Protein kinase C epsilon-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 Ca(2+) 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 Ca(2+) ([Ca(2+)](i)), and activates the novel, Ca(2+)-independent PKCs. Therefore, immunoprecipitation and western blotting experiments were used to examine the PKC and Ca(2+) dependence of PYK2 activation in NRVM. PYK2 was activated by ET (100 nM; 2-30 min) and phenylephrine (50 microM; 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 PKC epsilon and PKCdelta (but not PKCalpha) translocation, followed by downregulation of both novel PKC isoenzymes. Phorbol myristate acetate (PMA; 200 nM), a direct activator of Ca(2+)-dependent and Ca(2+)-independent PKCs, activated PYK2 within 10 min, and PYK2 phosphorylation remained elevated after 30 min of stimulation. Adv-mediated overexpression of caPKC epsilon increased PYK2 phosphorylation, whereas Adv-mediated overexpression of a kinase-inactive mutant of PKC epsilon 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 [Ca(2+)](i) with either nifedipine (10 microM) or BAPTA-AM (50 microM) decreased basal PYK2 phosphorylation, it did not prevent ET-induced PYK2 activation. Furthermore, increasing [Ca(2+)](i) with ionomycin (10 microM), K(+) depolarization, or BayK8644 (1 microM) was not sufficient to further activate PYK2. These data demonstrate that ET-induced PYK2 activation is Gq, PKC epsilon, and Src dependent, describing a distinct signaling pathway leading to agonist-induced PYK2 activation in cardiomyocytes.
Bayer
Allison L
AL
The Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, IL 60153, USA.
Heidkamp
Maria C
MC
Howes
Amy L
AL
Heller Brown
Joan
J
Byron
Kenneth L
KL
Samarel
Allen M
AM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002316:Cardiotonic Agents / Q000494:pharmacology; D019332:Endothelin-1 / Q000378:metabolism; D004789:Enzyme Activation; D006352:Heart Ventricles / Q000166:cytology*; D009154:Mutation; D032383:Myocytes, Cardiac / Q000201:enzymology*; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002316:Cardiotonic Agents; D019332:Endothelin-1; D010656:Phenylephrine; D011392:Proline; C497257:Prkce protein, rat; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate