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J. Biol. Chem., Vol. 279, Issue 26, 27482-27493, June 25, 2004

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Oxidative Stress Induces Protein Kinase C-mediated Activation Loop Phosphorylation and Nuclear Redistribution of Protein Kinase D^*

Richard T. Waldron, Recipient of National Institutes of Health Mentored Career Development Award K01 DK 02834-04, Osvaldo Rey, Elena Zhukova, and Enrique Rozengurt¶

From the Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, UCLA David Geffen School of Medicine, UCLA-CURE Digestive Diseases Research Center and Molecular Biology Institute, UCLA, Los Angeles, California 90095-1786

Oxidative stress induced by cell treatments with H₂O₂ activates protein kinase D (PKD) via a protein kinase C (PKC)-dependent signal transduction pathway (Waldron, R. T., and Rozengurt, E. (2000) J. Biol. Chem. 275, 17114–17121). Here we show that oxidative stress induces PKC-dependent activation loop Ser⁷⁴⁴ and Ser⁷⁴⁸ phosphorylation to mediate dose- and time-dependent activation of PKD, both endogenously expressed in Swiss 3T3 cells and stably overexpressed in Swiss 3T3-GFP.PKD cells. Although oxidative stress induced PKD activation loop phosphorylation and activation with identical kinetics, both were dose-dependently blocked by preincubation of cells with selective inhibitors of PKC (GF109203X and Gö6983) or c-Src (PP2). Inhibition of Src tyrosine kinase activity eliminated oxidative stress-induced direct PKD tyrosine phosphorylation, but only partially attenuated activation loop phosphorylation and activation. Mutation of a putative tyrosine phosphorylation site on PKD, Tyr⁴⁶⁹ to phenylalanine, had no effect on its activation by oxidative stress in transfected COS-7 cells. Similarly, a mutant with Tyr⁴⁶⁹ replaced by aspartic acid had increased basal activity but was also further activated by oxidative stress. Thus, PKD tyrosine phosphorylation at this site neither produced full activation by itself nor was required for oxidative stress-induced activation mediated by activation loop phosphorylation. In addition to PKD activation, activation loop phosphorylation in response to oxidative stress also redistributed activated PKD to cell nuclei, as revealed by PKD indirect immunofluorescence, imaging of a PKD-green fluorescent protein fusion construct (GFP-PKD), and analysis of nuclear pellets. Cell preincubation with Gö6983 strongly diminished H₂O₂-induced nuclear relocalization of GFP-PKD. Taken together, these results indicate that PKC-mediated PKD Ser⁷⁴⁴ and Ser⁷⁴⁸ phosphorylation induced by oxidative stress integrates PKD activation with redistribution to the nucleus.

Received for publication, March 15, 2004

^* This work was supported in part by National Institutes of Health Grants DK 55003 and DK 56930 and NCI Grant P50 CA90388 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of NCI Mentored Career Development Award K01CA097956-01 from the National Institutes of Health.

^¶ Ronald S. Hirshberg Professor of Cancer Research.

To whom correspondence should be addressed: 900 Veteran Ave., Warren Hall Room 11-115, Dept. of Medicine, The David Geffen School of Medicine, UCLA, Los Angeles, CA 90095-1786. Tel.: 310-794-6614; Fax: 310-267-1953; E-mail: rwaldron{at}mednet.ucla.edu.

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