PMC:1942070 / 0-6831
Annnotations
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 |
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 |
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 |
| 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 |
| 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 |
| R2277 | T2673 | T2674 | boundBy | two PKD isoforms,PKD1 |
| R2278 | T2673 | T2675 | boundBy | two PKD isoforms,PKD3 |
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 |
| 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 |
| 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 |
| 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 |
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 |
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 |
| 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 |
| 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 |
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 |
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 |
| 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 |
| 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. |
| 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. |
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 |
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 | ||
| 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 | ||
| 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 | ||
| 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 | ||
| 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 |
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 |
| 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 |
| 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 | ||
| 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 | ||
| 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 | ||
| 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 | ||
| 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 |
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 |
| 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 |
|---|---|---|---|---|---|---|
| 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 | ||
| 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 |