Cross-regulation of novel protein kinase C (PKC) isoform function in cardiomyocytes -: Role of PKCε in activation loop phosphorylations and PKCδ in hydrophobic motif phosphorylations

Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this, study identifies phorbol 12-myristoyl 13-acetate (PMA)- and norepinephrine-induced phosphorylations of PKCepsilon (at the C-terminal hydrophobic motif) and PKCdelta (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMA-dependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKCdelta is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKCdelta kinase activity is attenuated when PKCdelta activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKCdelta and PKCepsilon mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKCdelta and WT-PKCepsilon are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKCdelta is phosphorylated at the activation loop but not the hydrophobic motif, DN-PKCepsilon is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKCepsilon in nPKC activation loop phosphorylations and PKCdelta in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKCdelta kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.