Oxidative stress induces protein kinase D activation in intact cells - Involvement of Src and dependence on protein kinase C

Protein kinase D (PKD) is a protein serine kinase that is directly stimulat ed in vitro by phorbol esters and diacylglycerol in the presence of phospho lipids, and activated by phorbol esters, neuropeptides, and platelet-derive d growth factor via protein kinase C (PKC) in intact cells. Recently, oxida tive stress was shown to activate transfected PKC isoforms via tyrosine pho sphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H2O2 (0.15-10 mM) to quiescent S wiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, a s measured by autophosphorylation and phosphorylation of an exogenous subst rate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 c ells but not a kinase-deficient mutant PKD form or a PKD mutant with critic al activating serine residues 744 and 748 mutated to alanines. Genistein, o r the specific Src inhibitors PP-1 and PP-2 (1-10 mu M) inhibited H2O2-medi ated PKD activation by 45%, indicating that Src contributes to this signali ng pathway. PKD activation by H2O2 was also selectively potentiated by cotr ansfection of PKD together with an active form of Src (v-Src) in COS-7 cell s, as compared with PDB-mediated activation. The specific phospholipase C i nhibitor, U73122 partly blocked H2O2-mediated but not PDB-mediated PKD acti vation. In contrast, PKC inhibitors blocked H2O2 or PDB-mediated PKD activa tion essentially completely; suggesting that whereas Src mediates part of i ts effects via phospholipase C activation, PKC acts more proximally as an u pstream activator of PHD. Together, these studies reveal that oxidative str ess activates PKD by initiating distinct Src-dependent and -independent pat hways involving PKC.