Focal adhesion kinase (FAK) is a non-receptor 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 down-stream Y397pFAK is partially dependent on PKCε, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.
|Original language||English (US)|
|Journal||American Journal of Physiology - Heart and Circulatory Physiology|
|Issue number||4 54-4|
|State||Published - Oct 1 2003|
- Proline-rich tyrosine kinase 2
- Signal transduction
ASJC Scopus subject areas