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PMC:1942070 / 0-4551 JSONTXT 21 Projects

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Id Subject Object Predicate Lexical cue
T429 0-24 Protein denotes Protein kinase D enzymes
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
T431 81-99 Regulation denotes receptor-regulated
T430 100-104 Protein denotes NFκB
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
T439 553-563 Regulation denotes regulation
T438 567-596 Protein denotes class II histone deacetylases
T440 664-668 Protein denotes PKDs
T444 678-686 Positive_regulation denotes required
T443 690-698 Regulation denotes regulate
T441 699-704 Protein denotes HSP27
T442 705-720 Phosphorylation denotes phosphorylation
T445 805-816 Protein denotes PKD enzymes
T448 824-832 Regulation denotes regulate
T446 907-911 Protein denotes NFκB
T447 955-978 Protein denotes B cell antigen receptor
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
T1596 1168-1175 Gene_expression denotes express
T1594 1189-1192 Protein denotes PKD
T1595 1206-1209 Protein denotes PKD
T1597 1240-1249 Gene_expression denotes expressed
T1598 1412-1416 Protein denotes PKDs
T1599 1444-1448 Protein denotes PLCγ
T1603 1509-1519 Positive_regulation denotes stimulates
T1604 1509-1519 Positive_regulation denotes stimulates
T1600 1543-1560 Protein denotes protein kinase Cs
T1601 1562-1565 Protein denotes PKC
T1605 1572-1585 Phosphorylation denotes phosphorylate
T1606 1572-1585 Phosphorylation denotes phosphorylate
T1602 1647-1658 Protein denotes PKD kinases
T1607 1702-1713 Protein denotes PKD enzymes
T1611 1755-1762 Binding denotes binding
T1608 1766-1769 Protein denotes DAG
T1614 1775-1786 Positive_regulation denotes contributes
T1609 1790-1794 Protein denotes PKD1
T1612 1795-1805 Positive_regulation denotes activation
T1615 1821-1831 Regulation denotes regulating
T1613 1844-1852 Localization denotes location
T1610 1856-1867 Protein denotes PKD enzymes
T1616 1889-1892 Protein denotes PKD
T1618 2062-2072 Gene_expression denotes Expression
T1619 2125-2134 Positive_regulation denotes activated
T1620 2125-2134 Positive_regulation denotes activated
T1617 2135-2139 Protein denotes PKDs
T1621 2239-2243 Protein denotes PKDs
T1623 2275-2285 Positive_regulation denotes activation
T1622 2293-2318 Protein denotes NFκB transcription factor
T1624 2413-2417 Protein denotes PKD1
T1625 2554-2565 Protein denotes PKD enzymes
T1629 2569-2579 Regulation denotes regulating
T1630 2569-2579 Regulation denotes regulating
T1626 2580-2609 Protein denotes class II histone deacetylases
T1627 2611-2616 Protein denotes HDACs
T1628 2640-2644 Protein denotes MEF2
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
T1643 3193-3206 Positive_regulation denotes indispensable
T1641 3211-3216 Protein denotes HSP27
T1642 3217-3232 Phosphorylation denotes phosphorylation
T1644 3254-3257 Protein denotes PKD
T1648 3323-3327 Negative_regulation denotes loss
T1645 3359-3362 Protein denotes PKD
T1646 3435-3446 Protein denotes PKD kinases
T1647 3484-3488 Protein denotes NFκB
T1649 3570-3581 Protein denotes PKD kinases
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
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
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
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