Protein kinase Cε (PKCε) and src control PKCδ activation loop phosphorylation in cardiomyocytes

Protein kinase C delta(PKC delta) is unusual among AGC kinases in that it does not require activation loop (Thr(505)) phosphorylation for catalytic competence. Nevertheless, Thr(505) phosphorylation has been implicated as a mechanism that influences PKC delta activity. This study examines the controls of PKC delta- Thr(505) phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKC delta autophosphorylation in the priming maturational PKC delta-Thr(505) phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKC delta-Thr(505) phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the alpha(1)-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKC epsilon overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKC delta-Thr(505) autophosphorylation and PKC delta-Thr(505) phosphorylation in trans by PKC epsilon. Our studies show that adenovirus-mediated PKC delta overexpression leads to an increase in PKC delta-Thr(505) phosphorylation. However, this cannot be attributed to an effect of PKC epsilon to function as a direct PKC delta-Thr(505) kinase, since the PKC epsilon-dependent increase in PKC delta-Thr(505) phosphorylation is accompanied by ( and dependent upon) increased PKC phosphorylation at Tyr(311) and Tyr(332). Further studies implicate Src in this mechanism, showing that 1) PKC epsilon overexpression increases PKC delta-Thr(505) phosphorylation in cardiomyocytes and Src+ cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKC delta-Tyr(311)/Tyr(332) phosphorylation), and 2) in vitro PKC delta-Thr(505) autophosphorylation is augmented in assays performed with Src ( which promotes PKC delta-Tyr(311)/Tyr(332) phosphorylation). Collectively, these results identify a novel PKC delta-Thr(505) autophosphorylation mechanism that is triggered by PKC epsilon overexpression and involves Src-dependent PKC delta-Tyr(311)/Tyr(332) phosphorylation.