title: Phorbol 12-myristate 13-acetate-dependent protein kinase C delta-Tyr311 phosphorylation in cardiomyocyte caveolae.
abstract: Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) 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 PKCdelta to caveolae and increase PKCdelta-Tyr(311) 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 PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.
authors.affiliation: Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
authors.forename: Vitalyi O
authors.initials: VO
authors.lastname: Rybin
authors.forename: Jianfen
authors.initials: J
authors.lastname: Guo
authors.forename: Zoya
authors.initials: Z
authors.lastname: Gertsberg
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Feinmark
authors.forename: Susan F
authors.initials: SF
authors.lastname: Steinberg
mesh_terms: D000818:Animals; D003505:Cyclodextrins / Q000494:pharmacology; D016232:Endothelins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000737:chemistry*; D016315:Proto-Oncogene Proteins c-abl / Q000037:antagonists & inhibitors; D051381:Rats; D017208:Rats, Wistar; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D003505:Cyclodextrins; D016232:Endothelins; D004791:Enzyme Inhibitors; D014443:Tyrosine; D006861:Hydrogen Peroxide; D016315:Proto-Oncogene Proteins c-abl; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate; D009638:Norepinephrine