Abstract 351: Distinct Requirement of PKCδ Tyr311 Phosphorylation and Kinase Activity for Vascular Smooth Muscle Cell Hypertrophy Induced by Angiotensin II
Protein kinase C (PKC) activation is one of the earliest signaling events induced by angiotensin II (AngII) in vascular smooth muscle cells (VSMCs). However, the detailed role of each PKC isoform in mediating vascular remodeling remains unclear. We have previously shown that activation of PKCδ is required for VSMC migration induced by AngII. Here, we hypothesized the existence of an integrated PKCδ activation by AngII in which Tyr311 phosphorylation plays a major role in VSMC hypertrophy. In cultured VSMCs, PKC δ-Tyr311 phosphorylation by AngII peaked at 2 min and was totally blocked by an AT1 receptor antagonist. Interestingly, AngII-induced PKC δ-Tyr311 phosphorylation was markedly blocked by a Src kinase inhibitor, PP2, whereas an EGF receptor kinase inhibitor, AG1478, had no inhibitory effect. In contrast, AG1478, but not PP2, inhibited EGF receptor transactivation induced by AngII as indicated by its Tyr1068 phosphorylation. Thus, adenovirus encoding dominant-negative Src inhibited the PKCδ phosphorylation but not the EGFR phosphorylation. To explore the role of PKCδ-Tyr311 phosphorylation in VSMC hypertrophy induced by AngII, we established a VSMC cell line over-expressing PKCδ wild-type or PKCδ Y311F mutants by retrovirus infection. We observed that AngII-induced protein synthesis and cell volume were significantly enhanced in VSMCs expressing PKCδ wild-type compared to Y311F mutant. However, no AngII-induced proliferation was detected in these VSMCs. Protein synthesis as well as cell volume increase induced by AngII was significantly inhibited in VSMCs infected with adenovirus encoding kinase-inactive PKCδ K376A. Interestingly, in K376A expressing VSMCs, but not in Y311F VSMCs, phospho-rylations of Akt and p70S6 kinase but not ERK induced by AngII were markedly inhibited. By contrast, a gene array experiment demonstrated marked inhibition of COX2 gene induction by AngII in Y311F VSMCs but not in K376A VSMCs. Taken together, these data demonstrate that AngII induces PKCδ phosphorylation at Tyr311 through a Src kinase. Also, this PKCδ phosphorylation is in part involved in COX2-dependent VSMC hypertrophy induced by AngII through a different pathway from AngII-induced PKCδ kinase activation which mediates the Akt/p70S6 kinase pathway.