Stimulus-specific differences in protein kinase Cδ localization and activation mechanisms in cardiomyocytes

Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKCdelta actions in cardiomyocytes. Using immunoblot analysis with antibodies to total PKCdelta and PKCdelta-pY(311), we demonstrate that PKCdelta partitions between soluble and particulate fractions (with little Tyr(311) phosphorylation) in resting cardiomyocytes. Phorbol 12-myristate 13-acetate (PMA) promotes PKCdelta translocation to membranes and phosphorylation at Tyr(311). H2O2 also increases PKCdelta-pY(311) in association with its release from membranes. Both PMA- and H2O2-dependent increases in PKCdelta-pY(311) are mediated by Src family kinases, but they occur via different mechanisms. The H2O2-dependent increase in PKCdelta-pY(311) results from Src activation and increased Src-PKCdelta complex formation. The PMA-dependent increase in PKCdelta-pY(311) results from a lipid cofactor-induced conformational change that renders PKCdelta a better substrate for phosphorylation by precomplexed Src kinases (without Src activation). PKCalpha-Y-311 phosphorylation does not grossly alter the kinetics of PMA- dependent PKCdelta down-regulation. Rather, tyrosine phosphorylation regulates PKCdelta kinase activity. PKCdelta is recovered from the soluble fraction of H2O2-treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKCdelta phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKCdelta substrate specificity. The stimulus-specific modes for PKCdelta signaling identified in this study allow for distinct PKCdelta-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes.