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T14 0-135 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells
T1 0-135 Sentence denotes Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells
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
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
T2 138-146 Sentence denotes Abstract
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.
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.
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.
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.
T17 615-646 Sentence denotes Mol. Cell Biol. 26, 1569–1577].
T6 620-630 Sentence denotes Cell Biol.
T7 631-646 Sentence denotes 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.
T18 647-737 Sentence denotes We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells.
T8 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.
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.
T20 980-1036 Sentence denotes Thus, PKDs have a selective role in DT40 B-cell biology.
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.
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].
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].
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].
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].
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].
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].
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].
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].
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]).
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]).
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].
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].
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].
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].
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].
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].
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.
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.
T499 3067-3158 Sentence denotes Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1].
T22 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.
T23 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.
T24 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.
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.
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.
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
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].
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].
T1672 3926-4038 Sentence denotes Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1].
T30 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].
T31 4039-4117 Sentence denotes Chloramphenicol acetyl transferase assays have been described previously [29].
T32 4119-4137 Sentence denotes 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.
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.
T1866 4336-4421 Sentence denotes The cells were washed twice and fluorescent intensity was analysed by flow cytometry.
T34 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.
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