Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H₂O₂ but not tumor necrosis factor

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. Gö6083 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H ₂O₂-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 H₂O ₂-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 H ₂O₂-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 H ₂O₂-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.