Protein Kinase D Specifically Mediates Apoptosis Signal-regulating Kinase 1-JNK Signaling Induced by H2O2 but Not Tumor Necrosis Factor (2005)

Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Gö6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Gö6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Gö6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Gö6976 but not by Gö6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling. Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Gö6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Gö6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Gö6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Gö6976 but not by Gö6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling. Increases in inflammatory cytokines such as tumor necrosis factor-α (TNF) 1The abbreviations used are: TNF, tumor necrosis factor-α; ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; ASK1, apoptosis signal-regulating kinase 1; EC, endothelial cell(s); PKD, protein kinase D; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; TRAF2, TNF receptor-associated factor 2; PH, pleckstrin homology; BAEC, bovine aortic EC(s); siRNA, small interfering RNA; GST, glutathione S-transferase; DAPI, 4′,6-diamidino-2-phenylindole; WT, wild type. and reactive oxygen species (ROS) represent common mechanism of atherosclerosis. The primary cells limiting inflammation is vascular endothelial cell(s) (EC). The signal transduction and resulting gene expression in response to TNF/ROS in EC have been extensively studied. TNF and ROS (H2O2 in cultured cells) activate overlapping of transcriptional factors and induce a common array of gene expression including EC adhesion molecules, coagulation factors, and matrix proteins leading to EC activation/dysfunction (1Madge L.A. Pober J.S. Exp. Mol. Pathol. 2001; 70: 317-325Crossref PubMed Scopus (263) Google Scholar, 2Irani K. Circ. Res. 2000; 87: 179-183Crossref PubMed Scopus (691) Google Scholar). However, TNF and ROS do have distinct effects on EC and induce distinct intracellular signaling. For example, TNF induced EC death/apoptosis only in the presence of protein synthesis inhibitor cycloheximide, which blocks TNF-induced NF-κB-dependent gene expression of survival factors. In contrast, H2O2 alone is sufficient to induce EC death. This is due, at least in part, to the fact that H2O2 induces the activation of c-Jun N-terminal kinase (JNK) but not of NF-κB in EC (3Bradley J.R. Johnson D.R. Pober J.S. Am. J. Pathol. 1993; 142: 1598-1609PubMed Google Scholar). Thus, it appears that stress-activated kinase cascade (JNK/p38) can be activated by both TNF and ROS. However, the signal transduction pathways utilized by H2O2 have not been fully defined. Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAPK) kinase kinase family, is an upstream activator of JNK and p38 MAPK signaling cascades (4Ichijo H. Nishida E. Irie K. Ten D.-P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2037) Google Scholar). ASK1 can be activated in response to diverse stresses including proinflammatory cytokine TNF, ROS, and death receptor Fas, disruption of microtubule structures, protein aggregation in endoplasmic reticulum, and genotoxic stress from nucleus (4Ichijo H. Nishida E. Irie K. Ten D.-P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2037) Google Scholar, 5Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (534) Google Scholar, 6Charette S.J. Lambert H. Landry J. J. Biol. Chem. 2001; 276: 36071-36074Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 7Hatai T. Matsuzawa A. Inoshita S. Mochida Y. Kuroda T. Sakamaki K. Kuida K. Yonehara S. Ichijo H. Takeda K. J. Biol. Chem. 2000; 275: 26576-26581Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 8Nishitoh H. Matsuzawa A. Tobiume K. Saegusa K. Takeda K. Inoue K. Hori S. Kakizuka A. Ichijo H. Genes Dev. 2002; 16: 1345-1355Crossref PubMed Scopus (1153) Google Scholar). In vitro data suggest that activation of ASK1 triggers various biological responses such as apoptosis, inflammation, differentiation, and survival in different cell types. Studies from ASK1-deficient mice indicate that ASK1 is critical for TNF- and ROS-induced apoptosis signaling (9Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1016) Google Scholar), suggesting that TNF and ROS converge, at least, at the level of MAPK kinase kinase (ASK1). The mechanism by which TNF/ROS activates ASK1 is not fully understood. The identification of proteins associated with ASK1 has provided some insights. ASK1 is a 170-kDa protein that functionally is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain. Several cellular factors including thioredoxin, glutaredoxin, and 14-3-3 have been reported to interacts with different ASK1 domains and inhibit ASK1 activity (10Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2092) Google Scholar, 11Liu H. Nishitoh H. Ichijo H. Kyriakis J.M. Mol. Cell. Biol. 2000; 20: 2198-2208Crossref PubMed Scopus (450) Google Scholar, 12Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 13Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (314) Google Scholar, 14Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar). TNF and H2O2 activate ASK1, in part, by dissociating these cellular inhibitors from ASK1. Several ASK1 upstream activators have been also identified. In response to TNF, the C-terminal domain of ASK1 binds to TNF receptor-associated factor 2 (TRAF2) and this association is required for ASK1 activation by TRAF2 (5Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (534) Google Scholar, 8Nishitoh H. Matsuzawa A. Tobiume K. Saegusa K. Takeda K. Inoue K. Hori S. Kakizuka A. Ichijo H. Genes Dev. 2002; 16: 1345-1355Crossref PubMed Scopus (1153) Google Scholar). It has been shown that ASK1 binding to TRAF2 may facilitate dissociation of ASK1 from thioredoxin. TRAF2 in turn induces ASK1 oligomerization, leading to autophosphorylation of ASK1 at Thr-845 within the activation loop in the kinase domain (15Tobiume K. Saitoh M. Ichijo H. J. Cell. Physiol. 2002; 191: 95-104Crossref PubMed Scopus (300) Google Scholar). Activated ASK1 subsequently recruits and activates its downstream targets MAP2K (MKK3/7 and MKK4/7) and MAPK (JNK and p38). Thus, ASK1 activation appears to involve several sequential steps including association of upstream activators (such as TRAF2 in response to TNF), release of cellular inhibitors (such as thioredoxin, glutaredoxin, glutathione S-transferase Mu, heat shock proteins, and 14-3-3), ASK1 oligomerization/autophosphorylation at Thr-845, and scaffold protein-mediated association of ASK1 with downstream MKK and JNK (16Matsuura H. Nishitoh H. Takeda K. Matsuzawa A. Amagasa T. Ito M. Yoshioka K. Ichijo H. J. Biol. Chem. 2002; 277: 40703-40709Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). However, the upstream activator mediating H2O2-induced ASK1 activation has not been identified. Protein kinase C (PKC) has been in the signal transduction pathways a of biological including in cell differentiation, and J. A. Y. T. S. J.R. T. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is and on activation by protein PKD, a member of from by its the domain and J. A. Y. T. S. J.R. T. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The N-terminal of PKD a pleckstrin homology domain and the in suggest that PKD is of different intracellular signaling pathways. In this study, we pharmacological inhibitors and small interfering RNA and identified PKD as a critical upstream mediator in H2O2- but not in TNF-induced ASK1 activation. we that 14-3-3 binding of PKD critical in mediating H2O2-induced ASK1-JNK activation and EC apoptosis. expression for PKD and ASK1 H. A. Mol. PubMed Scopus Google Scholar, H. A. Mol. Cell. Biol. PubMed Scopus Google Scholar, A. EMBO J. PubMed Scopus Google Scholar, L. J.M. J. Min W. Circ. Res. PubMed Scopus Google Scholar, X. W. M. Min W. J. Clin. Investig. PubMed Scopus Google Scholar). The PKD was by to the and at the by ASK1 was provided by J. Lee of J.J. Lee Y.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and and and from and from was from and and from and endothelial cells and bovine aortic from endothelial cells cultured in bovine endothelial cell 2 and at in on as L. J.M. J. Min W. Circ. Res. PubMed Scopus Google Scholar, X. W. M. Min W. J. Clin. Investig. PubMed Scopus Google Scholar). used at of and PKD and inhibitor from of was by to the in a and of cultured EC as L. J.M. J. Min W. Circ. Res. PubMed Scopus Google Scholar). or was an and to to the and and as L. J.M. J. Min W. Circ. Res. PubMed Scopus Google Scholar, X. W. M. Min W. J. Clin. Investig. PubMed Scopus Google Scholar). various with and in of 1 1 1 2 1 for on For to protein in of cell was by with on at The with the first PKD from for 2 with of by at for by with The to by with the protein The was an to the of the For of proteins and proteins (PKD), and used for protein and as L. J.M. J. Min W. Circ. Res. PubMed Scopus Google Scholar, X. W. M. Min W. J. Clin. Investig. PubMed Scopus Google Scholar). proteins in on of cell PKD at with to in the The with the the of PKD proteins on and by with and ASK1 was J.J. Lee Y.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in and of of in to of the cells the gene of with no of for of PKD to and from the of PKD and The RNA used as a for activity was from of with or PKD with to provided by the and of and was as cultured on and with H2O2 in the or presence of the PKD inhibitors as with to and survival cells on the For apoptosis cell with and cell a X. W. M. Min W. J. Clin. Investig. PubMed Scopus Google Scholar, Y. Min W. Circ. Res. 2002; PubMed Scopus (314) Google Scholar). as For kinase and apoptosis and gene at least with of was for different by the of cells to with by The of as of PKD H2O2- but TNF-induced JNK both TNF and H2O2 induce activation of JNK kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. the signaling by H2O2 in EC, we first the effects of a variety of pharmacological inhibitors on H2O2-induced JNK activation. in Gö6983 inhibits of the and Gö6976 inhibits the and with 1 Gö6983 or Gö6976 for to with H2O2 for or TNF for JNK activation was by with a Gö6983 had no effects on TNF- or H2O2-induced JNK activation Interestingly, Gö6976 specifically blocked H2O2- but not TNF-induced JNK activation inhibits with inhibits as well as that but not specifically H2O2- but not TNF-induced JNK activation In contrast, inhibitor blocked both H2O2 and TNF-induced JNK activation not L. J.M. J. Min W. Circ. Res. PubMed Scopus Google Scholar). data suggest that PKD but not be critical for H2O2-induced JNK activation. H2O2 but TNF of whether H2O2 induces PKD activation in EC, with H2O2 or TNF for various PKD activation at the activation loop was by with a a JNK phosphorylation and of for NF-κB also that TNF induced the activation of JNK and of but failed to induce PKD activation at of the examined In contrast, H2O2 induced activation of PKD as well as JNK, which at with D.R. A. S. Pober J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), TNF induced of at and by In contrast, H2O2 not induce NF-κB in EC (3Bradley J.R. Johnson D.R. Pober J.S. Am. J. Pathol. 1993; 142: 1598-1609PubMed Google Scholar). also examined the responses of H2O2 for and TNF for on PKD activation. TNF failed to induce PKD activation at of the H2O2 activates PKD at and not PKD phosphorylation H2O2, but TNF, of PKD, to of PKD with JNK has been shown that PKD activation a variety of translocation on the effects of H2O2 on PKD with H2O2 for the and PKD was by with The of PKD is on the of H2O2 induced PKD to a sequential translocation including from the cytoplasm membrane in the and of PKD in the nucleus in a The of PKD translocation in EC is to that was in cells in response to H2O2 O. E. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, TNF not induce these of PKD the translocation of PKD, we by the cells with and shown in of PKD with was at of PKD with was at H2O2 The translocation of PKD to whether PKD is with ASK1, a MAPK kinase kinase in H2O2-induced JNK activation. PKD and by or with H2O2 for the of PKD was in the of PKD and ASK1 was by with and PKD is on ASK1 is in the cytoplasm of EC H2O2 induced of PKD and ASK1 in the cytoplasm the but not in the nucleus whether PKD and ASK1 a in response to H2O2, with H2O2 or TNF for of PKD with ASK1 was by a with by with and by with that H2O2, but not TNF, induced association of PKD with ASK1 The and the of PKD for ASK1 whether the kinase activity of PKD is critical for association with ASK1, or was and association of PKD with ASK1 was by by with but not associated with ASK1 a with a at suggesting that the kinase activity of PKD is critical for Interestingly, a of PKD with a of the also failed to associate with ASK1, that the domain of PKD is critical for ASK1 binding Thus, PKD binding to ASK1 both its domain and its kinase the critical domain in ASK1 for PKD ASK1 the N-terminal domain the kinase domain or the N-terminal was of ASK1 proteins with PKD was by with by with or not to However, the association of with PKD was in cells and was in response to H2O2 H2O2 induced the association of PKD and ASK1 via the domain of PKD and the C-terminal domain of ASK1 PKD binding to ASK1. of PKD in H2O2-induced ASK1 and EC of PKD with ASK1 to the of PKD in H2O2-induced ASK1 activation. first examined the effects of PKD inhibitors on H2O2-induced ASK1 activation. with Gö6976 or Gö6983 for to to H2O2 for and ASK1 activation at the activation loop was by with a to JNK activation, H2O2-induced ASK1 activation was specifically by Gö6976 but not by Gö6983 suggesting that ASK1 is a downstream of PKD in H2O2 signaling. whether Gö6976 also inhibits H2O2-induced EC death effects of ASK1 activation in with H2O2 for in the presence or of inhibitor Gö6976 or Gö6983. EC survival was by survival that Gö6976 blocked H2O2-induced death and Gö6983 failed to fact H2O2-induced leading to EC survival Gö6976 also blocked EC death not the of PKD in H2O2-induced ASK1-JNK activation and EC apoptosis, we to specifically with a or of PKD, and cells with H2O2 for PKD expression was first by with and that of PKD PKD in by on TRAF2 or ASK1 expression However, the activation of ASK1 and JNK by of PKD the of PKD on EC apoptosis, PKD cells induced to apoptosis by with H2O2 for or by ASK1 with an EC apoptosis was also by by by H2O2-induced EC apoptosis was in PKD EC with the cells EC apoptosis was also in PKD EC these data that PKD is critical in mediating H2O2-induced ASK1-JNK activation and EC apoptosis. Gö6976 H2O2-induced 14-3-3 to the mechanism by which PKD H2O2-induced ASK1-JNK signaling, we first examined Gö6976 PKD PKD can be at within its activation loop by in response to a variety of PKD also several autophosphorylation including and for 14-3-3 binding A. G. H. A. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the of Gö6976 on PKD with H2O2 for in the presence or of Gö6983 or PKD phosphorylation at was by with a Gö6983 blocked H2O2-induced by In contrast, Gö6976 had no effects on H2O2-induced the to and not we the association of PKD with 14-3-3 by an in vitro that association of 14-3-3 with PKD was not in cells but was induced by Gö6976 not with H2O2-induced association of 14-3-3 with PKD, by PKD kinase activity This is with the that 14-3-3 binding of PKD is on the kinase activity of PKD A. G. H. A. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). a we also the phosphorylation of ASK1 at with a a critical for ASK1 binding to with from our and L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (314) Google Scholar, 14Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar, X. W. M. Min W. J. Clin. Investig. PubMed Scopus Google Scholar, Chen L. Fu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), ASK1 was at binding to and H2O2 induced of ASK1 at However, but not H2O2-induced of ASK1 at with the inhibitory of on H2O2-induced ASK1-JNK activation. 14-3-3 of PKD for H2O2-induced and JNK the of 14-3-3 binding of PKD in ASK1-JNK signaling, we PKD with at or at with the wild or 14-3-3 binding was by the at and 14-3-3 binding of induced by H2O2 and level of phosphorylation at in response to However, and failed to to suggesting that phosphorylation at both and is required for 14-3-3 binding the effects of PKD on H2O2-induced association and JNK activation. with the wild or by with H2O2 for and association of PKD with ASK1 was by with by with but not or associated with ASK1 H2O2-induced JNK activation was also and that of or not blocked H2O2-induced JNK activation data that 14-3-3 binding of PKD a critical in H2O2-induced association and JNK activation. have shown that TNF induces gene expression of proinflammatory including adhesion cell adhesion and 1 in EC by at least JNK and NF-κB signaling In to TNF, it has been shown that H2O2 induces gene expression of and 1 but not that of or (3Bradley J.R. Johnson D.R. Pober J.S. Am. J. Pathol. 1993; 142: 1598-1609PubMed Google Scholar). data suggest that TNF and H2O2 have distinct biological in However, the for the distinct of TNF and H2O2 to activate EC have not been defined. that H2O2 induces distinct signaling pathways from by our in the we on JNK cascades known to be activated by both H2O2 and TNF to intracellular pathways. The of this is that we have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling. Several of this H2O2, but not TNF, induces phosphorylation of PKD and translocation of PKD from EC membrane to H2O2, but not TNF, induces association of PKD with ASK1. of PKD by pharmacological inhibitors or blocked H2O2-induced ASK1-JNK activation and EC apoptosis. we that H2O2, but not TNF, induces 14-3-3 binding via phosphorylation of PKD at and which is critical for ASK1-JNK signaling. The mechanism by which TNF and ROS activate ASK1 is not fully understood. It has been shown that both TNF and ROS induce the release of cellular inhibitors such as and 14-3-3 from ASK1 by ASK1 and autophosphorylation at Thr-845, leading to ASK1 activation However, the upstream mediating TNF- and H2O2-induced ASK1 activation have not been fully In response to TNF, the C-terminal domain of ASK1 binds to TRAF2 and this association is required for ASK1 activation by TRAF2 (5Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (534) Google Scholar, 8Nishitoh H. Matsuzawa A. Tobiume K. Saegusa K. Takeda K. Inoue K. Hori S. Kakizuka A. Ichijo H. Genes Dev. 2002; 16: 1345-1355Crossref PubMed Scopus (1153) Google Scholar). have identified a protein and a cascade involved in TNF-induced activation of associates with TRAF2 in response to TNF and in turn binds to ASK1 and the release of 14-3-3 from ASK1, leading to ASK1-JNK activation X. W. M. Min W. J. Clin. Investig. PubMed Scopus Google Scholar, H. Y. A. Pober J.S. Min W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of TRAF2 and in H2O2-induced ASK1-JNK signaling to be we have identified PKD as an upstream activator specifically involved in H2O2- but not TNF-induced ASK1 activation. suggest that and represent pathways induced by TNF and H2O2, This is by our data that H2O2 is to induce PKD activation in suggesting that TRAF2, a critical mediator for TNF-induced signaling, is not required for H2O2-induced PKD activation. S. and W. PKD and in a in the different pathways. in response to TNF, PKD is from membrane to cytoplasm where it binds to ASK1. association with ASK1, both PKD and ASK1 of ASK1 at leading to 14-3-3 release and ASK1-JNK activation. However, the mechanism by which and PKD induce of ASK1 at is not protein has been to of ASK1 Chen L. Fu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is that both and PKD a in or the leading to 14-3-3 release and ASK1-JNK activation. This is have been for PKD activation in response to various phosphorylation of PKD at in the activation association with 14-3-3 via phosphorylation at and of the domain of PKD with and release of the inhibitory N-terminal domain J. A. Y. T. S. J.R. T. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism by which H2O2 induces PKD activation has been Studies from have shown that H2O2-induced PKD phosphorylation at in cells can be by inhibitors of or C and by inhibitors of PKC, suggesting that phosphorylation of PKD at in response to H2O2 is E. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). have also shown that of PKD at by and at in the domain by in response to stress critical for PKD activation. The phosphorylation of PKD at is required for its association with kinase and NF-κB activation and cell survival in cells H. A. Mol. PubMed Scopus Google Scholar, H. A. Mol. Cell. Biol. PubMed Scopus Google Scholar, A. EMBO J. PubMed Scopus Google Scholar). However, our data suggest that PKD H2O2-induced ASK1-JNK signaling in a the inhibitors of and have no inhibitory effects on H2O2-induced signaling. In contrast, pharmacological inhibitors and and the of PKD H2O2-induced ASK1-JNK activation. Gö6983 phosphorylation of PKD at but had no effects on H2O2-induced 14-3-3 binding of PKD and ASK1-JNK activation. In contrast, Gö6976 not inhibit H2O2-induced phosphorylation of PKD at However, Gö6976 14-3-3 binding of PKD and blocks H2O2-induced ASK1-JNK activation. data indicate that the 14-3-3 binding but not the phosphorylation at of PKD is critical for H2O2-induced ASK1-JNK signaling. a J. J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that phosphorylation of PKD at is not critical for activation by protein data that PKD can be activated of and phosphorylation of PKD at Interestingly, phosphorylation of PKD at by has been shown to be critical for its association with JNK, leading to an of JNK activity by PKD E. 2002; PubMed Scopus Google Scholar). It is that phosphorylation of PKD at different its activity in signaling. it has been shown that member of kinase family, kinase binds to and activates ASK1 by ASK1 at K. Matsuzawa A. Nishitoh H. Tobiume K. S. J. Matsumoto K. Ichijo H. EMBO Rep. PubMed Scopus Google Scholar). It to whether PKD ASK1, the kinase activity is critical for in ASK1-JNK activation. we that the autophosphorylation of PKD at the 14-3-3 binding and is critical for its in mediating H2O2-induced ASK1-JNK signaling. Thus, our a mechanism for activation in response to and Pober for critical