Stimulus-specific Differences in Protein Kinase Cδ Localization and Activation Mechanisms in Cardiomyocytes

Abstract

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 PKCδ actions in cardiomyocytes. Using immunoblot analysis with antibodies to total PKCδ and PKCδ-py311, we demonstrate that PKCδ partitions between soluble and particulate fractions (with little Tyr311 phosphorylation) in resting cardiomyocytes. Phorbol 12-myristate 13-acetate (PMA) promotes PKCδ translocation to membranes and phosphorylation at Tyr311. H 2O2 also increases PKCδ-pY311 in association with its release from membranes. Both PMA- and H2O 2-dependent increases in PKCδ-pY311 are mediated by Src family kinases, but they occur via different mechanisms. The H 2O2-dependent increase in PKCδ-pY311 results from Src activation and increased Src-PKCδ complex formation. The PMA-dependent increase in PKCδ-pY311 results from a lipid cofactor-induced conformational change that renders PKCδ a better substrate for phosphorylation by precomplexed Src kinases (without Src activation). PKCδ-Y311 phosphorylation does not grossly alter the kinetics of PMA-dependent PKCδ down-regulation. Rather, tyrosine phosphorylation regulates PKCδ kinase activity. PKCδ is recovered from the soluble fraction of H2O2-treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKCδ 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 PKCδ substrate specificity. The stimulus-specific modes for PKCδ signaling identified in this study allow for distinct PKCδ-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes.