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

Richard T Waldron,Osvaldo Rey,Elena Zhukova,Enrique Rozengurt

doi:10.1074/jbc.m402875200

Richard T Waldron, Osvaldo Rey + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m402875200

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Abstract

Oxidative stress induced by cell treatments with H(2)O(2) 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 Ser(744) and Ser(748) 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, Tyr(469) to phenylalanine, had no effect on its activation by oxidative stress in transfected COS-7 cells. Similarly, a mutant with Tyr(469) 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 H(2)O(2)-induced nuclear relocalization of GFP-PKD. Taken together, these results indicate that PKC-mediated PKD Ser(744) and Ser(748) phosphorylation induced by oxidative stress integrates PKD activation with redistribution to the nucleus.

Highlights

Oxidative stress has been implicated in aging and in a wide range of diseases affecting the nervous, respiratory, cardiovascular, and gastrointestinal systems in humans [1, 2]
Oxidative Stress Induces protein kinase D (PKD) Activation and Activation Loop Phosphorylation with Similar Kinetics and Dose Dependence—To determine whether oxidative stress-mediated PKD activation occurs via phosphorylation of activation loop Ser744 and Ser748, we examined PKD activation and phosphorylation of these residues in response to oxidative stress in Swiss 3T3PKD.GFP cells that are stably transfected with exogenous wild type PKD [34]
Activation loop phosphorylation (Fig. 5C, middle panel), MARCKS phosphorylation (Fig. 5C, lower panel), and PKD activation (Fig. 5D) were all drastically reduced. These results indicate that oxidative stress-induced, protein kinase C (PKC)-dependent activation loop phosphorylation, rather than PKD tyrosine phosphorylation, is critical for PKD activation

Summary

Introduction

Oxidative stress has been implicated in aging and in a wide range of diseases affecting the nervous, respiratory, cardiovascular, and gastrointestinal systems in humans [1, 2]. To examine the role of PKD activation loop phosphorylation in oxidative stress-induced PKD activation, Swiss 3T3-PKD.GFP cells were either left unstimulated or stimulated with 1 mM H2O2 for different times and lysed.

Results

Conclusion