Activation of focal adhesion kinase by protein kinase Cϵ in neonatal rat ventricular myocytes

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET (10 nmol/l; 2–30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine (5 μmol/l; 1 h pretreatment). Adv-caPKCϵ, but not Adv-caPKCδ, activated FAK compared with a control Adv encoding β-galactosidase. Conversely, Adv-dnPKCϵ inhibited ET-induced FAK activation. Y-27632 (10 μmol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKCϵ-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 μmol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKCϵ-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 μmol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET-induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKCϵ, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src

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 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 μM; 2–30 min), which are both hypertrophic agonists that activate Gq-coupled receptors. Moreover, adenoviral (Adv)–mediated overexpression of constitutively active (ca) Gαq increased PYK2–Y 402 phosphorylation as early as 8 h post-infection, as compared to NRVM infected with a control Adv encoding β-galactosidase. caGαq overexpression also induced PKCε and PKCδ (but not PKCα) 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 caPKCε increased PYK2 phosphorylation, whereas Adv-mediated overexpression of a kinase-inactive mutant of PKCε 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 μM) or BAPTA-AM (50 μM) decreased basal PYK2 phosphorylation, it did not prevent ET-induced PYK2 activation. Furthermore, increasing [Ca 2+] i with ionomycin (10 μM), K + depolarization, or BayK8644 (1 μM) was not sufficient to further activate PYK2. These data demonstrate that ET-induced PYK2 activation is Gq, PKCε, and Src dependent, describing a distinct signaling pathway leading to agonist-induced PYK2 activation in cardiomyocytes

PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy

Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor protein tyrosine kinases. PYK2 has been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase cascades and cellular growth in a variety of cell types. To determine whether PYK2 expression and phosphorylation is altered in left ventricular (LV) myocardium undergoing LV hypertrophy (LVH) and heart failure in vivo, suprarenal abdominal aortic coarctation was performed in 160-g male Sprague-Dawley rats. Immunohistochemistry and Western blotting were performed on LV tissue 1, 8, and 24 wk after aortic banding. Aortic banding produced sustained hypertension and gradually developing LVH. PYK2 levels were increased 1.8 ± 0.2-, 2.7 ± 0.6-, and 2.0 ± 0.2-fold in 1-, 8-, and 24-wk banded animals compared with their respective sham-operated controls. The increase in PYK2 expression was paralleled by an increase in PYK2 phosphorylation, both of which preceded the development of LVH. Immunohistochemistry revealed that enhanced PYK2 expression occurred predominantly in the cardiomyocyte population. Furthermore, there was a high degree of correlation ( R = 0.75; P< 0.001) between the level of PYK2 and the degree of LVH in 24-wk sham and banded animals. In contrast, FAK levels and FAK phosphorylation were not increased before the development of LVH. However, there was a high degree of correlation (R = 0.68; P < 0.001) between the level of FAK and the degree of LVH in 24-wk sham and banded rats. There was also a significant increase in the ratio of phosphospecific anti-FAK to FAK at this time point. These data are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK have distinctly different roles in LVH progression