Protein kinase Cε-dependent activation of proline-rich tyrosine kinase 2 in neonatal rat ventricular myocytes

Proline-rich tyrosine kinase 2 (PYK2) is a nonreceptor protein tyrosine kinase that links G-protein-coupled receptors to activation of MAPK cascades and cellular growth. In smooth muscle and other cell types, PYK2 activation is dependent on either Ca2+ or protein kinase C (PKC), and we have previously shown that endothelin-1 (ET) activates PYK2 in adult and neonatal rat ventricular myocytes (NRVM). However, ET both alters intracellular Ca2+ ([Ca2+](i)), and activates the novel, Ca2+-independent PKCs. Therefore, immunoprecipitation and western blotting experiments were used to examine the PKC and Ca2+ dependence of PYK2 activation in NRVM. PYK2 was activated by ET (100 nM; 2-30 min) and phenylephrine (50 muM; 2-30 min), which are both hypertrophic agonists that activate Gq-coupled receptors. Moreover, adenoviral (Adv)-mediated overexpression of constitutively active (ca) Galphaq increased PYK2-Y-402 phosphorylation as early as 8 h post-infection, as compared to NRVM infected with a control Adv encoding beta-galactosidase. caGalphaq overexpression also induced PKCepsilon and PKCdelta (but not PKCalpha) translocation, followed by downregulation of both novel PKC isoenzymes. Phorbol myristate acetate (PMA; 200 nM), a direct activator of Ca2+-dependent and Ca2+-independent PKCs, activated PYK2 within 10 min, and PYK2 phosphorylation remained elevated after 30 min of stimulation. Adv-mediated overexpression of caPKCepsilon increased PYK2 phosphorylation, whereas Adv-mediated overexpression of a kinase-inactive mutant of PKCepsilon markedly inhibited ET-induced, but not basal PYK2 phosphorylation. In contrast, both basal and ET-induced PYK2 phosphorylation were blocked by treatment with the Src-family protein kinase inhibitor PP2. Although reducing [Ca2+](i) with either nifedipine (10muM) or BAPTA-AM (50muM) decreased basal PYK2 phosphorylation, it did not prevent ET-induced PYK2 activation. Furthermore, increasing [Ca2+](i) with ionomycin (10 muM), K+ depolarization, or BayK8644 (1 muM) was not sufficient to further activate PYK2. These data demonstrate that ET-induced PYK2 activation is Gq, PKCepsilon, and Src dependent, describing a distinct signaling pathway leading to agonist-induced PYK2 activation in cardiomyocytes. (C) 2003 Elsevier Ltd. All rights reserved.