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 stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB-mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB-mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and -independent pathways involving PKC.
Waldron
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RT
Laboratory of Signal Transduction and Growth Regulation, Division of Digestive Diseases, Department of Medicine, UCLA School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Rozengurt
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D016475:3T3 Cells; D000818:Animals; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D018004:Receptors, Bombesin / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
D018004:Receptors, Bombesin; D012694:Serine; D006861:Hydrogen Peroxide; C089212:protein kinase D; D015688:Oncogene Protein pp60(v-src); D011493:Protein Kinase C