccomp are,reveal
R6067 T7580 T7579 neg not,are
R6068 T7581 T7579 acomp essential,are
R6069 T7582 T7581 prep for,essential
R6070 T7583 T7586 amod oxidative,responses
R6071 T7584 T7585 compound stress,survival
R6072 T7585 T7586 compound survival,responses
R6073 T7586 T7582 pobj responses,for
R6074 T7587 T7579 cc nor,are
R6075 T7588 T7590 auxpass are,required
R6076 T7589 T7590 nsubjpass they,required
R6077 T7590 T7579 conj required,are
R6078 T7591 T7590 prep for,required
R6079 T7592 T7591 pobj activation,for
R6080 T7593 T7592 prep of,activation
R6081 T7594 T7596 compound NFκB,factors
R6082 T7595 T7596 compound transcription,factors
R6083 T7596 T7593 pobj factors,of
R6084 T7597 T7574 punct .,reveal
R6085 T7598 T7600 det These,findings
R6086 T7599 T7600 amod latter,findings
R6087 T7600 T7601 nsubj findings,are
R6088 T7601 T7601 ROOT are,are
R6089 T7602 T7601 prep in,are
R6090 T7603 T7604 amod striking,contrast
R6091 T7604 T7602 pobj contrast,in
R6092 T7605 T7604 prep to,contrast
R6093 T7606 T7607 amod previous,observations
R6094 T7607 T7605 pobj observations,to
R6095 T7608 T7607 prep in,observations
R6096 T7609 T7613 nmod HeLa,lines
R6097 T7610 T7609 cc and,HeLa
R6098 T7611 T7613 amod epithelial,lines
R6099 T7612 T7613 compound cell,lines
R6100 T7613 T7608 pobj lines,in
R6101 T7614 T7618 advmod where,implicated
R6102 T7615 T7616 compound overexpression/RNAi,approaches
R6103 T7616 T7618 nsubj approaches,implicated
R6104 T7617 T7618 aux have,implicated
R6105 T7618 T7613 relcl implicated,lines
R6106 T7619 T7618 dobj PKD1/2,implicated
R6107 T7620 T7618 prep in,implicated
R6108 T7621 T7622 det the,control
R6109 T7622 T7620 pobj control,in
R6110 T7623 T7622 prep of,control
R6111 T7624 T7623 pobj proliferation,of
R6112 T7625 T7624 punct ",",proliferation
R6113 T7626 T7624 conj survival,proliferation
R6114 T7627 T7626 cc and,survival
R6115 T7628 T7629 compound NFκB,activation
R6116 T7629 T7626 conj activation,survival
R6117 T7630 T7632 compound [,]
R6118 T7631 T7632 compound "20,23",]
R6119 T7632 T7622 appos ],control
R6120 T7633 T7601 punct .,are
R6121 T7634 T7639 advmod Hence,shows
R6122 T7635 T7639 punct ",",shows
R6123 T7636 T7638 det the,report
R6124 T7637 T7638 amod present,report
R6125 T7638 T7639 nsubj report,shows
R6126 T7639 T7639 ROOT shows,shows
R6127 T7640 T7664 mark that,are
R6128 T7641 T7643 det the,roles
R6129 T7642 T7643 amod proposed,roles
R6130 T7643 T7664 nsubj roles,are
R6131 T7644 T7643 prep for,roles
R6132 T7645 T7644 pobj PKDs,for
R6133 T7646 T7664 mark as,are
R6134 T7647 T7648 amod key,sensors
R6135 T7648 T7646 pobj sensors,as
R6136 T7649 T7650 nsubj that,modulate
R6137 T7650 T7648 relcl modulate,sensors
R6138 T7651 T7652 compound survival,pathways
R6139 T7652 T7650 dobj pathways,modulate
R6140 T7653 T7650 prep in,modulate
R6141 T7654 T7653 pobj response,in
R6142 T7655 T7654 prep to,response
R6143 T7656 T7657 amod oxidative,stress
R6144 T7657 T7655 pobj stress,to
R6145 T7658 T7650 cc and,modulate
R6146 T7659 T7650 conj regulate,modulate
R6147 T7660 T7661 compound cell,survival
R6148 T7661 T7659 dobj survival,regulate
R6149 T7662 T7661 cc and,survival
R6150 T7663 T7661 conj proliferation,survival
R6151 T7664 T7639 ccomp are,shows
R6152 T7665 T7664 neg not,are
R6153 T7666 T7664 acomp ubiquitous,are
R6154 T7667 T7664 cc and,are
R6155 T7668 T7670 aux may,restricted
R6156 T7669 T7670 auxpass be,restricted
R6157 T7670 T7664 conj restricted,are
R6158 T7671 T7670 prep to,restricted
R6159 T7672 T7674 amod certain,lineages
R6160 T7673 T7674 compound cell,lineages
R6161 T7674 T7671 pobj lineages,to
R6162 T7675 T7639 punct .,shows
R6163 T7676 T7681 advcl Taken,indicate
R6164 T7677 T7676 advmod together,Taken
R6165 T7678 T7681 punct ",",indicate
R6166 T7679 T7680 det these,data
R6167 T7680 T7681 nsubj data,indicate
R6168 T7681 T7681 ROOT indicate,indicate
R6169 T7682 T7693 mark that,impact
R6170 T7683 T7693 nsubj loss,impact
R6171 T7684 T7683 prep of,loss
R6172 T7685 T7684 pobj expression,of
R6173 T7686 T7685 prep of,expression
R6174 T7687 T7689 compound PKD,members
R6175 T7688 T7689 compound family,members
R6176 T7689 T7686 pobj members,of
R6177 T7690 T7693 aux does,impact
R1050 T1164 T1166 amod numerous,functions
R1051 T1165 T1166 amod cellular,functions
R1052 T1166 T1163 dobj functions,regulate
R1053 T1167 T1166 punct ",",functions
R1054 T1168 T1166 prep including,functions
R1055 T1169 T1170 compound cell,proliferation
R1056 T1170 T1168 pobj proliferation,including
R1057 T1171 T1170 dobj [,proliferation
R1058 T1172 T1171 nummod 13,[
R1059 T1173 T1174 nummod 16,]
R1060 T1174 T1171 appos ],[
R1061 T1175 T1177 punct ",",signals
R1131 T1245 T1249 det Another,substrate
R1132 T1246 T1247 advmod recently,proposed
R1133 T1247 T1249 amod proposed,substrate
R1134 T1248 T1249 compound PKD1,substrate
R1135 T1249 T1250 nsubj substrate,is
R1136 T1250 T1250 ROOT is,is
R1137 T1251 T1252 compound HSP27,[
R1138 T1252 T1250 attr [,is
R1139 T1253 T1254 nummod 24,]
R1140 T1254 T1252 appos ],[
R1141 T1255 T1254 punct ",",]
R1142 T1256 T1260 det a,protein
R1143 T1257 T1260 amod small,protein
R1144 T1258 T1259 compound heat,shock
R1145 T1259 T1260 compound shock,protein
R1146 T1260 T1254 appos protein,]
R1147 T1261 T1260 amod involved,protein
R1148 T1262 T1261 prep in,involved
R1149 T1263 T1262 pcomp regulating,in
R1150 T1264 T1265 compound cell,migration
R1151 T1265 T1263 dobj migration,regulating
R1152 T1266 T1265 cc and,migration
R1153 T1267 T1268 compound cell,survival
R1154 T1268 T1265 conj survival,migration
R1155 T1269 T1271 nmod [,]
R1156 T1270 T1271 nummod 25,]
R1157 T1271 T1265 appos ],migration
R1158 T1272 T1250 punct .,is
R1159 T1273 T1275 det An,role
R1160 T1274 T1275 amod essential,role
R1161 T1275 T1289 nsubj role,enzymes
R1162 T1276 T1275 prep for,role
R1163 T1277 T1278 compound PKD,enzymes
R1164 T1278 T1276 pobj enzymes,for
R1165 T1279 T1278 prep in,enzymes
R1166 T1280 T1279 pcomp regulating,in
R1167 T1281 T1284 nmod class,deacetylases
R1168 T1282 T1281 nummod II,class
R1169 T1283 T1284 compound histone,deacetylases
R1170 T1284 T1280 dobj deacetylases,regulating
R1171 T1285 T1284 punct (,deacetylases
R1172 T1286 T1284 appos HDACs,deacetylases
R1173 T1287 T1284 punct ),deacetylases
R1174 T1288 T1278 punct ",",enzymes
R1177 T1291 T1289 ccomp repress,enzymes
R1178 T1292 T1294 amod MEF2-dependent,transcription
R1179 T1293 T1294 compound gene,transcription
R1180 T1294 T1291 dobj transcription,repress
R1181 T1295 T1299 punct ",",demonstrated
R1182 T1296 T1299 aux has,demonstrated
R1183 T1297 T1299 advmod also,demonstrated
R1184 T1298 T1299 auxpass been,demonstrated
R1185 T1299 T1289 conj demonstrated,enzymes
R1186 T1300 T1301 compound [,"1,26"
R1187 T1301 T1299 dobj "1,26",demonstrated
R1188 T1302 T1303 nummod 28,]
R1189 T1303 T1299 npadvmod ],demonstrated
R1190 T1304 T1299 punct .,demonstrated
R1191 T1305 T1306 aux To,investigate
R1192 T1306 T1314 advcl investigate,generated
R1193 T1307 T1309 det the,role
R1194 T1308 T1309 amod biological,role
R1195 T1309 T1306 dobj role,investigate
R1260 T1448 T1449 amod transcriptional,activity
R1262 T1449 T1446 dobj activity,regulating
R1263 T1450 T1441 punct .,play
R1265 T1451 T1455 advmod Together,reveal
R1266 T1452 T1455 punct ",",reveal
R1268 T1453 T1454 det these,findings
R1269 T1454 T1455 nsubj findings,reveal
R1270 T1455 T1455 ROOT reveal,reveal
R1271 T1456 T1463 mark that,are
R1272 T1457 T1463 prep in,are
R1273 T1458 T1459 compound B,lymphocytes
R1274 T1459 T1457 pobj lymphocytes,in
R1276 T1460 T1463 punct ",",are
R1277 T1461 T1462 compound PKD,kinases
R1278 T1462 T1463 nsubj kinases,are
R1280 T1463 T1455 ccomp are,reveal
R1281 T1464 T1463 neg not,are
R1282 T1465 T1466 amod critical,regulators
R1283 T1466 T1463 attr regulators,are
R1285 T1467 T1466 prep of,regulators
R1286 T1468 T1467 pobj many,of
R1287 T1469 T1468 prep of,many
R1289 T1470 T1472 det the,processes
R1290 T1471 T1472 amod cellular,processes
R1291 T1472 T1469 pobj processes,of
R1293 T1473 T1474 advmod previously,ascribed
R1294 T1474 T1463 conj ascribed,are
R1295 T1475 T1474 prep to,ascribed
R1297 T1476 T1475 pobj them,to
R1502 T1742 T1744 nummod "Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 ",culture
R1503 T1743 T1744 compound Cell,culture
R1504 T1744 T1744 ROOT culture,culture
R1505 T1745 T1744 punct ",",culture
R1506 T1746 T1747 compound transient,transfections
R1507 T1747 T1744 appos transfections,culture
R1508 T1748 T1747 cc and,transfections
R1509 T1749 T1750 compound cell,stimulation
R1510 T1750 T1747 conj stimulation,transfections
R1511 T1751 T1752 det The,generation
R1512 T1752 T1765 nsubj generation,/
R1513 T1753 T1752 punct ",",generation
R1514 T1754 T1752 conj culture,generation
R1515 T1755 T1754 cc and,culture
R1046 T1160 T1161 auxpass been,proposed
R1047 T1161 T1161 ROOT proposed,proposed
R1048 T1162 T1163 aux to,regulate
R1049 T1163 T1161 xcomp regulate,proposed
R1128 T1242 T1243 nummod 23,]
R1129 T1243 T1243 ROOT ],]
R1130 T1244 T1223 punct .,linked
R1516 T1756 T1754 conj activation,culture
R1517 T1757 T1752 prep of,generation
R1518 T1758 T1761 nummod PKD1,−
R1672 T1948 T1949 nummod 106,cells
R1673 T1949 T1944 appos cells,cells
R1674 T1950 T1949 prep per,cells
R1675 T1951 T1950 pobj point,per
R1676 T1952 T1944 punct ),cells
R2447 T2898 T2897 prep upon,restored
R2448 T2899 T2898 pobj re-expression,upon
R2449 T2900 T2899 prep of,re-expression
R2450 T2901 T2904 det a,isoform
R2452 T2903 T2904 compound PKD,isoform
R2453 T2904 T2900 pobj isoform,of
R2454 T2905 T2907 nmod [,]
R2455 T2906 T2907 nummod 1,]
R2456 T2907 T2869 dobj ],demonstrated
R2457 T2908 T2869 punct .,demonstrated
R2458 T2909 T2913 det The,protein
R2459 T2910 T2913 amod small,protein
R2460 T2911 T2912 compound heat,shock
R2461 T2912 T2913 compound shock,protein
R2462 T2913 T2918 nsubj protein,proposed
R2463 T2914 T2913 appos HSP27,protein
R2464 T2915 T2918 aux has,proposed
R2465 T2916 T2918 advmod recently,proposed
R2466 T2917 T2918 auxpass been,proposed
R2467 T2918 T2918 ROOT proposed,proposed
R2468 T2919 T2918 prep as,proposed
R2469 T2920 T2922 det a,substrate
R2470 T2921 T2922 compound PKD1,substrate
R2471 T2922 T2919 pobj substrate,as
R2472 T2923 T2925 nmod [,]
R2473 T2924 T2925 nummod 24,]
R2474 T2925 T2922 dep ],substrate
R2475 T2926 T2922 cc and,substrate
R2476 T2927 T2929 nsubj we,assessed
R2477 T2928 T2929 advmod accordingly,assessed
R2478 T2929 T2922 conj assessed,substrate
R2479 T2930 T2934 mark whether,have
R2480 T2931 T2932 compound PKD-null,DT40
R2481 T2932 T2933 compound DT40,cells
R2482 T2933 T2934 nsubj cells,have
R2483 T2934 T2929 ccomp have,assessed
R2484 T2935 T2936 amod defective,phosphorylation
R2485 T2936 T2934 dobj phosphorylation,have
R2486 T2937 T2936 prep of,phosphorylation
R2487 T2938 T2937 pobj HSP27,of
R3651 T4452 T4450 conj proliferation,survival
R3652 T4453 T4452 prep of,proliferation
R3653 T4454 T4455 compound DT40,B
R3654 T4455 T4456 compound B,cells
R3655 T4456 T4453 pobj cells,of
R3656 T4457 T4444 punct .,are
R3657 T4458 T4459 compound PKD,enzymes
R175 T212 T215 compound Protein,enzymes
R176 T213 T215 compound kinase,enzymes
R177 T214 T215 compound D,enzymes
R178 T215 T216 nsubj enzymes,are
R179 T216 T216 ROOT are,are
R180 T217 T216 acomp dispensable,are
R181 T218 T217 prep for,dispensable
R182 T219 T226 nmod proliferation,activity
R183 T220 T219 punct ",",proliferation
R184 T221 T219 conj survival,proliferation
R185 T222 T221 cc and,survival
R186 T223 T221 conj antigen,survival
R187 T224 T226 amod receptor-regulated,activity
R188 T225 T226 compound NFκB,activity
R189 T226 T218 pobj activity,for
R190 T227 T226 prep in,activity
R191 T228 T229 amod vertebrate,B-cells
R192 T229 T227 pobj B-cells,in
R193 T230 T231 aux To,investigate
R194 T231 T216 advcl investigate,are
R195 T232 T233 det the,importance
R196 T233 T231 dobj importance,investigate
R197 T234 T233 prep of,importance
R198 T235 T234 pobj protein,of
R199 T236 T237 compound kinase,D
R200 T237 T235 appos D,protein
R201 T238 T237 punct (,D
R202 T239 T237 appos PKD,D
R203 T240 T237 punct ),D
R4850 T5997 T6001 nsubj interpretation,is
R4851 T5998 T5997 prep of,interpretation
R4852 T5999 T6000 det this,data
R4853 T6000 T5998 pobj data,of
R4931 T6078 T6079 compound Flag-PKD3,+
R4932 T6079 T6074 appos +,−
R4933 T6080 T6074 punct ),−
R4934 T6081 T6082 amod B,cells
R4935 T6082 T6085 nsubj cells,impact
R4936 T6083 T6085 aux may,impact
R4937 T6084 T6085 nsubj itself,impact
R4938 T6085 T6085 ROOT impact,impact
R5079 T6226 T6227 nsubj that,contain
R5080 T6227 T6225 relcl contain,proteins
R5081 T6228 T6229 compound LxRxxpS/T,motifs
R5082 T6229 T6227 dobj motifs,contain
R5083 T6230 T6220 punct .,disrupt
R5084 T6231 T6232 det This,result
R5085 T6232 T6233 nsubj result,is
R5086 T6233 T6233 ROOT is,is
R5087 T6234 T6236 advmod perhaps,surprising
R5088 T6235 T6236 neg not,surprising
R5089 T6236 T6233 acomp surprising,is
R5090 T6237 T6241 mark as,act
R5091 T6238 T6239 compound LxRxxS/T,motifs
R5092 T6239 T6241 nsubj motifs,act
R5093 T6240 T6241 advmod also,act
R5094 T6241 T6236 advcl act,surprising
R5095 T6242 T6241 prep as,act
R5096 T6243 T6244 amod good,substrates
R5097 T6244 T6242 pobj substrates,as
R5098 T6245 T6244 prep for,substrates
R5099 T6246 T6248 amod other,kinases
R5100 T6247 T6248 amod serine/threonine,kinases
R5101 T6248 T6245 pobj kinases,for
R5102 T6249 T6250 amod such,as
R5103 T6250 T6248 prep as,kinases
R5104 T6251 T6250 pobj MAPKAPK2,as
R5105 T6252 T6233 punct .,is
R5106 T6253 T6257 advmod However,provide
R5107 T6254 T6255 det these,experiments
R5108 T6255 T6257 nsubj experiments,provide
R5109 T6256 T6257 aux do,provide
R5110 T6257 T6257 ROOT provide,provide
R5111 T6258 T6259 amod further,evidence
R5112 T6259 T6257 dobj evidence,provide
R5113 T6260 T6263 mark that,are
R5114 T6261 T6262 amod phosphospecific,antisera
R5115 T6262 T6263 nsubj antisera,are
R5116 T6263 T6259 acl are,evidence
R5117 T6264 T6263 neg not,are
R5118 T6265 T6266 advmod sufficiently,selective
R5119 T6266 T6263 acomp selective,are
R5120 T6267 T6269 aux to,designated
R5121 T6268 T6269 auxpass be,designated
R5122 T6269 T6266 xcomp designated,selective
R5123 T6270 T6269 oprd kinase,designated
R5124 T6271 T6273 amod specific,antisera
R5125 T6272 T6273 compound substrate,antisera
R5126 T6273 T6270 appos antisera,kinase
R5127 T6274 T6257 punct .,provide
R5128 T6275 T6277 amod BCR-induced,pathways
R5129 T6276 T6277 amod signalling,pathways
R5130 T6277 T6278 nsubj pathways,culminate
R5131 T6278 T6278 ROOT culminate,culminate
R5308 T6452 T6453 compound Fig.,4A
R5310 T6453 T6442 npadvmod 4A,abolished
R5311 T6454 T6442 punct ),abolished
R5312 T6455 T6442 punct ",",abolished
R5313 T6456 T6473 mark although,observed
R5315 T6457 T6458 amod strong,activation
R5316 T6458 T6473 nsubjpass activation,observed
R5318 T6459 T6458 prep of,activation
R5319 T6460 T6461 compound PKD,kinases
R5320 T6461 T6459 pobj kinases,of
R5322 T6462 T6464 punct (,assessed
R5323 T6463 T6464 mark as,assessed
R5325 T6464 T6473 advcl assessed,observed
R5326 T6465 T6464 agent by,assessed
R5327 T6466 T6465 pobj autophosphorylation,by
R5328 T6467 T6466 prep of,autophosphorylation
R5329 T6468 T6467 pobj PKD1,of
R5330 T6469 T6466 prep at,autophosphorylation
R5331 T6470 T6469 pobj S916,at
R5332 T6471 T6464 punct ),assessed
R5333 T6472 T6473 auxpass was,observed
R5335 T6473 T6442 advcl observed,abolished
R5336 T6474 T6473 prep in,observed
R5338 T6475 T6477 det the,−
R5339 T6476 T6477 compound PKCβ,−
R5340 T6477 T6474 pobj −,in
R5341 T6478 T6442 conj /,abolished
R5343 T6479 T6480 nummod −,cells
R5344 T6480 T6478 dobj cells,/
R5345 T6481 T6483 punct (,4B
R5347 T6482 T6483 compound Fig.,4B
R5348 T6483 T6478 parataxis 4B,/
R5349 T6484 T6483 punct ),4B
R5351 T6485 T6442 punct .,abolished
R5303 T6503 T6500 pobj cells,in
R5306 T6504 T6496 cc and,mediate
R5307 T6505 T6508 advmod hence,participate
R5309 T6506 T6508 aux do,participate
R5314 T6507 T6508 neg not,participate
R5317 T6508 T6496 conj participate,mediate
R5321 T6509 T6508 prep in,participate
R5324 T6510 T6509 pobj DAG/PKC,in
R5334 T6511 T6490 conj mediated,are
R5337 T6512 T6511 dobj control,mediated
R5342 T6513 T6512 prep of,control
R5346 T6514 T6513 pobj NFκB,of
R5350 T6515 T6490 punct .,are
R5353 T6486 T6490 advmod Thus,are
R5354 T6487 T6490 punct ",",are
R5355 T6488 T6489 compound PKD,kinases
R5356 T6489 T6490 nsubj kinases,are
R5357 T6490 T6490 ROOT are,are
R5358 T6491 T6492 advmod neither,essential
R5359 T6492 T6490 acomp essential,are
R5360 T6493 T6492 cc nor,essential
R5361 T6494 T6492 conj sufficient,essential
R5362 T6495 T6496 aux to,mediate
R5363 T6496 T6494 xcomp mediate,sufficient
R5364 T6497 T6499 amod BCR-induced,activation
R5365 T6498 T6499 compound NFκB,activation
R5366 T6499 T6496 dobj activation,mediate
R5367 T6500 T6499 prep in,activation
R5368 T6501 T6502 compound DT40,B
R5369 T6502 T6503 compound B,cells
R5864 T7377 T7381 amod "40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway. To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera. BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor. Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38]. In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored. The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB. 4 Discussion Protein",kinases
R5865 T7378 T7381 compound kinase,kinases
R5866 T7379 T7381 compound D,kinases
R5867 T7380 T7381 compound serine,kinases
R5868 T7381 T7384 nsubjpass kinases,proposed
R5869 T7382 T7384 aux have,proposed
R5878 T7391 T7392 det the,phosphorylation
R5879 T7392 T7390 pobj phosphorylation,including
R5880 T7393 T7392 cc and,phosphorylation
R5881 T7394 T7395 amod nuclear,localisation
R5882 T7395 T7392 conj localisation,phosphorylation
R5883 T7396 T7395 prep of,localisation
R5884 T7397 T7399 compound class,HDACs
R5885 T7398 T7399 compound II,HDACs
R5886 T7399 T7396 pobj HDACs,of
R5887 T7400 T7399 cc and,HDACs
R5888 T7401 T7402 det the,phosphorylation
R5889 T7402 T7399 conj phosphorylation,HDACs
R5890 T7403 T7402 prep of,phosphorylation
R5891 T7404 T7403 pobj HSP27,of
R5892 T7405 T7384 punct .,proposed
R5893 T7406 T7410 nsubj It,suggested
R5894 T7407 T7410 aux has,suggested
R5895 T7408 T7410 advmod also,suggested
R5896 T7409 T7410 auxpass been,suggested
R5897 T7410 T7410 ROOT suggested,suggested
R5898 T7411 T7413 mark that,act
R5899 T7412 T7413 nsubj PKDs,act
R5900 T7413 T7410 ccomp act,suggested
R5901 T7414 T7413 prep as,act
R5902 T7415 T7416 amod mitochondrial,sensors
R5903 T7416 T7414 pobj sensors,as
R5904 T7417 T7416 prep for,sensors
R5905 T7418 T7419 amod oxidative,stress
R5906 T7419 T7417 pobj stress,for
R5907 T7420 T7413 cc and,act
R5908 T7421 T7413 conj play,act
R5909 T7422 T7423 det a,role
R5910 T7423 T7421 dobj role,play
R5911 T7424 T7423 prep in,role
R5912 T7425 T7424 pcomp regulating,in
R5940 T7453 T7448 dobj mutants,express
R5941 T7454 T7453 cc or,mutants
R5942 T7455 T7456 det that,use
R5943 T7456 T7453 conj use,mutants
R5944 T7457 T7456 dobj RNAi,use
R5945 T7458 T7459 aux to,reduce
R5946 T7459 T7456 xcomp reduce,use
R5947 T7460 T7461 compound PKD,expression
R5948 T7461 T7459 dobj expression,reduce
R5949 T7462 T7443 punct .,come
R5950 T7463 T7465 nsubj We,used
R5951 T7464 T7465 aux have,used
R5952 T7465 T7465 ROOT used,used
R5953 T7466 T7465 dobj gene,used
R5954 T7467 T7466 acl targeting,gene
R5955 T7468 T7470 aux to,delete
R5956 T7469 T7470 advmod specifically,delete
R5957 T7470 T7467 xcomp delete,targeting
R5958 T7471 T7472 compound PKD,alleles
R5959 T7472 T7470 dobj alleles,delete
R5960 T7473 T7470 prep in,delete
R5961 T7474 T7477 compound DT40,cells
R5962 T7475 T7476 compound chicken,B
R5963 T7476 T7477 compound B,cells
R5964 T7477 T7473 pobj cells,in
R5965 T7478 T7470 cc and,delete
R5966 T7479 T7481 aux can,use
R5967 T7480 T7481 advmod thus,use
R6178 T7691 T7693 neg not,impact
R6179 T7692 T7693 advmod globally,impact
R6180 T7693 T7681 ccomp impact,indicate
R6181 T7694 T7693 prep on,impact
R6182 T7695 T7698 amod early,pathways
R6183 T7696 T7698 amod BCR-regulated,pathways
R6184 T7697 T7698 compound signalling,pathways
R6185 T7698 T7694 pobj pathways,on
R6186 T7699 T7681 punct .,indicate

UBERON-AE

Id Subject Object Predicate Lexical cue
T9 117-127 http://purl.obolibrary.org/obo/UBERON_3010224 denotes vertebrate
T1863 4244-4249 http://purl.obolibrary.org/obo/UBERON_0001977 denotes serum
T3782 8575-8580 http://purl.obolibrary.org/obo/UBERON_0001977 denotes serum

GO-BP

Id Subject Object Predicate Lexical cue
T504 1364-1374 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T505 2014-2021 http://purl.obolibrary.org/obo/GO_0023052 denotes signals
T506 1364-1382 http://purl.obolibrary.org/obo/GO_0007165 denotes signalling pathway
T507 1562-1565 http://purl.obolibrary.org/obo/GO_0004697 denotes PKC
T508 1941-1949 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T509 3342-3350 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T510 3625-3633 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T511 3681-3689 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T512 1971-1989 http://purl.obolibrary.org/obo/GO_0008283 denotes cell proliferation
T513 2044-2055 http://purl.obolibrary.org/obo/GO_0032502 denotes development
T514 2172-2185 http://purl.obolibrary.org/obo/GO_0007155 denotes cell adhesion
T515 2190-2203 http://purl.obolibrary.org/obo/GO_0048870 denotes cell motility
T516 2298-2311 http://purl.obolibrary.org/obo/GO_0006351 denotes transcription
T517 2660-2673 http://purl.obolibrary.org/obo/GO_0006351 denotes transcription
T518 3489-3504 http://purl.obolibrary.org/obo/GO_0006351 denotes transcriptional
T519 2451-2469 http://purl.obolibrary.org/obo/GO_0006986 denotes heat shock protein
T520 2451-2469 http://purl.obolibrary.org/obo/GO_0034620 denotes heat shock protein
T521 2451-2469 http://purl.obolibrary.org/obo/GO_0042026 denotes heat shock protein
T522 2493-2507 http://purl.obolibrary.org/obo/GO_0016477 denotes cell migration
T523 3132-3142 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T524 3217-3232 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T525 3390-3416 http://purl.obolibrary.org/obo/GO_1902883 denotes oxidative stress responses
T526 3390-3416 http://purl.obolibrary.org/obo/GO_0006979 denotes oxidative stress responses
T527 3390-3416 http://purl.obolibrary.org/obo/GO_0097468 denotes oxidative stress responses
T528 3390-3416 http://purl.obolibrary.org/obo/GO_1902884 denotes oxidative stress responses
T529 3390-3416 http://purl.obolibrary.org/obo/GO_0034599 denotes oxidative stress responses
T530 3400-3416 http://purl.obolibrary.org/obo/GO_0006950 denotes stress responses
T531 3625-3643 http://purl.obolibrary.org/obo/GO_0009987 denotes cellular processes
T2272 4561-4576 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2273 5322-5337 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2274 5680-5695 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2275 5807-5822 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2276 5996-6011 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2277 6109-6124 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2278 6221-6236 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2279 6475-6490 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2280 6657-6672 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2281 6731-6746 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T2282 5532-5550 http://purl.obolibrary.org/obo/GO_0006986 denotes heat shock protein
T2283 5532-5550 http://purl.obolibrary.org/obo/GO_0034620 denotes heat shock protein
T3833 8362-8390 http://purl.obolibrary.org/obo/GO_1902884 denotes response to oxidative stress
T3834 8362-8390 http://purl.obolibrary.org/obo/GO_0034599 denotes response to oxidative stress
T3835 8525-8557 http://purl.obolibrary.org/obo/GO_1990046 denotes response to mitochondrial stress
T6532 10874-10889 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T6533 10994-11009 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T6534 11430-11445 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T6535 9682-9686 http://purl.obolibrary.org/obo/GO_0004707 denotes MAPK
T6536 9682-9697 http://purl.obolibrary.org/obo/GO_0000165 denotes MAPK signalling
T6537 9823-9831 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T6538 11013-11021 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T7016 13237-13245 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T7017 13270-13285 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T7018 13337-13352 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T7019 14168-14183 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T7020 13298-13310 http://purl.obolibrary.org/obo/GO_0051179 denotes localisation
T7021 13485-13498 http://purl.obolibrary.org/obo/GO_0006351 denotes transcription
T7022 14468-14481 http://purl.obolibrary.org/obo/GO_0006351 denotes transcription
T7023 13653-13657 http://purl.obolibrary.org/obo/GO_0016246 denotes RNAi
T7024 14618-14622 http://purl.obolibrary.org/obo/GO_0016246 denotes RNAi
T7025 13903-13929 http://purl.obolibrary.org/obo/GO_1902883 denotes oxidative stress responses
T7026 14843-14871 http://purl.obolibrary.org/obo/GO_1902883 denotes response to oxidative stress
T7027 13903-13929 http://purl.obolibrary.org/obo/GO_0006979 denotes oxidative stress responses
T7028 14843-14871 http://purl.obolibrary.org/obo/GO_0006979 denotes response to oxidative stress
T7029 13903-13929 http://purl.obolibrary.org/obo/GO_0097468 denotes oxidative stress responses
T7030 14843-14871 http://purl.obolibrary.org/obo/GO_0097468 denotes response to oxidative stress
T7031 13903-13929 http://purl.obolibrary.org/obo/GO_1902884 denotes oxidative stress responses
T7032 14843-14871 http://purl.obolibrary.org/obo/GO_1902884 denotes response to oxidative stress
T7033 13903-13929 http://purl.obolibrary.org/obo/GO_0034599 denotes oxidative stress responses
T7034 14843-14871 http://purl.obolibrary.org/obo/GO_0034599 denotes response to oxidative stress
T7035 13913-13929 http://purl.obolibrary.org/obo/GO_0006950 denotes stress responses
T7036 13939-13949 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T7037 14056-14066 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T7038 14256-14259 http://purl.obolibrary.org/obo/GO_0004707 denotes p38
T7039 14269-14279 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T7040 15114-15124 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T7041 14269-14287 http://purl.obolibrary.org/obo/GO_0007165 denotes signalling cascade
T7042 15114-15133 http://purl.obolibrary.org/obo/GO_0007165 denotes signalling pathways
T21 553-563 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T22 705-720 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T23 839-847 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T24 839-857 http://purl.obolibrary.org/obo/GO_0009987 denotes cellular processes
T25 912-927 http://purl.obolibrary.org/obo/GO_0006351 denotes transcriptional
T3820 6963-6973 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T3821 6963-6995 http://purl.obolibrary.org/obo/GO_0030888 denotes regulation of cell proliferation
T3822 6963-6995 http://purl.obolibrary.org/obo/GO_0051140 denotes regulation of cell proliferation
T3823 6963-6995 http://purl.obolibrary.org/obo/GO_0060251 denotes regulation of cell proliferation
T3824 6963-6995 http://purl.obolibrary.org/obo/GO_0070666 denotes regulation of cell proliferation
T3825 6963-6995 http://purl.obolibrary.org/obo/GO_1904073 denotes regulation of cell proliferation
T3826 6963-6995 http://purl.obolibrary.org/obo/GO_1904697 denotes regulation of cell proliferation
T3827 6963-6995 http://purl.obolibrary.org/obo/GO_1904441 denotes regulation of cell proliferation
T3828 6977-6995 http://purl.obolibrary.org/obo/GO_0008283 denotes cell proliferation
T3829 7271-7277 http://purl.obolibrary.org/obo/GO_0040007 denotes growth
T3830 8362-8390 http://purl.obolibrary.org/obo/GO_1902883 denotes response to oxidative stress
T3831 8362-8390 http://purl.obolibrary.org/obo/GO_0006979 denotes response to oxidative stress
T3832 8362-8390 http://purl.obolibrary.org/obo/GO_0097468 denotes response to oxidative stress
T2284 5532-5550 http://purl.obolibrary.org/obo/GO_0042026 denotes heat shock protein
T2285 5787-5797 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T2286 6455-6465 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T2287 6068-6075 http://purl.obolibrary.org/obo/GO_0023052 denotes signals
T6516 8969-8979 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T6517 9135-9145 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T6518 9490-9500 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T6519 9687-9697 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T6520 10081-10091 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T6521 10338-10348 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T6522 10683-10693 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T6523 11816-11826 http://purl.obolibrary.org/obo/GO_0023052 denotes signalling
T6524 12178-12185 http://purl.obolibrary.org/obo/GO_0023052 denotes signals
T6525 8969-8988 http://purl.obolibrary.org/obo/GO_0007165 denotes signalling pathways
T6526 9687-9706 http://purl.obolibrary.org/obo/GO_0007165 denotes signalling pathways
T6527 9490-9509 http://purl.obolibrary.org/obo/GO_0007165 denotes signalling cascades
T6528 9653-9676 http://purl.obolibrary.org/obo/GO_0006468 denotes protein phosphorylation
T6529 9661-9676 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T6530 9795-9810 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T6531 10424-10439 http://purl.obolibrary.org/obo/GO_0016310 denotes phosphorylation
T6539 11449-11457 http://purl.obolibrary.org/obo/GO_0007349 denotes cellular
T6540 10053-10063 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T6541 10769-10779 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T6542 12498-12508 http://purl.obolibrary.org/obo/GO_0065007 denotes regulation
T6543 10310-10316 http://purl.obolibrary.org/obo/GO_0040007 denotes growth
T6544 10669-10701 http://purl.obolibrary.org/obo/GO_0030522 denotes intracellular signalling pathway
T6545 10669-10701 http://purl.obolibrary.org/obo/GO_0035556 denotes intracellular signalling pathway
T6546 10669-10701 http://purl.obolibrary.org/obo/GO_1902532 denotes intracellular signalling pathway
T6547 10669-10701 http://purl.obolibrary.org/obo/GO_1902531 denotes intracellular signalling pathway
T6548 10669-10701 http://purl.obolibrary.org/obo/GO_1902533 denotes intracellular signalling pathway
T6549 11872-11885 http://purl.obolibrary.org/obo/GO_0006351 denotes transcription
T6550 12029-12042 http://purl.obolibrary.org/obo/GO_0006351 denotes transcription
T6551 12074-12087 http://purl.obolibrary.org/obo/GO_0006351 denotes transcription
T6552 12566-12581 http://purl.obolibrary.org/obo/GO_0006351 denotes transcriptional
T6553 12761-12776 http://purl.obolibrary.org/obo/GO_0006351 denotes transcriptional
T6554 13121-13124 http://purl.obolibrary.org/obo/GO_0004697 denotes PKC

GO-MF

Id Subject Object Predicate Lexical cue
T1483 1562-1565 http://purl.obolibrary.org/obo/GO_0004697 denotes PKC
T1484 1729-1736 http://purl.obolibrary.org/obo/GO_0005488 denotes binding
T1485 1755-1762 http://purl.obolibrary.org/obo/GO_0005488 denotes binding
T2014 4289-4297 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T7700 14256-14259 http://purl.obolibrary.org/obo/GO_0004707 denotes p38
T6581 9643-9660 http://purl.obolibrary.org/obo/GO_0003735 denotes ribosomal protein
T6582 9682-9686 http://purl.obolibrary.org/obo/GO_0004707 denotes MAPK
T6583 10839-10847 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T6584 11058-11066 http://purl.obolibrary.org/obo/GO_0003823 denotes antibody
T6585 13121-13124 http://purl.obolibrary.org/obo/GO_0004697 denotes PKC

GO-CC

Id Subject Object Predicate Lexical cue
T1499 2156-2161 http://purl.obolibrary.org/obo/GO_0005794 denotes Golgi
T1500 3625-3643 http://purl.obolibrary.org/obo/GO_0042995 denotes cellular processes
T1816 3773-3777 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1817 3879-3883 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T2015 4145-4150 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T2016 4160-4165 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T2017 4340-4345 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T2018 4289-4297 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T2019 4289-4297 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T4613 6869-6874 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4614 7159-7164 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4615 7310-7315 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4616 7383-7388 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4617 7475-7480 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4618 7563-7568 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4619 7647-7652 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4620 7701-7706 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4621 7826-7831 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4622 8076-8081 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4623 8516-8521 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T4624 8870-8874 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T4625 8643-8645 http://purl.obolibrary.org/obo/GO_0005783 denotes ER
T6555 9008-9013 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6556 9186-9191 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6557 9314-9319 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6558 9349-9354 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6559 9429-9434 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6560 9549-9554 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6561 9747-9752 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6562 9957-9962 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6563 10127-10132 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6564 10157-10162 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6565 10483-10488 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6566 10598-10603 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6567 10800-10805 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6568 11106-11111 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6569 11218-11223 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6570 12387-12392 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6571 12651-12656 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6572 12818-12823 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6573 12958-12963 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6574 13079-13084 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T6575 9867-9880 http://purl.obolibrary.org/obo/GO_0005622 denotes intracellular
T6576 10669-10682 http://purl.obolibrary.org/obo/GO_0005622 denotes intracellular
T6577 10839-10847 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T6578 11058-11066 http://purl.obolibrary.org/obo/GO_0019815 denotes antibody
T6579 10839-10847 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T6580 11058-11066 http://purl.obolibrary.org/obo/GO_0042571 denotes antibody
T1486 1157-1161 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1487 1971-1975 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1488 2172-2176 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1489 2190-2194 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1490 2337-2341 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1491 2493-2497 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1492 2512-2516 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1493 2781-2785 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T1494 2930-2934 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T3137 4587-4592 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3138 4641-4646 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3139 5098-5103 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3140 5163-5168 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3141 5451-5456 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3142 5659-5664 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3143 5849-5854 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3144 6039-6044 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3145 6164-6169 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3146 6272-6277 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3147 6416-6421 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3148 6591-6596 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T3149 6761-6766 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T7701 13765-13770 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T7702 13802-13807 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T7703 14010-14015 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T7704 14328-14333 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T7705 14586-14590 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T7706 14885-14889 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T7707 14969-14973 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T399 130-135 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T400 376-381 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T401 731-736 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T402 793-797 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T403 957-961 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T404 1023-1027 http://purl.obolibrary.org/obo/GO_0005623 denotes cell
T405 839-857 http://purl.obolibrary.org/obo/GO_0042995 denotes cellular processes
T1495 3148-3153 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T1496 3238-3243 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T1497 3270-3275 http://purl.obolibrary.org/obo/GO_0005623 denotes cells
T1498 3422-3427 http://purl.obolibrary.org/obo/GO_0005623 denotes cells

sentences

Id Subject Object Predicate Lexical cue
T17 615-646 Sentence denotes Mol. Cell Biol. 26, 1569–1577].
T489 635-1151 Sentence denotes 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3.
T490 1152-1351 Sentence denotes Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3].
T491 1352-1665 Sentence denotes A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6].
T492 1666-1888 Sentence denotes The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12].
T493 1889-2061 Sentence denotes PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19].
T494 2062-2223 Sentence denotes Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]).
T495 2224-2386 Sentence denotes In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23].
T496 2387-2531 Sentence denotes Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25].
T497 2532-2712 Sentence denotes An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28].
T498 2713-3066 Sentence denotes To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members.
T499 3067-3158 Sentence denotes Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1].
T500 3159-3244 Sentence denotes Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells.
T501 3245-3312 Sentence denotes However, PKD-null DT40 B cells are viable and proliferate normally.
T502 3313-3514 Sentence denotes Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity.
T503 3515-3698 Sentence denotes Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems.
T1670 0-3789 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation
T1671 3790-3925 Sentence denotes The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1].
T1672 3926-4038 Sentence denotes Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1].
T1673 4039-4117 Sentence denotes Chloramphenicol acetyl transferase assays have been described previously [29].
T1864 0-4137 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining
T1865 4138-4335 Sentence denotes DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice.
T1866 4336-4421 Sentence denotes The cells were washed twice and fluorescent intensity was analysed by flow cytometry.
T1867 4422-4528 Sentence denotes All results shown are representative of at two to four independent experiments unless otherwise indicated.
T2259 0-4633 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases
T2260 4634-4825 Sentence denotes DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1].
T2261 4826-5011 Sentence denotes In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter.
T2262 5012-5283 Sentence denotes Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A).
T2263 5284-5521 Sentence denotes Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1].
T2264 5522-5756 Sentence denotes The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence.
T2265 5757-5883 Sentence denotes We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3.
T2266 5884-6045 Sentence denotes As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells.
T2267 6046-6180 Sentence denotes BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B).
T2268 6181-6319 Sentence denotes However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C).
T2269 6320-6501 Sentence denotes Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C).
T2270 6502-6683 Sentence denotes In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D).
T2271 6684-6816 Sentence denotes Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases.
T3808 0-6915 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases
T3809 6916-7028 Sentence denotes PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]).
T3810 7029-7192 Sentence denotes To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−:
T3811 7193-7278 Sentence denotes Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth.
T3812 7279-7432 Sentence denotes As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation.
T3813 7433-7586 Sentence denotes Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown).
T3814 7587-7974 Sentence denotes It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes.
T3815 7975-8082 Sentence denotes Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells.
T3816 8083-8274 Sentence denotes PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32].
T3817 8275-8416 Sentence denotes We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli.
T3818 8417-8805 Sentence denotes As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs.
T3819 8806-8935 Sentence denotes Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli.
T5007 0-9013 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells
T5008 9014-9192 Sentence denotes To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells.
T5009 9193-9384 Sentence denotes Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown).
T5010 9385-9565 Sentence denotes Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B).
T5011 9566-9763 Sentence denotes Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B).
T5012 9764-9980 Sentence denotes Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown).
T5013 9981-10173 Sentence denotes We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B).
T5014 10174-10254 Sentence denotes One interpretation of this data is that PKD enzymes may modulate Erk activation.
T5015 10255-10398 Sentence denotes Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35].
T5016 10399-10702 Sentence denotes However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway.
T5017 10703-10941 Sentence denotes To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36].
T5018 10942-11156 Sentence denotes As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes.
T5019 11157-11371 Sentence denotes However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs.
T5020 11372-11497 Sentence denotes Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs.
T5021 11498-11635 Sentence denotes This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2.
T5022 11636-11803 Sentence denotes However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera.
T5023 11804-11950 Sentence denotes BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function.
T5024 11951-12095 Sentence denotes In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor.
T5025 12096-12284 Sentence denotes Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37].
T5026 12285-12381 Sentence denotes Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38].
T5027 12382-12531 Sentence denotes In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored.
T5028 12532-12712 Sentence denotes The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation.
T5029 12713-12974 Sentence denotes In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B).
T5030 12975-13150 Sentence denotes Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB.
T7007 237-13362 Sentence denotes 40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway. To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera. BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor. Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38]. In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored. The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB. 4 Discussion Protein kinase D serine kinases have been proposed to regulate diverse cellular functions including the phosphorylation and nuclear localisation of class II HDACs and the phosphorylation of HSP27.
T7008 13363-13512 Sentence denotes It has also been suggested that PKDs act as mitochondrial sensors for oxidative stress and play a role in regulating NFκB transcription factors [41].
T7009 13513-13683 Sentence denotes Most of the data about the function of PKDs has come from experiments that ectopically express active or inhibitory PKD mutants or that use RNAi to reduce PKD expression.
T7010 13684-13965 Sentence denotes We have used gene targeting to specifically delete PKD alleles in DT40 chicken B cells and can thus use PKD-null DT40 cells to assess the relative contribution of individual PKD isoforms in class II HDAC control versus oxidative stress responses and NFκB regulation in lymphocytes.
T7011 13966-14293 Sentence denotes We have previously used these PKD-null DT40 cells to define an essential role for PKDs in regulation of class II HDACs, the present report now describes an indispensable role for PKDs in regulating the phosphorylation of HSP27 on serine 82, a site previously identified as a target for the p38-MAPKAPK2 signalling cascade [42].
T7012 14294-14490 Sentence denotes However, studies of PKD-null DT40 cells reveal that PKD family kinases are not essential for oxidative stress survival responses nor are they required for activation of NFκB transcription factors.
T7013 14491-14727 Sentence denotes These latter findings are in striking contrast to previous observations in HeLa and epithelial cell lines where overexpression/RNAi approaches have implicated PKD1/2 in the control of proliferation, survival and NFκB activation [20,23].
T7014 14728-14983 Sentence denotes Hence, the present report shows that the proposed roles for PKDs as key sensors that modulate survival pathways in response to oxidative stress and regulate cell survival and proliferation are not ubiquitous and may be restricted to certain cell lineages.
T7015 14984-15134 Sentence denotes Taken together, these data indicate that loss of expression of PKD family members does not globally impact on early BCR-regulated signalling pathways.
T14 0-135 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells
T15 147-263 Sentence denotes To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line.
T16 264-614 Sentence denotes Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes.
T18 647-737 Sentence denotes We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells.
T19 738-979 Sentence denotes However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor.
T20 980-1036 Sentence denotes Thus, PKDs have a selective role in DT40 B-cell biology.
T1 0-135 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells
T2 138-146 Sentence denotes Abstract
T3 147-263 Sentence denotes To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line.
T4 264-614 Sentence denotes Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes.
T5 615-619 Sentence denotes Mol.
T6 620-630 Sentence denotes Cell Biol.
T7 631-646 Sentence denotes 26, 1569–1577].
T8 647-737 Sentence denotes We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells.
T9 738-979 Sentence denotes However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor.
T10 980-1036 Sentence denotes Thus, PKDs have a selective role in DT40 B-cell biology.
T11 1038-1053 Sentence denotes 1 Introduction
T12 1054-1151 Sentence denotes The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3.
T13 1152-1351 Sentence denotes Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3].
T14 1352-1665 Sentence denotes A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6].
T15 1666-1888 Sentence denotes The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12].
T16 1889-2061 Sentence denotes PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19].
T17 2062-2223 Sentence denotes Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]).
T18 2224-2386 Sentence denotes In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23].
T19 2387-2531 Sentence denotes Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25].
T20 2532-2712 Sentence denotes An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28].
T21 2713-3066 Sentence denotes To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members.
T22 3067-3158 Sentence denotes Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1].
T23 3159-3244 Sentence denotes Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells.
T24 3245-3312 Sentence denotes However, PKD-null DT40 B cells are viable and proliferate normally.
T25 3313-3514 Sentence denotes Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity.
T26 3515-3698 Sentence denotes Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems.
T27 3700-3724 Sentence denotes 2 Materials and methods
T28 3726-3789 Sentence denotes 2.1 Cell culture, transient transfections and cell stimulation
T29 3790-3925 Sentence denotes The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1].
T30 3926-4038 Sentence denotes Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1].
T31 4039-4117 Sentence denotes Chloramphenicol acetyl transferase assays have been described previously [29].
T32 4119-4137 Sentence denotes 2.2 sIgM staining
T33 4138-4335 Sentence denotes DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice.
T34 4336-4421 Sentence denotes The cells were washed twice and fluorescent intensity was analysed by flow cytometry.
T35 4422-4528 Sentence denotes All results shown are representative of at two to four independent experiments unless otherwise indicated.
T36 4530-4540 Sentence denotes 3 Results
T37 4542-4633 Sentence denotes 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases
T38 4634-4825 Sentence denotes DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1].
T39 4826-5011 Sentence denotes In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter.
T40 5012-5283 Sentence denotes Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A).
T41 5284-5521 Sentence denotes Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1].
T42 5522-5756 Sentence denotes The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence.
T43 5757-5883 Sentence denotes We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3.
T44 5884-6045 Sentence denotes As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells.
T45 6046-6180 Sentence denotes BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B).
T46 6181-6319 Sentence denotes However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C).
T47 6320-6501 Sentence denotes Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C).
T48 6502-6683 Sentence denotes In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D).
T49 6684-6816 Sentence denotes Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases.
T50 6818-6915 Sentence denotes 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases
T51 6916-7028 Sentence denotes PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]).
T52 7029-7192 Sentence denotes To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−:
T53 7193-7278 Sentence denotes Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth.
T54 7279-7432 Sentence denotes As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation.
T55 7433-7586 Sentence denotes Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown).
T56 7587-7974 Sentence denotes It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes.
T57 7975-8082 Sentence denotes Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells.
T58 8083-8274 Sentence denotes PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32].
T59 8275-8416 Sentence denotes We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli.
T60 8417-8805 Sentence denotes As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs.
T61 8806-8935 Sentence denotes Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli.
T62 8937-9013 Sentence denotes 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells
T63 9014-9192 Sentence denotes To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells.
T64 9193-9384 Sentence denotes Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown).
T65 9385-9565 Sentence denotes Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B).
T66 9566-9763 Sentence denotes Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B).
T67 9764-9980 Sentence denotes Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown).
T68 9981-10173 Sentence denotes We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B).
T69 10174-10254 Sentence denotes One interpretation of this data is that PKD enzymes may modulate Erk activation.
T70 10255-10398 Sentence denotes Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35].
T71 10399-10702 Sentence denotes However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway.
T72 10703-10941 Sentence denotes To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36].
T73 10942-11156 Sentence denotes As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes.
T74 11157-11371 Sentence denotes However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs.
T75 11372-11497 Sentence denotes Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs.
T76 11498-11635 Sentence denotes This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2.
T77 11636-11803 Sentence denotes However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera.
T78 11804-11950 Sentence denotes BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function.
T79 11951-12095 Sentence denotes In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor.
T80 12096-12284 Sentence denotes Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37].
T81 12285-12381 Sentence denotes Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38].
T82 12382-12531 Sentence denotes In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored.
T83 12532-12712 Sentence denotes The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation.
T84 12713-12974 Sentence denotes In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B).
T85 12975-13150 Sentence denotes Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB.
T86 13152-13165 Sentence denotes 4 Discussion
T87 13166-13362 Sentence denotes Protein kinase D serine kinases have been proposed to regulate diverse cellular functions including the phosphorylation and nuclear localisation of class II HDACs and the phosphorylation of HSP27.
T88 13363-13512 Sentence denotes It has also been suggested that PKDs act as mitochondrial sensors for oxidative stress and play a role in regulating NFκB transcription factors [41].
T89 13513-13683 Sentence denotes Most of the data about the function of PKDs has come from experiments that ectopically express active or inhibitory PKD mutants or that use RNAi to reduce PKD expression.
T90 13684-13965 Sentence denotes We have used gene targeting to specifically delete PKD alleles in DT40 chicken B cells and can thus use PKD-null DT40 cells to assess the relative contribution of individual PKD isoforms in class II HDAC control versus oxidative stress responses and NFκB regulation in lymphocytes.
T91 13966-14293 Sentence denotes We have previously used these PKD-null DT40 cells to define an essential role for PKDs in regulation of class II HDACs, the present report now describes an indispensable role for PKDs in regulating the phosphorylation of HSP27 on serine 82, a site previously identified as a target for the p38-MAPKAPK2 signalling cascade [42].
T92 14294-14490 Sentence denotes However, studies of PKD-null DT40 cells reveal that PKD family kinases are not essential for oxidative stress survival responses nor are they required for activation of NFκB transcription factors.
T93 14491-14727 Sentence denotes These latter findings are in striking contrast to previous observations in HeLa and epithelial cell lines where overexpression/RNAi approaches have implicated PKD1/2 in the control of proliferation, survival and NFκB activation [20,23].
T94 14728-14983 Sentence denotes Hence, the present report shows that the proposed roles for PKDs as key sensors that modulate survival pathways in response to oxidative stress and regulate cell survival and proliferation are not ubiquitous and may be restricted to certain cell lineages.
T95 14984-15134 Sentence denotes Taken together, these data indicate that loss of expression of PKD family members does not globally impact on early BCR-regulated signalling pathways.

ICD10

Id Subject Object Predicate Lexical cue
T3136 5537-5542 http://purl.bioontology.org/ontology/ICD10/R57.9 denotes shock
T1482 2456-2461 http://purl.bioontology.org/ontology/ICD10/R57.9 denotes shock
T4612 8206-8212 http://purl.bioontology.org/ontology/ICD10/T14.9 denotes injury

events-check-again

Id Subject Object Predicate Lexical cue Negation Speculation
T3563 5132-5136 Protein denotes PKD3
T3564 5439-5443 Protein denotes PKD1
T3565 5444-5445 Protein denotes 3
T3566 5551-5556 Protein denotes HSP27
T3567 5589-5593 Protein denotes PKD1
T3568 5670-5679 Negative_regulation denotes defective
T3569 5680-5695 Phosphorylation denotes phosphorylation
T3570 5699-5704 Protein denotes HSP27
T3571 5708-5717 Entity denotes serine 82
T3572 5732-5736 Protein denotes PKD1
T3573 5787-5797 Regulation denotes regulation true
T3574 5801-5806 Protein denotes HSP27
T3575 5807-5822 Phosphorylation denotes phosphorylation
T3576 5855-5862 Negative_regulation denotes lacking
T3577 5855-5862 Negative_regulation denotes lacking
T3578 5870-5874 Protein denotes PKD1
T3579 5878-5882 Protein denotes PKD3
T3580 5966-5975 Positive_regulation denotes increased
T3581 5990-5995 Protein denotes HSP27
T3582 5996-6011 Phosphorylation denotes phosphorylation
T3583 6015-6018 Entity denotes S82
T3584 6094-6102 Positive_regulation denotes increase
T3585 6103-6108 Protein denotes HSP27
T3586 6109-6124 Phosphorylation denotes phosphorylation
T3587 6128-6132 Protein denotes PKD1
T3588 6136-6140 Protein denotes PKD3
T3589 6148-6156 Negative_regulation denotes knockout
T3590 6148-6156 Negative_regulation denotes knockout
T3591 6213-6220 Positive_regulation denotes induced
T3592 6221-6236 Phosphorylation denotes phosphorylation
T3593 6240-6245 Protein denotes HSP27
T3594 6249-6252 Entity denotes S82
T3595 6257-6266 Negative_regulation denotes abolished
T3596 6257-6266 Negative_regulation denotes abolished
T3597 6283-6289 Negative_regulation denotes lacked
T3598 6283-6289 Negative_regulation denotes lacked
T3599 6295-6299 Protein denotes PKD1
T3600 6304-6308 Protein denotes PKD3
T3601 6347-6354 Positive_regulation denotes induced
T3602 6355-6365 Gene_expression denotes expression
T3603 6378-6382 Protein denotes PKD3
T3604 6426-6447 Positive_regulation denotes sufficient to restore
T3605 6455-6465 Regulation denotes regulation
T3606 6469-6474 Protein denotes HSP27
T3607 6475-6490 Phosphorylation denotes phosphorylation
T3608 6515-6525 Gene_expression denotes expression
T3609 6538-6547 Negative_regulation denotes deficient
T3610 6548-6552 Protein denotes PKD3
T3611 6613-6620 Positive_regulation denotes restore true
T3612 6643-6650 Positive_regulation denotes induced
T3613 6651-6656 Protein denotes HSP27
T3614 6657-6672 Phosphorylation denotes phosphorylation
T3615 6691-6695 Protein denotes PKD3
T3616 6707-6711 Protein denotes PKD1
T3617 6716-6724 Regulation denotes regulate
T3618 6716-6724 Regulation denotes regulate
T3619 6725-6730 Protein denotes HSP27
T3620 6731-6746 Phosphorylation denotes phosphorylation
T3621 6802-6807 Protein denotes HSP27
T4751 7182-7186 Protein denotes PKD1
T4752 7187-7188 Protein denotes 3
T4753 7198-7202 Protein denotes PKD3
T4754 7219-7223 Protein denotes PKD1
T4755 7224-7225 Protein denotes 3
T4756 7300-7304 Protein denotes PKD1
T4757 7305-7306 Protein denotes 3
T4758 7347-7360 Gene_expression denotes re-expression
T4759 7369-7373 Protein denotes PKD3
T4760 7463-7467 Protein denotes PKD1
T4761 7468-7469 Protein denotes 3
T4762 7637-7641 Protein denotes PKD1
T4763 7642-7643 Protein denotes 3
T4764 7761-7765 Protein denotes PKD3
T4765 7766-7779 Gene_expression denotes re-expression
T4766 7816-7820 Protein denotes PKD1
T4767 7821-7822 Protein denotes 3
T4768 8109-8113 Protein denotes PKD1
T4769 8118-8122 Protein denotes PKD2
T4770 8438-8442 Negative_regulation denotes loss
T4771 8438-8442 Negative_regulation denotes loss
T4772 8446-8450 Protein denotes PKD1
T4773 8451-8452 Protein denotes 3
T4774 8453-8463 Gene_expression denotes expression
T4775 8453-8463 Gene_expression denotes expression
T6905 9272-9276 Protein denotes PKD1
T6906 9277-9278 Protein denotes 3
T6907 9290-9294 Protein denotes PKD1
T6908 9295-9296 Protein denotes 3
T6909 9305-9309 Protein denotes PKD3
T6910 9419-9423 Protein denotes PKD1
T6911 9424-9425 Protein denotes 3
T6912 9577-9584 Positive_regulation denotes induced
T6913 9577-9584 Positive_regulation denotes induced
T6914 9577-9584 Positive_regulation denotes induced
T6915 9585-9595 Positive_regulation denotes activation
T6916 9585-9595 Positive_regulation denotes activation
T6917 9585-9595 Positive_regulation denotes activation
T6918 9603-9606 Protein denotes Akt
T6919 9608-9612 Protein denotes mTOR
T6920 9640-9660 Protein denotes S6 ribosomal protein
T6921 9661-9676 Phosphorylation denotes phosphorylation
T6922 9719-9729 Negative_regulation denotes detectable true
T6923 9719-9729 Negative_regulation denotes detectable true
T6924 9719-9729 Negative_regulation denotes detectable true
T6925 9719-9729 Negative_regulation denotes detectable true
T6926 9719-9729 Negative_regulation denotes detectable true
T6927 9719-9729 Negative_regulation denotes detectable true
T6928 9733-9737 Protein denotes PKD1
T6929 9738-9739 Protein denotes 3
T6930 9740-9744 Negative_regulation denotes null
T6931 9740-9744 Negative_regulation denotes null
T6932 9943-9947 Protein denotes PKD1
T6933 9948-9949 Protein denotes 3
T6934 10071-10075 Protein denotes Erk1
T6935 10076-10080 Protein denotes RSK1
T6942 10564-10565 Protein denotes 3
T6943 10574-10578 Protein denotes PKD1
T425 678-686 Positive_regulation denotes required
T426 690-698 Regulation denotes regulate
T427 699-704 Protein denotes HSP27
T428 705-720 Phosphorylation denotes phosphorylation
T1575 1131-1135 Protein denotes PKD1
T1576 1137-1141 Protein denotes PKD2
T1577 1146-1150 Protein denotes PKD3
T1578 1755-1762 Binding denotes binding
T1579 1775-1786 Positive_regulation denotes contributes
T1580 1790-1794 Protein denotes PKD1
T1581 1795-1805 Positive_regulation denotes activation
T1582 2413-2417 Protein denotes PKD1
T1583 2431-2436 Protein denotes HSP27
T1584 2640-2644 Protein denotes MEF2
T1585 3193-3206 Positive_regulation denotes indispensable
T1586 3211-3216 Protein denotes HSP27
T1587 3217-3232 Phosphorylation denotes phosphorylation
T3540 4547-4551 Negative_regulation denotes Loss true
T3541 4555-4560 Protein denotes HSP27
T3542 4561-4576 Phosphorylation denotes phosphorylation
T3543 4647-4654 Gene_expression denotes express
T3544 4647-4654 Gene_expression denotes express
T3545 4673-4677 Protein denotes PKD1
T3546 4682-4686 Protein denotes PKD3
T3547 4766-4770 Negative_regulation denotes lack
T3548 4766-4770 Negative_regulation denotes lack
T3549 4771-4781 Gene_expression denotes expression
T3550 4771-4781 Gene_expression denotes expression
T3551 4792-4796 Protein denotes PKD1
T3552 4800-4804 Protein denotes PKD3
T3553 4887-4891 Protein denotes PKD1
T3554 4902-4906 Protein denotes PKD3
T3555 4925-4934 Gene_expression denotes expressed
T3556 4942-4946 Protein denotes PKD3
T3557 5056-5060 Protein denotes PKD3
T3558 5061-5071 Gene_expression denotes expression
T3559 5075-5079 Protein denotes PKD1
T3560 5080-5081 Protein denotes 3
T3561 5082-5097 Negative_regulation denotes double knockout
T3562 5082-5097 Negative_regulation denotes double knockout
T6936 10115-10119 Protein denotes PKD1
T6937 10120-10121 Protein denotes 3
T6938 10460-10464 Protein denotes PKD1
T6939 10465-10466 Protein denotes 3
T6940 10475-10479 Protein denotes PKD3
T6941 10559-10563 Protein denotes PKD1
T6944 10579-10580 Protein denotes 3
T6945 10589-10593 Protein denotes PKD3
T6946 10783-10787 Protein denotes PKD1
T6947 10788-10789 Protein denotes 3
T6948 11096-11100 Protein denotes PKD1
T6949 11101-11102 Protein denotes 3
T6950 11201-11205 Protein denotes PKD1
T6951 11206-11207 Protein denotes 3
T6952 11377-11381 Negative_regulation denotes loss
T6953 11377-11381 Negative_regulation denotes loss
T6954 11385-11389 Protein denotes PKD1
T6955 11390-11391 Protein denotes 3
T6956 11626-11634 Protein denotes MAPKAPK2
T6957 12250-12266 Protein denotes Bcr-Abl oncogene
T6958 12298-12324 Positive_regulation denotes expression of an activated
T6959 12325-12329 Protein denotes PKD1
T6960 12346-12350 Protein denotes HPK1
T6961 12436-12440 Protein denotes PKCβ
T6962 12634-12638 Protein denotes PKD1
T6963 12639-12640 Protein denotes 3
T6964 12803-12808 Protein denotes PKCβ−
T6965 12893-12912 Phosphorylation denotes autophosphorylation
T6966 12916-12920 Protein denotes PKD1
T6967 12924-12928 Entity denotes S916
T6968 12934-12942 Positive_regulation denotes observed
T6969 12950-12955 Protein denotes PKCβ−
T7775 13183-13189 Entity denotes serine
T7776 13220-13228 Regulation denotes regulate true
T7777 13337-13352 Phosphorylation denotes phosphorylation
T7778 13356-13361 Protein denotes HSP27
T1851 3832-3836 Protein denotes PKD1
T1852 3841-3845 Protein denotes PKD3
T1853 3853-3857 Protein denotes PKD1
T1854 3858-3859 Protein denotes 3
T1855 3863-3871 Negative_regulation denotes knockout
T1856 3863-3871 Negative_regulation denotes knockout
T1857 3863-3871 Negative_regulation denotes knockout
T1858 3863-3871 Negative_regulation denotes knockout
T7779 14153-14163 Regulation denotes regulating
T7780 14168-14183 Phosphorylation denotes phosphorylation
T7781 14187-14192 Protein denotes HSP27
T7782 14196-14205 Entity denotes serine 82
T7783 14241-14247 Regulation denotes target
T7784 14260-14268 Protein denotes MAPKAPK2
T7785 14650-14654 Protein denotes PKD1
T7786 14655-14656 Protein denotes 2
R2918 T3541 T3542 themeOf HSP27,phosphorylation
R2925 T3542 T3540 themeOf phosphorylation,Loss
R2926 T3545 T3543 themeOf PKD1,express
R2927 T3546 T3544 themeOf PKD3,express
R2929 T3550 T3548 themeOf expression,lack
R2930 T3551 T3550 themeOf PKD1,expression
R2941 T3587 T3590 themeOf PKD1,knockout
R2942 T3588 T3589 themeOf PKD3,knockout
R2944 T3591 T3595 themeOf induced,abolished
R2945 T3591 T3596 themeOf induced,abolished
R2946 T3592 T3591 themeOf phosphorylation,induced
R2948 T3594 T3592 themeOf S82,phosphorylation
R2949 T3594 T3593 partOf S82,HSP27
R2950 T3597 T3596 causeOf lacked,abolished
R2951 T3598 T3595 causeOf lacked,abolished
R2952 T3599 T3598 themeOf PKD1,lacked
R2953 T3600 T3597 themeOf PKD3,lacked
R2954 T3601 T3604 causeOf induced,sufficient to restore
R2955 T3602 T3601 themeOf expression,induced
R2957 T3603 T3602 themeOf PKD3,expression
R2958 T3605 T3604 themeOf regulation,sufficient to restore
R2959 T3606 T3607 themeOf HSP27,phosphorylation
R2960 T3607 T3605 themeOf phosphorylation,regulation
R2962 T3608 T3611 causeOf expression,restore
R2963 T3610 T3608 themeOf PKD3,expression
R2964 T3610 T3609 themeOf PKD3,deficient
R2966 T3612 T3611 themeOf induced,restore
R2967 T3613 T3614 themeOf HSP27,phosphorylation
R2968 T3614 T3612 themeOf phosphorylation,induced
R2969 T3615 T3618 causeOf PKD3,regulate
R2971 T3616 T3617 causeOf PKD1,regulate
R2973 T3619 T3620 themeOf HSP27,phosphorylation
R2974 T3620 T3618 themeOf phosphorylation,regulate
R2975 T3620 T3617 themeOf phosphorylation,regulate
R3843 T4759 T4758 themeOf PKD3,re-expression
R3844 T4764 T4765 themeOf PKD3,re-expression
R3845 T4772 T4774 themeOf PKD1,expression
R3846 T4773 T4775 themeOf 3,expression
R3847 T4774 T4771 themeOf expression,loss
R3848 T4775 T4770 themeOf expression,loss
R5508 T6931 T6926 causeOf null,detectable
R5509 T6965 T6968 themeOf autophosphorylation,observed
R5510 T6967 T6965 themeOf S916,autophosphorylation
R5511 T6967 T6966 partOf S916,PKD1
R6224 T7775 T7777 themeOf serine,phosphorylation
R6225 T7775 T7778 partOf serine,HSP27
R6226 T7777 T7776 themeOf phosphorylation,regulate
R6227 T7780 T7779 themeOf phosphorylation,regulating
R6228 T7782 T7780 themeOf serine 82,phosphorylation
R6229 T7782 T7781 partOf serine 82,HSP27
R6230 T7782 T7783 themeOf serine 82,target
R410 T426 T425 themeOf regulate,required
R411 T427 T428 themeOf HSP27,phosphorylation
R412 T428 T426 themeOf phosphorylation,regulate
R1400 T1578 T1579 causeOf binding,contributes
R1402 T1580 T1578 themeOf PKD1,binding
R1403 T1580 T1581 themeOf PKD1,activation
R1404 T1581 T1579 themeOf activation,contributes
R1405 T1586 T1587 themeOf HSP27,phosphorylation
R1406 T1587 T1585 themeOf phosphorylation,indispensable
R2928 T3549 T3547 themeOf expression,lack
R2931 T3579 T3577 themeOf PKD3,lacking
R2932 T3552 T3549 themeOf PKD3,expression
R2933 T3582 T3580 themeOf phosphorylation,increased
R2934 T3556 T3555 themeOf PKD3,expressed
R2935 T3583 T3582 themeOf S82,phosphorylation
R2936 T3583 T3581 partOf S82,HSP27
R2937 T3585 T3586 themeOf HSP27,phosphorylation
R2938 T3586 T3584 themeOf phosphorylation,increase
R2939 T3557 T3558 themeOf PKD3,expression
R2940 T3559 T3562 themeOf PKD1,double knockout
R2943 T3560 T3561 themeOf 3,double knockout
R2947 T3569 T3568 themeOf phosphorylation,defective
R2956 T3571 T3569 themeOf serine 82,phosphorylation
R2961 T3571 T3570 partOf serine 82,HSP27
R2965 T3574 T3575 themeOf HSP27,phosphorylation
R2970 T3575 T3573 themeOf phosphorylation,regulation
R2972 T3578 T3576 themeOf PKD1,lacking
R1592 T1851 T1855 themeOf PKD1,knockout
R1593 T1852 T1856 themeOf PKD3,knockout
R1594 T1853 T1857 themeOf PKD1,knockout
R1595 T1854 T1858 themeOf 3,knockout
R5479 T6912 T6922 themeOf induced,detectable
R5486 T6912 T6924 themeOf induced,detectable
R5487 T6913 T6927 themeOf induced,detectable
R5488 T6913 T6926 themeOf induced,detectable
R5489 T6914 T6923 themeOf induced,detectable
R5490 T6914 T6925 themeOf induced,detectable
R5491 T6915 T6914 themeOf activation,induced
R5492 T6916 T6912 themeOf activation,induced
R5493 T6917 T6913 themeOf activation,induced
R5494 T6918 T6917 themeOf Akt,activation
R5495 T6919 T6916 themeOf mTOR,activation
R5496 T6920 T6921 themeOf S6 ribosomal protein,phosphorylation
R5497 T6921 T6915 themeOf phosphorylation,activation
R5498 T6928 T6931 themeOf PKD1,null
R5499 T6929 T6930 themeOf 3,null
R5500 T6930 T6927 causeOf null,detectable
R5501 T6954 T6953 themeOf PKD1,loss
R5502 T6930 T6924 causeOf null,detectable
R5503 T6930 T6925 causeOf null,detectable
R5504 T6955 T6952 themeOf 3,loss
R5505 T6931 T6922 causeOf null,detectable
R5506 T6931 T6923 causeOf null,detectable
R5507 T6959 T6958 themeOf PKD1,expression of an activated

bionlp-st-ge-2016-reference-tees

Id Subject Object Predicate Lexical cue
T429 0-24 Protein denotes Protein kinase D enzymes
T430 100-104 Protein denotes NFκB
T431 81-99 Regulation denotes receptor-regulated
T432 180-196 Protein denotes protein kinase D
T433 198-201 Protein denotes PKD
T434 226-229 Protein denotes PKD
T435 294-298 Protein denotes PKDs
T436 336-365 Protein denotes class II histone deacetylases
T437 514-545 Protein denotes protein kinase D family kinases
T438 567-596 Protein denotes class II histone deacetylases
T439 553-563 Regulation denotes regulation
T440 664-668 Protein denotes PKDs
T441 699-704 Protein denotes HSP27
T442 705-720 Phosphorylation denotes phosphorylation
T443 690-698 Regulation denotes regulate
T444 678-686 Positive_regulation denotes required
T445 805-816 Protein denotes PKD enzymes
T446 907-911 Protein denotes NFκB
T447 955-978 Protein denotes B cell antigen receptor
T448 824-832 Regulation denotes regulate
T449 986-990 Protein denotes PKDs
T1588 1058-1074 Protein denotes protein kinase D
T1589 1076-1079 Protein denotes PKD
T1590 1081-1111 Protein denotes serine/threonine kinase family
T1591 1131-1135 Protein denotes PKD1
T1592 1137-1141 Protein denotes PKD2
T1593 1146-1150 Protein denotes PKD3
T1594 1189-1192 Protein denotes PKD
T1595 1206-1209 Protein denotes PKD
T1596 1168-1175 Gene_expression denotes express
T1597 1240-1249 Gene_expression denotes expressed
T1598 1412-1416 Protein denotes PKDs
T1599 1444-1448 Protein denotes PLCγ
T1600 1543-1560 Protein denotes protein kinase Cs
T1601 1562-1565 Protein denotes PKC
T1602 1647-1658 Protein denotes PKD kinases
T1603 1509-1519 Positive_regulation denotes stimulates
T1604 1509-1519 Positive_regulation denotes stimulates
T1605 1572-1585 Phosphorylation denotes phosphorylate
T1606 1572-1585 Phosphorylation denotes phosphorylate
T1607 1702-1713 Protein denotes PKD enzymes
T1608 1766-1769 Protein denotes DAG
T1609 1790-1794 Protein denotes PKD1
T1610 1856-1867 Protein denotes PKD enzymes
T1611 1755-1762 Binding denotes binding
T1612 1795-1805 Positive_regulation denotes activation
T1613 1844-1852 Localization denotes location
T1614 1775-1786 Positive_regulation denotes contributes
T1615 1821-1831 Regulation denotes regulating
T1616 1889-1892 Protein denotes PKD
T1617 2135-2139 Protein denotes PKDs
T1618 2062-2072 Gene_expression denotes Expression
T1619 2125-2134 Positive_regulation denotes activated
T1620 2125-2134 Positive_regulation denotes activated
T1621 2239-2243 Protein denotes PKDs
T1622 2293-2318 Protein denotes NFκB transcription factor
T1623 2275-2285 Positive_regulation denotes activation
T1624 2413-2417 Protein denotes PKD1
T1625 2554-2565 Protein denotes PKD enzymes
T1626 2580-2609 Protein denotes class II histone deacetylases
T1627 2611-2616 Protein denotes HDACs
T1628 2640-2644 Protein denotes MEF2
T1629 2569-2579 Regulation denotes regulating
T1630 2569-2579 Regulation denotes regulating
T1631 2751-2755 Protein denotes PKDs
T1632 2843-2846 Protein denotes PKD
T1633 2905-2908 Protein denotes PKD
T1634 2928-2951 Protein denotes B cell antigen receptor
T1635 2953-2956 Protein denotes BCR
T1636 3047-3065 Protein denotes PKD family members
T1637 3100-3104 Protein denotes PKDs
T1638 3127-3131 Protein denotes HDAC
T1639 3132-3142 Regulation denotes regulation
T1640 3179-3183 Protein denotes PKDs
T1641 3211-3216 Protein denotes HSP27
T1642 3217-3232 Phosphorylation denotes phosphorylation
T1643 3193-3206 Positive_regulation denotes indispensable
T1644 3254-3257 Protein denotes PKD
T1645 3359-3362 Protein denotes PKD
T1646 3435-3446 Protein denotes PKD kinases
T1647 3484-3488 Protein denotes NFκB
T1648 3323-3327 Negative_regulation denotes loss
T1649 3570-3581 Protein denotes PKD kinases
T3622 4555-4560 Protein denotes HSP27
T3623 4580-4586 Protein denotes DT40 B
T3624 4615-4625 Protein denotes PKD family
T3625 4561-4576 Phosphorylation denotes phosphorylation
T3626 4601-4611 Gene_expression denotes expression
T3627 0-4551 Negative_regulation denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss
T3628 4593-4600 Negative_regulation denotes lacking
T3629 4634-4640 Protein denotes DT40 B
T3630 4659-4662 Protein denotes PKD
T3631 4673-4677 Protein denotes PKD1
T3632 4682-4686 Protein denotes PKD3
T3633 4792-4796 Protein denotes PKD1
T3634 4800-4804 Protein denotes PKD3
T3635 4647-4654 Gene_expression denotes express
T3636 4647-4654 Gene_expression denotes express
T3637 4647-4654 Gene_expression denotes express
T3638 4771-4781 Gene_expression denotes expression
T3639 4771-4781 Gene_expression denotes expression
T3640 4766-4770 Negative_regulation denotes lack
T3641 4766-4770 Negative_regulation denotes lack
T3642 4887-4896 Protein denotes PKD1 loci
T3643 4902-4906 Protein denotes PKD3
T3644 4937-4941 Protein denotes Flag
T3645 4942-4946 Protein denotes PKD3
T3646 4925-4934 Gene_expression denotes expressed
T3647 4925-4934 Gene_expression denotes expressed
T3648 5051-5055 Protein denotes Flag
T3649 5056-5060 Protein denotes PKD3
T3650 5075-5081 Protein denotes PKD1/3
T3651 5132-5136 Protein denotes PKD3
T3652 5061-5071 Gene_expression denotes expression
T3653 5061-5071 Gene_expression denotes expression
T3654 5363-5392 Protein denotes class II histone deacetylases
T3655 5394-5399 Protein denotes HDACs
T3656 5408-5411 Protein denotes BCR
T3657 5439-5443 Protein denotes PKD1
T3658 5322-5337 Phosphorylation denotes phosphorylation
T3659 5322-5337 Phosphorylation denotes phosphorylation
T3660 5412-5422 Binding denotes engagement
T3661 5465-5473 Positive_regulation denotes restored
T3662 5465-5473 Positive_regulation denotes restored
T3663 5526-5550 Protein denotes small heat shock protein
T3664 5551-5556 Protein denotes HSP27
T3665 5589-5593 Protein denotes PKD1
T3666 5645-5648 Protein denotes PKD
T3667 5699-5704 Protein denotes HSP27
T3668 5732-5736 Protein denotes PKD1
T3669 5708-5714 Entity denotes serine
T3670 5680-5695 Phosphorylation denotes phosphorylation
T3671 5670-5679 Negative_regulation denotes defective
T3672 5801-5806 Protein denotes HSP27
T3673 5870-5874 Protein denotes PKD1
T3675 5807-5822 Phosphorylation denotes phosphorylation
T3676 5855-5862 Negative_regulation denotes lacking
T3677 5855-5862 Negative_regulation denotes lacking
T3678 5787-5797 Regulation denotes regulation
T3679 5896-5899 Protein denotes Fig
T3680 5923-5926 Protein denotes BCR
T3681 5990-5995 Protein denotes HSP27
T3682 5905-5915 Positive_regulation denotes activation
T3683 5966-5975 Positive_regulation denotes increased
T3684 5996-6011 Phosphorylation denotes phosphorylation
T3685 6046-6049 Protein denotes BCR
T3686 6103-6108 Protein denotes HSP27
T3687 6128-6132 Protein denotes PKD1
T3688 6136-6140 Protein denotes PKD3
T3689 6109-6124 Phosphorylation denotes phosphorylation
T3690 6148-6156 Negative_regulation denotes knockout
T3691 6148-6156 Negative_regulation denotes knockout
T3692 6094-6102 Positive_regulation denotes increase
T3693 6190-6193 Protein denotes BCR
T3694 6240-6245 Protein denotes HSP27
T3695 6295-6299 Protein denotes PKD1
T3696 6304-6308 Protein denotes PKD3
T3697 6310-6317 Protein denotes Fig. 1C
T3698 6249-6252 Entity denotes S82
T3699 6283-6289 Negative_regulation denotes lacked
T3700 6283-6289 Negative_regulation denotes lacked
T3701 6283-6289 Negative_regulation denotes lacked
T3702 6221-6236 Phosphorylation denotes phosphorylation
T3703 6221-6236 Phosphorylation denotes phosphorylation
T3704 6257-6266 Negative_regulation denotes abolished
T3705 6257-6266 Negative_regulation denotes abolished
T3706 6373-6377 Protein denotes Flag
T3707 6378-6382 Protein denotes PKD3
T3708 6469-6474 Protein denotes HSP27
T3709 6355-6365 Gene_expression denotes expression
T3710 6355-6365 Gene_expression denotes expression
T3711 6475-6490 Phosphorylation denotes phosphorylation
T3712 6335-6354 Positive_regulation denotes doxycycline-induced
T3713 6335-6354 Positive_regulation denotes doxycycline-induced
T3714 6455-6465 Regulation denotes regulation
T3715 6440-6447 Positive_regulation denotes restore
T3716 6548-6552 Protein denotes PKD3
T3717 6651-6656 Protein denotes HSP27
T3718 6515-6525 Gene_expression denotes expression
T3719 6531-6547 Negative_regulation denotes kinase-deficient
T3720 6657-6672 Phosphorylation denotes phosphorylation
T3721 6613-6620 Positive_regulation denotes restore
T3722 6637-6650 Positive_regulation denotes ester-induced
T3723 6691-6695 Protein denotes PKD3
T3724 6707-6711 Protein denotes PKD1
T3725 6725-6730 Protein denotes HSP27
T3726 6802-6815 Protein denotes HSP27 kinases
T3727 6731-6746 Phosphorylation denotes phosphorylation
T3728 6716-6724 Regulation denotes regulate
T3729 6716-6724 Regulation denotes regulate
T4793 7463-7467 Protein denotes PKD1
T4794 7637-7641 Protein denotes PKD1
T4795 7761-7765 Protein denotes PKD3
T4796 7816-7820 Protein denotes PKD1
T4797 7962-7973 Protein denotes PKD enzymes
T4798 7766-7779 Gene_expression denotes re-expression
T4799 7954-7958 Negative_regulation denotes loss
T4800 7981-7999 Protein denotes PKD family enzymes
T4801 8069-8075 Protein denotes DT40 B
T4802 8083-8094 Protein denotes PKD enzymes
T4803 8109-8113 Protein denotes PKD1
T4804 8118-8122 Protein denotes PKD2
T4805 8310-8328 Protein denotes PKD family kinases
T4806 8429-8432 Protein denotes Fig
T4807 8446-8450 Protein denotes PKD1
T4808 8451-8452 Protein denotes 3
T4809 8509-8515 Protein denotes DT40 B
T4810 8794-8804 Protein denotes I/II HDACs
T4811 8453-8463 Gene_expression denotes expression
T4812 8453-8463 Gene_expression denotes expression
T4813 8775-8784 Negative_regulation denotes inhibitor
T4814 8438-8442 Negative_regulation denotes loss
T4815 8438-8442 Negative_regulation denotes loss
T4816 8812-8823 Protein denotes PKD kinases
T7787 237-13197 Protein denotes 40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway. To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera. BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor. Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38]. In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored. The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB. 4 Discussion Protein kinase D serine kinases
T7788 13314-13328 Protein denotes class II HDACs
T7789 13356-13361 Protein denotes HSP27
T7790 13270-13285 Phosphorylation denotes phosphorylation
T7791 13337-13352 Phosphorylation denotes phosphorylation
T7792 13220-13228 Regulation denotes regulate
T7793 13220-13228 Regulation denotes regulate
T7794 13395-13399 Protein denotes PKDs
T7795 13480-13484 Protein denotes NFκB
T7796 13469-13479 Regulation denotes regulating
T7797 13552-13556 Protein denotes PKDs
T7798 13629-13640 Protein denotes PKD mutants
T7799 13649-13657 Protein denotes use RNAi
T7800 13668-13671 Protein denotes PKD
T7801 13600-13607 Gene_expression denotes express
T7802 13600-13607 Gene_expression denotes express
T7803 13672-13682 Gene_expression denotes expression
T7804 13661-13667 Negative_regulation denotes reduce
T7805 13735-13746 Protein denotes PKD alleles
T7806 13788-13791 Protein denotes PKD
T7807 13858-13870 Protein denotes PKD isoforms
T7808 13934-13938 Protein denotes NFκB
T7809 13939-13949 Regulation denotes regulation
T7810 13996-13999 Protein denotes PKD
T7811 14048-14052 Protein denotes PKDs
T7812 14070-14084 Protein denotes class II HDACs
T7813 14145-14149 Protein denotes PKDs
T7814 14187-14192 Protein denotes HSP27
T7815 14256-14259 Protein denotes p38
T7816 14260-14268 Protein denotes MAPKAPK2
T7817 14196-14202 Entity denotes serine
T7818 14241-14247 Regulation denotes target
T7819 14168-14183 Phosphorylation denotes phosphorylation
T7820 14153-14163 Regulation denotes regulating
T7821 14314-14317 Protein denotes PKD
T7822 14346-14364 Protein denotes PKD family kinases
T7823 14463-14489 Protein denotes NFκB transcription factors
T7824 14449-14459 Positive_regulation denotes activation
T7825 14618-14622 Protein denotes RNAi
T7826 14650-14656 Protein denotes PKD1/2
T7827 14703-14707 Protein denotes NFκB
T7828 14603-14617 Gene_expression denotes overexpression
T7829 14603-14617 Positive_regulation denotes overexpression
T7830 14788-14792 Protein denotes PKDs
T7831 15047-15065 Protein denotes PKD family members
T7832 15033-15043 Gene_expression denotes expression
T7833 15025-15029 Negative_regulation denotes loss
T4776 6897-6907 Protein denotes PKD family
T4777 6883-6893 Gene_expression denotes expression
T4778 6875-6882 Negative_regulation denotes lacking
T4779 6916-6919 Protein denotes PKD
T4780 7068-7079 Protein denotes PKD kinases
T4781 7150-7153 Protein denotes PKD
T4782 7182-7186 Protein denotes PKD1
T4783 7193-7197 Protein denotes Flag
T4784 7198-7202 Protein denotes PKD3
T4785 7219-7223 Protein denotes PKD1
T4786 7060-7064 Negative_regulation denotes loss
T4787 7291-7294 Protein denotes Fig
T4788 7300-7304 Protein denotes PKD1
T4789 7364-7368 Protein denotes Flag
T4790 7369-7373 Protein denotes PKD3
T4791 7347-7360 Gene_expression denotes re-expression
T4792 7347-7360 Gene_expression denotes re-expression
T3674 5878-5882 Protein denotes PKD3
T1859 3832-3836 Protein denotes PKD1
T1860 3841-3845 Protein denotes PKD3
T1861 3853-3857 Protein denotes PKD1
T1862 4039-4073 Protein denotes Chloramphenicol acetyl transferase
T2020 4262-4297 Protein denotes anti-chicken M1 monoclonal antibody
R1413 T1609 T1612 themeOf PKD1,activation
R1414 T1610 T1613 themeOf PKD enzymes,location
R1415 T1611 T1614 causeOf binding,contributes
R1416 T1612 T1614 themeOf activation,contributes
R1417 T1613 T1615 themeOf location,regulating
R1418 T1617 T1618 themeOf PKDs,Expression
R1419 T1617 T1619 themeOf PKDs,activated
R1420 T1618 T1620 themeOf Expression,activated
R1421 T1622 T1623 themeOf NFκB transcription factor,activation
R1422 T1626 T1629 themeOf class II histone deacetylases,regulating
R1423 T1627 T1630 themeOf HDACs,regulating
R1424 T1638 T1639 themeOf HDAC,regulation
R1425 T1640 T1643 causeOf PKDs,indispensable
R1426 T1641 T1642 themeOf HSP27,phosphorylation
R1427 T1642 T1643 themeOf phosphorylation,indispensable
R1428 T1645 T1648 themeOf PKD,loss
R2976 T3625 T3627 themeOf phosphorylation,"Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells Abstract To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss"
R2977 T3626 T3628 themeOf expression,lacking
R2978 T3630 T3635 themeOf PKD,express
R2979 T3631 T3636 themeOf PKD1,express
R2980 T3622 T3625 themeOf HSP27,phosphorylation
R2981 T3632 T3637 themeOf PKD3,express
R2982 T3624 T3626 themeOf PKD family,expression
R2983 T3633 T3638 themeOf PKD1,expression
R2984 T3634 T3639 themeOf PKD3,expression
R2985 T3638 T3640 themeOf expression,lack
R2986 T3674 T3677 themeOf PKD3,lacking
R2987 T3675 T3678 themeOf phosphorylation,regulation
R2988 T3639 T3641 themeOf expression,lack
R2989 T3644 T3646 themeOf Flag,expressed
R2990 T3680 T3682 themeOf BCR,activation
R2991 T3681 T3684 themeOf HSP27,phosphorylation
R2992 T3682 T3683 themeOf activation,increased
R2993 T3686 T3689 themeOf HSP27,phosphorylation
R2994 T3645 T3647 themeOf PKD3,expressed
R2995 T3648 T3652 themeOf Flag,expression
R2999 T3649 T3653 themeOf PKD3,expression
R3001 T3654 T3658 themeOf class II histone deacetylases,phosphorylation
R3003 T3694 T3703 themeOf HSP27,phosphorylation
R3004 T3694 T3698 partOf HSP27,S82
R3005 T3695 T3699 themeOf PKD1,lacked
R3006 T3696 T3700 themeOf PKD3,lacked
R3007 T3697 T3701 themeOf Fig. 1C,lacked
R3008 T3698 T3702 Site S82,phosphorylation
R3009 T3698 T3703 Site S82,phosphorylation
R3010 T3655 T3659 themeOf HDACs,phosphorylation
R3011 T3702 T3704 themeOf phosphorylation,abolished
R3012 T3656 T3660 themeOf BCR,engagement
R3013 T3703 T3705 themeOf phosphorylation,abolished
R3014 T3706 T3709 themeOf Flag,expression
R3015 T3707 T3710 themeOf PKD3,expression
R3016 T3658 T3661 themeOf phosphorylation,restored
R3017 T3708 T3711 themeOf HSP27,phosphorylation
R3018 T3709 T3712 themeOf expression,doxycycline-induced
R3019 T3659 T3662 themeOf phosphorylation,restored
R3020 T3667 T3670 themeOf HSP27,phosphorylation
R3021 T3710 T3713 themeOf expression,doxycycline-induced
R3022 T3711 T3714 themeOf phosphorylation,regulation
R3023 T3714 T3715 themeOf regulation,restore
R3024 T3716 T3718 themeOf PKD3,expression
R3025 T3717 T3720 themeOf HSP27,phosphorylation
R3026 T3667 T3669 partOf HSP27,serine
R3027 T3718 T3719 themeOf expression,kinase-deficient
R3028 T3718 T3721 causeOf expression,restore
R3029 T3668 T3669 partOf PKD1,serine
R3030 T3720 T3721 themeOf phosphorylation,restore
R3031 T3720 T3722 themeOf phosphorylation,ester-induced
R3032 T3669 T3670 Site serine,phosphorylation
R3033 T3723 T3728 causeOf PKD3,regulate
R3034 T3670 T3671 themeOf phosphorylation,defective
R3035 T3724 T3729 causeOf PKD1,regulate
R3036 T3725 T3727 themeOf HSP27,phosphorylation
R3037 T3672 T3675 themeOf HSP27,phosphorylation
R3038 T3727 T3728 themeOf phosphorylation,regulate
R3039 T3727 T3729 themeOf phosphorylation,regulate
R3040 T3673 T3676 themeOf PKD1,lacking
R3849 T4776 T4777 themeOf PKD family,expression
R3850 T4777 T4778 themeOf expression,lacking
R3851 T4780 T4786 themeOf PKD kinases,loss
R3852 T4789 T4791 themeOf Flag,re-expression
R3853 T4790 T4792 themeOf PKD3,re-expression
R3854 T4795 T4798 themeOf PKD3,re-expression
R3855 T4797 T4799 themeOf PKD enzymes,loss
R3856 T4807 T4811 themeOf PKD1,expression
R3857 T4808 T4812 themeOf 3,expression
R3858 T4810 T4813 themeOf I/II HDACs,inhibitor
R3859 T4811 T4814 themeOf expression,loss
R3860 T4812 T4815 themeOf expression,loss
R6231 T7788 T7790 themeOf class II HDACs,phosphorylation
R6232 T7789 T7791 themeOf HSP27,phosphorylation
R6233 T7790 T7792 themeOf phosphorylation,regulate
R6234 T7791 T7793 themeOf phosphorylation,regulate
R6235 T7794 T7796 causeOf PKDs,regulating
R6236 T7795 T7796 themeOf NFκB,regulating
R6237 T7798 T7801 themeOf PKD mutants,express
R6238 T7799 T7802 themeOf use RNAi,express
R6239 T7800 T7803 themeOf PKD,expression
R6240 T7803 T7804 themeOf expression,reduce
R6241 T7808 T7809 themeOf NFκB,regulation
R6242 T7814 T7819 themeOf HSP27,phosphorylation
R6243 T7814 T7817 partOf HSP27,serine
R6244 T7816 T7818 themeOf MAPKAPK2,target
R6245 T7817 T7819 Site serine,phosphorylation
R6246 T7819 T7820 themeOf phosphorylation,regulating
R6247 T7823 T7824 themeOf NFκB transcription factors,activation
R6248 T7825 T7828 themeOf RNAi,overexpression
R6249 T7828 T7829 themeOf overexpression,overexpression
R6250 T7831 T7832 themeOf PKD family members,expression
R6251 T7832 T7833 themeOf expression,loss
R1401 T1594 T1596 themeOf PKD,express
R1407 T1595 T1597 themeOf PKD,expressed
R1408 T1600 T1603 themeOf protein kinase Cs,stimulates
R1409 T1600 T1605 themeOf protein kinase Cs,phosphorylate
R1410 T1601 T1604 themeOf PKC,stimulates
R1411 T1601 T1606 themeOf PKC,phosphorylate
R1412 T1608 T1611 themeOf DAG,binding
R362 T430 T431 themeOf NFκB,receptor-regulated
R363 T438 T439 themeOf class II histone deacetylases,regulation
R364 T440 T443 causeOf PKDs,regulate
R365 T440 T444 causeOf PKDs,required
R366 T441 T442 themeOf HSP27,phosphorylation
R367 T442 T443 themeOf phosphorylation,regulate
R368 T443 T444 themeOf regulate,required
R369 T445 T448 causeOf PKD enzymes,regulate
R370 T447 T448 themeOf B cell antigen receptor,regulate
R2996 T3687 T3690 themeOf PKD1,knockout
R2997 T3688 T3691 themeOf PKD3,knockout
R2998 T3689 T3692 themeOf phosphorylation,increase
R3000 T3693 T3702 themeOf BCR,phosphorylation
R3002 T3693 T3698 partOf BCR,S82

bionlp-st-ge-2016-reference

Id Subject Object Predicate Lexical cue Negation Speculation
T2250 6651-6656 Protein denotes HSP27
T2251 6657-6672 Phosphorylation denotes phosphorylation
T2252 6691-6695 Protein denotes PKD3
T2253 6707-6711 Protein denotes PKD1
T2254 6716-6724 Regulation denotes regulate
T2255 6716-6724 Regulation denotes regulate
T2256 6725-6730 Protein denotes HSP27
T2257 6731-6746 Phosphorylation denotes phosphorylation
T2258 6802-6807 Protein denotes HSP27
T4955 9603-9606 Protein denotes Akt
T4956 9608-9612 Protein denotes mTOR
T4957 9640-9660 Protein denotes S6 ribosomal protein
T4958 9661-9676 Phosphorylation denotes phosphorylation
T4959 9719-9729 Negative_regulation denotes detectable true
T4960 9719-9729 Negative_regulation denotes detectable true
T4961 9719-9729 Negative_regulation denotes detectable true
T4962 9719-9729 Negative_regulation denotes detectable true
T4963 9719-9729 Negative_regulation denotes detectable true
T4964 9719-9729 Negative_regulation denotes detectable true
T4965 9733-9737 Protein denotes PKD1
T4966 9738-9739 Protein denotes 3
T4967 9740-9744 Negative_regulation denotes null
T4968 9740-9744 Negative_regulation denotes null
T4969 9943-9947 Protein denotes PKD1
T4970 9948-9949 Protein denotes 3
T4971 10071-10075 Protein denotes Erk1
T4972 10076-10080 Protein denotes RSK1
T4973 10115-10119 Protein denotes PKD1
T4974 10120-10121 Protein denotes 3
T4975 10460-10464 Protein denotes PKD1
T4976 10465-10466 Protein denotes 3
T4977 10475-10479 Protein denotes PKD3
T4978 10559-10563 Protein denotes PKD1
T4979 10564-10565 Protein denotes 3
T4980 10574-10578 Protein denotes PKD1
T4981 10579-10580 Protein denotes 3
T4982 10589-10593 Protein denotes PKD3
T4983 10783-10787 Protein denotes PKD1
T4984 10788-10789 Protein denotes 3
T4985 11096-11100 Protein denotes PKD1
T4986 11101-11102 Protein denotes 3
T4987 11201-11205 Protein denotes PKD1
T4988 11206-11207 Protein denotes 3
T4989 11377-11381 Negative_regulation denotes loss
T4990 11377-11381 Negative_regulation denotes loss
T4991 11385-11389 Protein denotes PKD1
T4992 11390-11391 Protein denotes 3
T4993 11626-11634 Protein denotes MAPKAPK2
T4994 12250-12266 Protein denotes Bcr-Abl oncogene
T4995 12298-12324 Positive_regulation denotes expression of an activated
T4996 12325-12329 Protein denotes PKD1
T4997 12346-12350 Protein denotes HPK1
T4998 12436-12440 Protein denotes PKCβ
T4999 12634-12638 Protein denotes PKD1
T5000 12639-12640 Protein denotes 3
T5001 12803-12808 Protein denotes PKCβ−
T5002 12893-12912 Phosphorylation denotes autophosphorylation
T5003 12916-12920 Protein denotes PKD1
T5004 12924-12928 Entity denotes S916
T5005 12934-12942 Positive_regulation denotes observed
T5006 12950-12955 Protein denotes PKCβ−
T10 678-686 Positive_regulation denotes required
T11 690-698 Regulation denotes regulate
T12 699-704 Protein denotes HSP27
T13 705-720 Phosphorylation denotes phosphorylation
T2177 4547-4551 Negative_regulation denotes Loss true
T2178 4555-4560 Protein denotes HSP27
T2179 4561-4576 Phosphorylation denotes phosphorylation
T2180 4647-4654 Gene_expression denotes express
T2181 4647-4654 Gene_expression denotes express
T2182 4673-4677 Protein denotes PKD1
T2183 4682-4686 Protein denotes PKD3
T2184 4766-4770 Negative_regulation denotes lack
T2185 4766-4770 Negative_regulation denotes lack
T2186 4771-4781 Gene_expression denotes expression
T2187 4771-4781 Gene_expression denotes expression
T2188 4792-4796 Protein denotes PKD1
T2189 4800-4804 Protein denotes PKD3
T2190 4887-4891 Protein denotes PKD1
T2191 4902-4906 Protein denotes PKD3
T2192 4925-4934 Gene_expression denotes expressed
T2193 4942-4946 Protein denotes PKD3
T2194 5056-5060 Protein denotes PKD3
T2195 5061-5071 Gene_expression denotes expression
T2196 5075-5079 Protein denotes PKD1
T2197 5080-5081 Protein denotes 3
T2198 5082-5097 Negative_regulation denotes double knockout
T2199 5082-5097 Negative_regulation denotes double knockout
T2200 5132-5136 Protein denotes PKD3
T2201 5439-5443 Protein denotes PKD1
T2202 5444-5445 Protein denotes 3
T2203 5551-5556 Protein denotes HSP27
T2204 5589-5593 Protein denotes PKD1
T2205 5670-5679 Negative_regulation denotes defective
T2206 5680-5695 Phosphorylation denotes phosphorylation
T2207 5699-5704 Protein denotes HSP27
T2208 5708-5717 Entity denotes serine 82
T2209 5732-5736 Protein denotes PKD1
T2210 5787-5797 Regulation denotes regulation true
T2211 5801-5806 Protein denotes HSP27
T2212 5807-5822 Phosphorylation denotes phosphorylation
T2213 5855-5862 Negative_regulation denotes lacking
T2214 5855-5862 Negative_regulation denotes lacking
T2215 5870-5874 Protein denotes PKD1
T2216 5878-5882 Protein denotes PKD3
T2217 5966-5975 Positive_regulation denotes increased
T2218 5990-5995 Protein denotes HSP27
T2219 5996-6011 Phosphorylation denotes phosphorylation
T2220 6015-6018 Entity denotes S82
T2221 6094-6102 Positive_regulation denotes increase
T2222 6103-6108 Protein denotes HSP27
T2223 6109-6124 Phosphorylation denotes phosphorylation
T2224 6128-6132 Protein denotes PKD1
T2225 6136-6140 Protein denotes PKD3
T2226 6148-6156 Negative_regulation denotes knockout
T2227 6148-6156 Negative_regulation denotes knockout
T2228 6213-6220 Positive_regulation denotes induced
T2229 6221-6236 Phosphorylation denotes phosphorylation
T2230 6240-6245 Protein denotes HSP27
T2231 6249-6252 Entity denotes S82
T2232 6257-6266 Negative_regulation denotes abolished
T2233 6257-6266 Negative_regulation denotes abolished
T2234 6283-6289 Negative_regulation denotes lacked
T2235 6283-6289 Negative_regulation denotes lacked
T2236 6295-6299 Protein denotes PKD1
T2237 6304-6308 Protein denotes PKD3
T2238 6347-6354 Positive_regulation denotes induced
T2239 6355-6365 Gene_expression denotes expression
T2240 6378-6382 Protein denotes PKD3
T2241 6426-6447 Positive_regulation denotes sufficient to restore
T2242 6455-6465 Regulation denotes regulation
T2243 6469-6474 Protein denotes HSP27
T2244 6475-6490 Phosphorylation denotes phosphorylation
T2245 6515-6525 Gene_expression denotes expression
T2246 6538-6547 Negative_regulation denotes deficient
T2247 6548-6552 Protein denotes PKD3
T2248 6613-6620 Positive_regulation denotes restore true
T2249 6643-6650 Positive_regulation denotes induced
T3783 7182-7186 Protein denotes PKD1
T3784 7187-7188 Protein denotes 3
T3785 7198-7202 Protein denotes PKD3
T3786 7219-7223 Protein denotes PKD1
T3787 7224-7225 Protein denotes 3
T3788 7300-7304 Protein denotes PKD1
T3789 7305-7306 Protein denotes 3
T3790 7347-7360 Gene_expression denotes re-expression
T3791 7369-7373 Protein denotes PKD3
T3792 7463-7467 Protein denotes PKD1
T3793 7468-7469 Protein denotes 3
T3794 7637-7641 Protein denotes PKD1
T3795 7642-7643 Protein denotes 3
T3796 7761-7765 Protein denotes PKD3
T3797 7766-7779 Gene_expression denotes re-expression
T3798 7816-7820 Protein denotes PKD1
T3799 7821-7822 Protein denotes 3
T3800 8109-8113 Protein denotes PKD1
T3801 8118-8122 Protein denotes PKD2
T3802 8438-8442 Negative_regulation denotes loss
T3803 8438-8442 Negative_regulation denotes loss
T3804 8446-8450 Protein denotes PKD1
T3805 8451-8452 Protein denotes 3
T3806 8453-8463 Gene_expression denotes expression
T3807 8453-8463 Gene_expression denotes expression
T476 1131-1135 Protein denotes PKD1
T477 1137-1141 Protein denotes PKD2
T478 1146-1150 Protein denotes PKD3
T479 1755-1762 Binding denotes binding
T480 1775-1786 Positive_regulation denotes contributes
T481 1790-1794 Protein denotes PKD1
T482 1795-1805 Positive_regulation denotes activation
T483 2413-2417 Protein denotes PKD1
T484 2431-2436 Protein denotes HSP27
T485 2640-2644 Protein denotes MEF2
T486 3193-3206 Positive_regulation denotes indispensable
T487 3211-3216 Protein denotes HSP27
T488 3217-3232 Phosphorylation denotes phosphorylation
T1662 3832-3836 Protein denotes PKD1
T1663 3841-3845 Protein denotes PKD3
T1664 3853-3857 Protein denotes PKD1
T1665 3858-3859 Protein denotes 3
T1666 3863-3871 Negative_regulation denotes knockout
T1667 3863-3871 Negative_regulation denotes knockout
T1668 3863-3871 Negative_regulation denotes knockout
T1669 3863-3871 Negative_regulation denotes knockout
T6995 13183-13189 Entity denotes serine
T6996 13220-13228 Regulation denotes regulate true
T6997 13337-13352 Phosphorylation denotes phosphorylation
T6998 13356-13361 Protein denotes HSP27
T6999 14153-14163 Regulation denotes regulating
T7000 14168-14183 Phosphorylation denotes phosphorylation
T7001 14187-14192 Protein denotes HSP27
T7002 14196-14205 Entity denotes serine 82
T7003 14241-14247 Regulation denotes target
T7004 14260-14268 Protein denotes MAPKAPK2
T7005 14650-14654 Protein denotes PKD1
T7006 14655-14656 Protein denotes 2
T4942 9272-9276 Protein denotes PKD1
T4943 9277-9278 Protein denotes 3
T4944 9290-9294 Protein denotes PKD1
T4945 9295-9296 Protein denotes 3
T4946 9305-9309 Protein denotes PKD3
T4947 9419-9423 Protein denotes PKD1
T4948 9424-9425 Protein denotes 3
T4949 9577-9584 Positive_regulation denotes induced
T4950 9577-9584 Positive_regulation denotes induced
T4951 9577-9584 Positive_regulation denotes induced
T4952 9585-9595 Positive_regulation denotes activation
T4953 9585-9595 Positive_regulation denotes activation
T4954 9585-9595 Positive_regulation denotes activation
R1883 T2253 T2254 causeOf PKD1,regulate
R1884 T2256 T2257 themeOf HSP27,phosphorylation
R1885 T2257 T2255 themeOf phosphorylation,regulate
R1886 T2257 T2254 themeOf phosphorylation,regulate
R371 T11 T10 themeOf regulate,required
R372 T12 T13 themeOf HSP27,phosphorylation
R373 T13 T11 themeOf phosphorylation,regulate
R1839 T2186 T2184 themeOf expression,lack
R1842 T2189 T2186 themeOf PKD3,expression
R1843 T2193 T2192 themeOf PKD3,expressed
R1844 T2194 T2195 themeOf PKD3,expression
R1845 T2196 T2199 themeOf PKD1,double knockout
R1846 T2197 T2198 themeOf 3,double knockout
R1847 T2206 T2205 themeOf phosphorylation,defective
R1848 T2208 T2206 themeOf serine 82,phosphorylation
R1849 T2208 T2207 partOf serine 82,HSP27
R1850 T2211 T2212 themeOf HSP27,phosphorylation
R1851 T2212 T2210 themeOf phosphorylation,regulation
R1852 T2215 T2213 themeOf PKD1,lacking
R1853 T2216 T2214 themeOf PKD3,lacking
R1854 T2219 T2217 themeOf phosphorylation,increased
R1855 T2220 T2219 themeOf S82,phosphorylation
R1856 T2220 T2218 partOf S82,HSP27
R1857 T2222 T2223 themeOf HSP27,phosphorylation
R1858 T2223 T2221 themeOf phosphorylation,increase
R1835 T2178 T2179 themeOf HSP27,phosphorylation
R1836 T2179 T2177 themeOf phosphorylation,Loss
R1837 T2182 T2180 themeOf PKD1,express
R1838 T2183 T2181 themeOf PKD3,express
R1840 T2187 T2185 themeOf expression,lack
R1841 T2188 T2187 themeOf PKD1,expression
R1859 T2224 T2227 themeOf PKD1,knockout
R1860 T2225 T2226 themeOf PKD3,knockout
R1861 T2228 T2232 themeOf induced,abolished
R1862 T2228 T2233 themeOf induced,abolished
R1863 T2229 T2228 themeOf phosphorylation,induced
R1864 T2231 T2229 themeOf S82,phosphorylation
R1865 T2231 T2230 partOf S82,HSP27
R1866 T2234 T2233 causeOf lacked,abolished
R1867 T2235 T2232 causeOf lacked,abolished
R1868 T2236 T2235 themeOf PKD1,lacked
R1869 T2237 T2234 themeOf PKD3,lacked
R1870 T2238 T2241 causeOf induced,sufficient to restore
R1871 T2239 T2238 themeOf expression,induced
R1872 T2240 T2239 themeOf PKD3,expression
R1873 T2242 T2241 themeOf regulation,sufficient to restore
R1874 T2243 T2244 themeOf HSP27,phosphorylation
R1875 T2244 T2242 themeOf phosphorylation,regulation
R1876 T2245 T2248 causeOf expression,restore
R1877 T2247 T2245 themeOf PKD3,expression
R1878 T2247 T2246 themeOf PKD3,deficient
R1879 T2249 T2248 themeOf induced,restore
R1880 T2250 T2251 themeOf HSP27,phosphorylation
R1881 T2251 T2249 themeOf phosphorylation,induced
R1882 T2252 T2255 causeOf PKD3,regulate
R3050 T3791 T3790 themeOf PKD3,re-expression
R3051 T3796 T3797 themeOf PKD3,re-expression
R3052 T3804 T3806 themeOf PKD1,expression
R3053 T3805 T3807 themeOf 3,expression
R3054 T3806 T3803 themeOf expression,loss
R3055 T3807 T3802 themeOf expression,loss
R424 T479 T480 causeOf binding,contributes
R425 T481 T479 themeOf PKD1,binding
R426 T481 T482 themeOf PKD1,activation
R427 T482 T480 themeOf activation,contributes
R428 T487 T488 themeOf HSP27,phosphorylation
R429 T488 T486 themeOf phosphorylation,indispensable
R1433 T1662 T1666 themeOf PKD1,knockout
R1434 T1663 T1667 themeOf PKD3,knockout
R1435 T1664 T1668 themeOf PKD1,knockout
R1436 T1665 T1669 themeOf 3,knockout
R5524 T6995 T6997 themeOf serine,phosphorylation
R5525 T6995 T6998 partOf serine,HSP27
R5526 T6997 T6996 themeOf phosphorylation,regulate
R5527 T7000 T6999 themeOf phosphorylation,regulating
R5528 T7002 T7000 themeOf serine 82,phosphorylation
R5529 T7002 T7001 partOf serine 82,HSP27
R5530 T7002 T7003 themeOf serine 82,target
R3904 T4949 T4959 themeOf induced,detectable
R3905 T4949 T4961 themeOf induced,detectable
R3906 T4950 T4964 themeOf induced,detectable
R3907 T4950 T4963 themeOf induced,detectable
R3908 T4951 T4960 themeOf induced,detectable
R3909 T4951 T4962 themeOf induced,detectable
R3910 T4952 T4951 themeOf activation,induced
R3911 T4953 T4949 themeOf activation,induced
R3912 T4954 T4950 themeOf activation,induced
R3913 T4955 T4954 themeOf Akt,activation
R3914 T4956 T4953 themeOf mTOR,activation
R3915 T4957 T4958 themeOf S6 ribosomal protein,phosphorylation
R3916 T4958 T4952 themeOf phosphorylation,activation
R3917 T4965 T4968 themeOf PKD1,null
R3918 T4966 T4967 themeOf 3,null
R3919 T4967 T4964 causeOf null,detectable
R3920 T4967 T4961 causeOf null,detectable
R3921 T4967 T4962 causeOf null,detectable
R3922 T4968 T4959 causeOf null,detectable
R3923 T4968 T4960 causeOf null,detectable
R3924 T4968 T4963 causeOf null,detectable
R3925 T4991 T4990 themeOf PKD1,loss
R3926 T4992 T4989 themeOf 3,loss
R3927 T4996 T4995 themeOf PKD1,expression of an activated
R3928 T5002 T5005 themeOf autophosphorylation,observed
R3929 T5004 T5002 themeOf S916,autophosphorylation
R3930 T5004 T5003 partOf S916,PKD1

bionlp-st-ge-2016-uniprot

Id Subject Object Predicate Lexical cue
T1737 3853-3857 P98161 denotes PKD1
T2676 4555-4560 P04792 denotes HSP27
T2677 4673-4677 P98161 denotes PKD1
T2678 4682-4686 Q99853 denotes PKD3
T2679 4792-4796 P98161 denotes PKD1
T2680 4800-4804 Q99853 denotes PKD3
T2681 4887-4891 P98161 denotes PKD1
T2682 4902-4906 Q99853 denotes PKD3
T2683 4942-4946 Q99853 denotes PKD3
T2684 5056-5060 Q99853 denotes PKD3
T2685 5075-5079 P98161 denotes PKD1
T2686 5132-5136 Q99853 denotes PKD3
T2687 5408-5411 P11274 denotes BCR
T2688 5439-5443 P98161 denotes PKD1
T2689 5551-5556 P04792 denotes HSP27
T2690 5589-5593 P98161 denotes PKD1
T2691 5699-5704 P04792 denotes HSP27
T2692 5732-5736 P98161 denotes PKD1
T2693 5801-5806 P04792 denotes HSP27
T4193 7182-7186 P98161 denotes PKD1
T4194 7198-7202 Q99853 denotes PKD3
T4195 7219-7223 P98161 denotes PKD1
T4196 7300-7304 P98161 denotes PKD1
T4197 7369-7373 Q99853 denotes PKD3
T4198 7463-7467 P98161 denotes PKD1
T4199 7637-7641 P98161 denotes PKD1
T4200 7761-7765 Q99853 denotes PKD3
T4201 7816-7820 P98161 denotes PKD1
T4202 8109-8113 P98161 denotes PKD1
T4203 8446-8450 P98161 denotes PKD1
T4204 8643-8645 P03372 denotes ER
T5705 9121-9124 P11274 denotes BCR
T5706 9253-9256 P11274 denotes BCR
T5707 9272-9276 P98161 denotes PKD1
T5708 9290-9294 P98161 denotes PKD1
T5709 9305-9309 Q99853 denotes PKD3
T5710 9399-9402 P11274 denotes BCR
T5711 9419-9423 P98161 denotes PKD1
T5712 9573-9576 P11274 denotes BCR
T5713 9603-9606 P31751 denotes Akt
T5714 9603-9606 Q9Y243 denotes Akt
T5715 9603-9606 P31749 denotes Akt
T5716 9608-9612 P42345 denotes mTOR
T5717 9733-9737 P98161 denotes PKD1
T5718 9924-9927 P11274 denotes BCR
T5719 9943-9947 P98161 denotes PKD1
T5720 10017-10020 P11274 denotes BCR
T5721 10071-10075 P27361 denotes Erk1
T5722 10115-10119 P98161 denotes PKD1
T5723 10408-10411 P11274 denotes BCR
T5724 10460-10464 P98161 denotes PKD1
T5725 10475-10479 Q99853 denotes PKD3
T5726 10530-10533 P11274 denotes BCR
T5727 10559-10563 P98161 denotes PKD1
T5728 10574-10578 P98161 denotes PKD1
T5729 10589-10593 Q99853 denotes PKD3
T5730 10783-10787 P98161 denotes PKD1
T5731 10982-10985 P11274 denotes BCR
T5732 11096-11100 P98161 denotes PKD1
T5733 11201-11205 P98161 denotes PKD1
T5734 11385-11389 P98161 denotes PKD1
T5735 11804-11807 P11274 denotes BCR
T5736 12250-12253 P11274 denotes Bcr
T5737 12254-12257 P00519 denotes Abl
T5738 12325-12329 P98161 denotes PKD1
T5739 12634-12638 P98161 denotes PKD1
T5740 12696-12699 P11274 denotes BCR
T5741 12726-12729 P11274 denotes BCR
T5742 12916-12920 P98161 denotes PKD1
T5743 13041-13044 P11274 denotes BCR
T7359 13356-13361 P04792 denotes HSP27
T7360 13822-13830 Q04864 denotes relative
T7361 14187-14192 P04792 denotes HSP27
T7362 14256-14259 Q16539 denotes p38
T7363 14256-14259 Q15759 denotes p38
T7364 14256-14259 P53778 denotes p38
T7365 14256-14259 O15264 denotes p38
T7366 14650-14654 P98161 denotes PKD1
T7367 15100-15103 P11274 denotes BCR
T986 1131-1135 P98161 denotes PKD1
T987 1146-1150 Q99853 denotes PKD3
T988 1790-1794 P98161 denotes PKD1
T989 2413-2417 P98161 denotes PKD1
T990 2431-2436 P04792 denotes HSP27
T991 2953-2956 P11274 denotes BCR
T992 3211-3216 P04792 denotes HSP27
T1735 3832-3836 P98161 denotes PKD1
T1736 3841-3845 Q99853 denotes PKD3
T204 699-704 P04792 denotes HSP27
T2694 5870-5874 P98161 denotes PKD1
T2695 5878-5882 Q99853 denotes PKD3
T2696 5923-5926 P11274 denotes BCR
T2697 5990-5995 P04792 denotes HSP27
T2698 6046-6049 P11274 denotes BCR
T2699 6086-6090 P00519 denotes able
T2700 6103-6108 P04792 denotes HSP27
T2701 6128-6132 P98161 denotes PKD1
T2702 6136-6140 Q99853 denotes PKD3
T2703 6190-6193 P11274 denotes BCR
T2704 6240-6245 P04792 denotes HSP27
T2705 6295-6299 P98161 denotes PKD1
T2706 6304-6308 Q99853 denotes PKD3
T2707 6378-6382 Q99853 denotes PKD3
T2708 6469-6474 P04792 denotes HSP27
T2709 6548-6552 Q99853 denotes PKD3
T2710 6605-6609 P00519 denotes able
T2711 6621-6624 P11274 denotes BCR
T2712 6651-6656 P04792 denotes HSP27
T2713 6691-6695 Q99853 denotes PKD3
T2714 6707-6711 P98161 denotes PKD1
T2715 6725-6730 P04792 denotes HSP27
T2716 6802-6807 P04792 denotes HSP27

test2

Id Subject Object Predicate Lexical cue Negation Speculation
T3755 7182-7186 Protein denotes PKD1
T3756 7187-7188 Protein denotes 3
T5 678-686 Positive_regulation denotes required
T6 690-698 Regulation denotes regulate
T7 699-704 Protein denotes HSP27
T8 705-720 Phosphorylation denotes phosphorylation
T463 1131-1135 Protein denotes PKD1
T464 1137-1141 Protein denotes PKD2
T465 1146-1150 Protein denotes PKD3
T466 1755-1762 Binding denotes binding
T467 1775-1786 Positive_regulation denotes contributes
T468 1790-1794 Protein denotes PKD1
T469 1795-1805 Positive_regulation denotes activation
T470 2413-2417 Protein denotes PKD1
T471 2431-2436 Protein denotes HSP27
T472 2640-2644 Protein denotes MEF2
T473 3193-3206 Positive_regulation denotes indispensable
T474 3211-3216 Protein denotes HSP27
T475 3217-3232 Phosphorylation denotes phosphorylation
T1658 3832-3836 Protein denotes PKD1
T1659 3841-3845 Protein denotes PKD3
T1660 3853-3857 Protein denotes PKD1
T1661 3858-3859 Protein denotes 3
T2103 4555-4560 Protein denotes HSP27 true
T2104 4561-4576 Phosphorylation denotes phosphorylation
T2105 4647-4654 Gene_expression denotes express
T2106 4673-4677 Protein denotes PKD1
T2107 4682-4686 Protein denotes PKD3
T2108 4766-4770 Negative_regulation denotes lack
T2109 4771-4781 Gene_expression denotes expression
T2110 4792-4796 Protein denotes PKD1
T2111 4800-4804 Protein denotes PKD3
T2112 4887-4891 Protein denotes PKD1
T2113 4902-4906 Protein denotes PKD3
T2114 4925-4934 Gene_expression denotes expressed
T2115 4942-4946 Protein denotes PKD3
T2116 5056-5060 Protein denotes PKD3
T2117 5061-5071 Gene_expression denotes expression true
T2118 5075-5079 Protein denotes PKD1
T2119 5080-5081 Protein denotes 3
T2120 5089-5097 Negative_regulation denotes knockout
T2121 5132-5136 Protein denotes PKD3
T2122 5439-5443 Protein denotes PKD1
T2123 5444-5445 Protein denotes 3
T2124 5551-5556 Protein denotes HSP27
T2125 5589-5593 Protein denotes PKD1
T2126 5670-5679 Negative_regulation denotes defective
T2127 5680-5695 Phosphorylation denotes phosphorylation
T2128 5699-5704 Protein denotes HSP27
T2129 5708-5714 Entity denotes serine
T2130 5732-5736 Protein denotes PKD1
T2131 5787-5797 Regulation denotes regulation
T2132 5801-5806 Protein denotes HSP27
T2133 5807-5822 Phosphorylation denotes phosphorylation
T2134 5833-5841 Negative_regulation denotes knockout
T2135 5855-5862 Negative_regulation denotes lacking
T2136 5870-5874 Protein denotes PKD1
T2137 5878-5882 Protein denotes PKD3
T2138 5966-5975 Positive_regulation denotes increased
T2139 5990-5995 Protein denotes HSP27
T2140 5996-6011 Phosphorylation denotes phosphorylation true
T2141 6015-6018 Entity denotes S82
T2142 6094-6102 Positive_regulation denotes increase
T2143 6103-6108 Protein denotes HSP27
T2144 6109-6124 Phosphorylation denotes phosphorylation
T2145 6128-6132 Protein denotes PKD1
T2146 6136-6140 Protein denotes PKD3
T2147 6148-6156 Negative_regulation denotes knockout
T2148 6213-6220 Positive_regulation denotes induced
T2149 6221-6236 Phosphorylation denotes phosphorylation
T2150 6240-6245 Protein denotes HSP27
T2151 6249-6252 Entity denotes S82
T2152 6257-6266 Negative_regulation denotes abolished
T2153 6283-6289 Negative_regulation denotes lacked
T2154 6295-6299 Protein denotes PKD1
T2155 6304-6308 Protein denotes PKD3
T2156 6347-6354 Positive_regulation denotes induced
T2157 6355-6365 Gene_expression denotes expression
T2158 6378-6382 Protein denotes PKD3
T2159 6455-6465 Regulation denotes regulation
T2160 6469-6474 Protein denotes HSP27
T2161 6475-6490 Phosphorylation denotes phosphorylation
T2162 6515-6525 Gene_expression denotes expression
T2163 6531-6537 Negative_regulation denotes kinase
T2164 6538-6547 Negative_regulation denotes deficient
T2165 6548-6552 Protein denotes PKD3
T2166 6613-6620 Positive_regulation denotes restore
T2167 6643-6650 Positive_regulation denotes induced
T2168 6651-6656 Protein denotes HSP27
T2169 6657-6672 Phosphorylation denotes phosphorylation
T2170 6691-6695 Protein denotes PKD3
T2171 6707-6711 Protein denotes PKD1
T2172 6716-6724 Regulation denotes regulate
T2173 6725-6730 Protein denotes HSP27
T2174 6731-6746 Phosphorylation denotes phosphorylation
T2175 6789-6798 Negative_regulation denotes redundant
T2176 6802-6807 Protein denotes HSP27
T3757 7198-7202 Protein denotes PKD3
T3758 7219-7223 Protein denotes PKD1
T3759 7224-7225 Protein denotes 3
T3760 7300-7304 Protein denotes PKD1
T3761 7305-7306 Protein denotes 3
T3762 7347-7349 Positive_regulation denotes re
T3763 7349-7350 Gene_expression denotes -
T3764 7350-7360 Gene_expression denotes expression
T3765 7369-7373 Protein denotes PKD3
T3766 7463-7467 Protein denotes PKD1
T3767 7468-7469 Protein denotes 3
T3768 7637-7641 Protein denotes PKD1
T3769 7642-7643 Protein denotes 3
T3770 7761-7765 Protein denotes PKD3
T3771 7766-7769 Gene_expression denotes re-
T3772 7769-7779 Gene_expression denotes expression
T3773 7816-7820 Protein denotes PKD1
T3774 7821-7822 Protein denotes 3
T3775 8109-8113 Protein denotes PKD1
T3776 8118-8122 Protein denotes PKD2
T3777 8438-8442 Negative_regulation denotes loss
T3778 8446-8450 Protein denotes PKD1
T3779 8451-8452 Protein denotes 3
T3780 8453-8463 Gene_expression denotes expression
T3781 8775-8784 Negative_regulation denotes inhibitor
T4882 9235-9245 Gene_expression denotes expression
T4883 9261-9268 Negative_regulation denotes reduced
T4884 9272-9276 Protein denotes PKD1
T4885 9277-9278 Protein denotes 3
T4886 9290-9294 Protein denotes PKD1
T4887 9295-9296 Protein denotes 3
T4888 9305-9309 Protein denotes PKD3
T4889 9419-9423 Protein denotes PKD1 true
T4890 9424-9425 Protein denotes 3
T4891 9577-9584 Positive_regulation denotes induced true
T4892 9585-9595 Positive_regulation denotes activation
T4893 9603-9606 Protein denotes Akt true
T4894 9608-9612 Protein denotes mTOR true
T4895 9640-9660 Protein denotes S6 ribosomal protein true
T4896 9661-9676 Phosphorylation denotes phosphorylation true
T4897 9733-9737 Protein denotes PKD1
T4898 9738-9739 Protein denotes 3
T4899 9740-9744 Negative_regulation denotes null
T4900 9943-9947 Protein denotes PKD1
T4901 9948-9949 Protein denotes 3
T4902 9950-9954 Negative_regulation denotes null
T4903 10053-10063 Regulation denotes regulation
T4904 10071-10075 Protein denotes Erk1
T4905 10076-10080 Protein denotes RSK1
T4906 10104-10111 Negative_regulation denotes reduced
T4907 10115-10119 Protein denotes PKD1
T4908 10120-10121 Protein denotes 3
T4909 10412-10419 Positive_regulation denotes induced
T4910 10424-10439 Phosphorylation denotes phosphorylation
T4911 10449-10456 Negative_regulation denotes reduced
T4912 10460-10464 Protein denotes PKD1
T4913 10465-10466 Protein denotes 3
T4914 10475-10479 Protein denotes PKD3
T4915 10559-10563 Protein denotes PKD1
T4916 10564-10565 Protein denotes 3
T4917 10574-10578 Protein denotes PKD1
T4918 10579-10580 Protein denotes 3
T4919 10589-10593 Protein denotes PKD3
T4920 10783-10787 Protein denotes PKD1
T4921 10788-10789 Protein denotes 3
T4922 11096-11100 Protein denotes PKD1
T4923 11101-11102 Protein denotes 3
T4924 11201-11205 Protein denotes PKD1
T4925 11206-11207 Protein denotes 3
T4926 11377-11381 Negative_regulation denotes loss
T4927 11385-11389 Protein denotes PKD1
T4928 11390-11391 Protein denotes 3
T4929 11626-11634 Protein denotes MAPKAPK2
T4930 12250-12266 Protein denotes Bcr-Abl oncogene
T4931 12315-12324 Positive_regulation denotes activated
T4932 12325-12329 Protein denotes PKD1
T4933 12346-12350 Protein denotes HPK1
T4934 12414-12423 Regulation denotes regulated
T4935 12436-12440 Protein denotes PKCβ
T4936 12634-12638 Protein denotes PKD1
T4937 12639-12640 Protein denotes 3
T4938 12803-12808 Protein denotes PKCβ−
T4939 12893-12912 Phosphorylation denotes autophosphorylation
T4940 12916-12920 Protein denotes PKD1
T4941 12950-12955 Protein denotes PKCβ−
T6982 13270-13285 Phosphorylation denotes phosphorylation true
T6983 13290-13297 Entity denotes nuclear
T6984 13298-13310 Localization denotes localisation
T6985 13337-13352 Phosphorylation denotes phosphorylation
T6986 13356-13361 Protein denotes HSP27
T6987 14136-14140 Regulation denotes role
T6988 14153-14163 Regulation denotes regulating
T6989 14168-14183 Phosphorylation denotes phosphorylation
T6990 14187-14192 Protein denotes HSP27
T6991 14196-14205 Entity denotes serine 82
T6992 14260-14268 Protein denotes MAPKAPK2
T6993 14650-14654 Protein denotes PKD1
T6994 14655-14656 Protein denotes 2
R1820 T2154 T2153 themeOf PKD1,lacked
R1821 T2155 T2153 themeOf PKD3,lacked
R1822 T2157 T2156 themeOf expression,induced
R1823 T2158 T2157 themeOf PKD3,expression
R1825 T2161 T2159 themeOf phosphorylation,regulation
R4 T6 T5 themeOf regulate,required
R5 T7 T8 themeOf HSP27,phosphorylation
R6 T8 T6 themeOf phosphorylation,regulate
R419 T466 T467 causeOf binding,contributes
R420 T468 T469 themeOf PKD1,activation
R421 T469 T467 themeOf activation,contributes
R422 T474 T475 themeOf HSP27,phosphorylation
R423 T475 T473 themeOf phosphorylation,indispensable
R1790 T2103 T2104 themeOf HSP27,phosphorylation
R1791 T2106 T2105 themeOf PKD1,express
R1792 T2107 T2105 themeOf PKD3,express
R1793 T2109 T2108 themeOf expression,lack
R1794 T2110 T2109 themeOf PKD1,expression
R1795 T2111 T2109 themeOf PKD3,expression
R1796 T2115 T2114 themeOf PKD3,expressed
R1797 T2116 T2117 themeOf PKD3,expression
R1798 T2118 T2120 themeOf PKD1,knockout
R1799 T2127 T2126 themeOf phosphorylation,defective
R1800 T2132 T2133 themeOf HSP27,phosphorylation
R1801 T2133 T2131 themeOf phosphorylation,regulation
R1802 T2134 T2131 causeOf knockout,regulation
R1803 T2135 T2131 causeOf lacking,regulation
R1804 T2136 T2135 themeOf PKD1,lacking
R1805 T2137 T2135 themeOf PKD3,lacking
R1806 T2139 T2140 themeOf HSP27,phosphorylation
R1807 T2140 T2138 themeOf phosphorylation,increased
R1808 T2141 T2140 themeOf S82,phosphorylation
R1809 T2141 T2139 partOf S82,HSP27
R1810 T2143 T2144 themeOf HSP27,phosphorylation
R1811 T2144 T2142 themeOf phosphorylation,increase
R1812 T2145 T2147 themeOf PKD1,knockout
R1813 T2146 T2147 themeOf PKD3,knockout
R1814 T2148 T2152 themeOf induced,abolished
R1815 T2149 T2148 themeOf phosphorylation,induced
R1816 T2149 T2152 themeOf phosphorylation,abolished
R1817 T2151 T2149 themeOf S82,phosphorylation
R1818 T2151 T2150 partOf S82,HSP27
R1819 T2153 T2152 causeOf lacked,abolished
R1824 T2160 T2161 themeOf HSP27,phosphorylation
R1826 T2165 T2164 themeOf PKD3,deficient
R1827 T2165 T2162 themeOf PKD3,expression
R1828 T2167 T2166 themeOf induced,restore
R1829 T2168 T2169 themeOf HSP27,phosphorylation
R1830 T2169 T2167 themeOf phosphorylation,induced
R1831 T2170 T2172 causeOf PKD3,regulate
R1832 T2171 T2172 causeOf PKD1,regulate
R1833 T2173 T2174 themeOf HSP27,phosphorylation
R1834 T2174 T2172 themeOf phosphorylation,regulate
R3047 T3778 T3780 themeOf PKD1,expression
R3048 T3779 T3780 themeOf 3,expression
R3049 T3780 T3777 themeOf expression,loss
R3888 T4882 T4883 themeOf expression,reduced
R3889 T4892 T4891 themeOf activation,induced
R3890 T4893 T4892 themeOf Akt,activation
R3891 T4894 T4892 themeOf mTOR,activation
R3892 T4895 T4896 themeOf S6 ribosomal protein,phosphorylation
R3893 T4896 T4892 themeOf phosphorylation,activation
R3894 T4897 T4899 themeOf PKD1,null
R3895 T4898 T4899 themeOf 3,null
R3896 T4900 T4902 themeOf PKD1,null
R3897 T4901 T4902 themeOf 3,null
R3898 T4909 T4911 themeOf induced,reduced
R3899 T4910 T4909 themeOf phosphorylation,induced
R3900 T4910 T4911 themeOf phosphorylation,reduced
R3901 T4927 T4926 themeOf PKD1,loss
R3902 T4935 T4934 causeOf PKCβ,regulated
R3903 T4940 T4939 themeOf PKD1,autophosphorylation
R5519 T6983 T6984 locationOf nuclear,localisation
R5520 T6986 T6985 themeOf HSP27,phosphorylation
R5521 T6989 T6988 themeOf phosphorylation,regulating
R5522 T6991 T6990 partOf serine 82,HSP27
R5523 T6991 T6989 themeOf serine 82,phosphorylation