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J. Biol. Chem., Vol. 283, Issue 26, 17777-17788, June 27, 2008

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Phorbol 12-Myristate 13-Acetate-dependent Protein Kinase C-Tyr³¹¹ Phosphorylation in Cardiomyocyte Caveolae^*

From the Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Protein kinase C (PKC) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKC also is dynamically regulated through tyrosine phosphorylation in H₂O₂- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H₂O₂ activates Src and related Src-family kinases (SFKs), which function as dual PKC-Tyr³¹¹ and -Tyr³³² kinases in vitro and contribute to H₂O₂-dependent PKC-Tyr³¹¹/Tyr³³² phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H₂O₂-dependent PKC-Tyr³¹¹/Tyr³³² phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKC-Tyr³¹¹ phosphorylation, but this is not associated with SFK activation or PKC-Tyr³³² phosphorylation. Rather, PMA increases PKC-Tyr³¹¹ phosphorylation by delivering PKC to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKC-Tyr³¹¹ phosphorylation, without blocking H₂O₂-dependent PKC-Tyr³¹¹ phosphorylation. The enzyme that acts as a PKC-Tyr³¹¹ kinase without increasing PKC phosphorylation at Tyr³³² in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKC-Tyr³¹¹ kinase, PMA-dependent PKC-Tyr³¹¹ phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKC to caveolae and increase PKC-Tyr³¹¹ phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKC exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKC localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.

Received for publication, January 14, 2008 , and in revised form, April 1, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant HL77860 from NHLBI. 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.

¹ To whom correspondence should be addressed: 630 West 168 St., New York, NY 10032. Tel.: 212-305-4297; Fax: 212-305-8780; E-mail: sfs1{at}columbia.edu.

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