PMC:1942070 / 0-4551 JSONTXT 21 Projects

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Id Subject Object Predicate Lexical cue
T212 0-7 NN denotes Protein
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
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
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
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
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
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 .
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 .
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 .
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 .
T2731 4547-4551 NN denotes Loss
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
R1046 T1160 T1161 auxpass been,proposed
R1047 T1161 T1161 ROOT proposed,proposed
R1048 T1162 T1163 aux to,regulate
R1049 T1163 T1161 xcomp regulate,proposed
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
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
R1128 T1242 T1243 nummod 23,]
R1129 T1243 T1243 ROOT ],]
R1130 T1244 T1223 punct .,linked
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
R1175 T1289 T1289 ROOT enzymes,enzymes
R1176 T1290 T1291 mark that,repress
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
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
R1260 T1448 T1449 amod transcriptional,activity
R1261 T1374 T1375 aux have,shown
R1262 T1449 T1446 dobj activity,regulating
R1263 T1450 T1441 punct .,play
R1264 T1375 T1375 ROOT shown,shown
R1265 T1451 T1455 advmod Together,reveal
R1266 T1452 T1455 punct ",",reveal
R1267 T1376 T1378 mark that,are
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
R1275 T1377 T1378 nsubj PKDs,are
R1276 T1460 T1463 punct ",",are
R1277 T1461 T1462 compound PKD,kinases
R1278 T1462 T1463 nsubj kinases,are
R1279 T1378 T1375 ccomp are,shown
R1280 T1463 T1455 ccomp are,reveal
R1281 T1464 T1463 neg not,are
R1282 T1465 T1466 amod critical,regulators
R1283 T1466 T1463 attr regulators,are
R1284 T1379 T1378 acomp indispensable,are
R1285 T1467 T1466 prep of,regulators
R1286 T1468 T1467 pobj many,of
R1287 T1469 T1468 prep of,many
R1288 T1380 T1378 prep for,are
R1289 T1470 T1472 det the,processes
R1290 T1471 T1472 amod cellular,processes
R1291 T1472 T1469 pobj processes,of
R1292 T1381 T1382 compound HDAC,regulation
R1293 T1473 T1474 advmod previously,ascribed
R1294 T1474 T1463 conj ascribed,are
R1295 T1475 T1474 prep to,ascribed
R1296 T1382 T1380 pobj regulation,for
R1297 T1476 T1475 pobj them,to
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
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
R1516 T1756 T1754 conj activation,culture
R1517 T1757 T1752 prep of,generation
R1518 T1758 T1761 nummod PKD1,−
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
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
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
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
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
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
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