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 mumol/l; 1 h pretreatment). AdvcaPKCepsilon, but not Adv-caPKCdelta, activated FAK compared with a control Adv encoding beta-galactosidase. Conversely, Adv-dnPKCepsilon inhibited ET-induced FAK activation. Y-27632 ( 10 mumol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKCepsilon-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 mumol/l, 1 h pretreatment) also inhibited ET- induced Y397pFAK and cofilin phosphorylation and caPKCepsilon-induced Y397pFAK. Neither inhibitor, however, interfered with ET- induced ERK1/2 activation. Finally, PP2 (50 mumol/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 PKCepsilon, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.