Phorbol 12-myristate 13-acetate-dependent protein kinase Cδ-Tyr311 phosphorylation in cardiomyocyte caveolae

Protein kinase C delta(PKC delta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKC delta 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 delta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H2O2- dependent PKC delta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H2O2- dependent PKC delta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells ( deficient in SFKs) and restored by Src re-expression. PMA also promotes PKC delta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKC delta-Tyr(332) phosphorylation. Rather, PMA increases PKC delta-Tyr(311) phosphorylation by delivering PKC delta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKC delta-Tyr(311) phosphorylation, without blocking H2O2 dependent PKC delta-Tyr(311) phosphorylation. The enzyme that acts as a PKC delta-Tyr(311) kinase without increasing PKC delta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKC delta-Tyr(311) kinase, PMA-dependent PKC delta-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 PKC delta to caveolae and increase PKC delta-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 PKC delta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKC delta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.