title: Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H2O2 but not tumor necrosis factor.
abstract: 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. Go6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Go6976 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 Go6976 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 Go6976 but not by Go6983. 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.
authors.affiliation: Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Shusen
authors.initials: S
authors.lastname: Zheng
authors.forename: Peter
authors.initials: P
authors.lastname: Storz
authors.forename: Wang
authors.initials: W
authors.lastname: Min
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002227:Carbazoles / Q000494:pharmacology; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007211:Indoles; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D008301:Maleimides; D008954:Models, Biological; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D048948:14-3-3 Proteins; C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D002227:Carbazoles; D007211:Indoles; D008301:Maleimides; D009281:Naphthalenes; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C061465:calphostin complex; D012694:Serine; D006861:Hydrogen Peroxide; C089212:protein kinase D; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; D019311:Staurosporine; D003545:Cysteine