Activation of focal adhesion kinase by protein kinase C epsilon 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 micromol/l; 1 h pretreatment). Adv-caPKC epsilon, but not Adv-caPKC delta, activated FAK compared with a control Adv encoding beta-galactosidase. Conversely, Adv-dnPKC epsilon inhibited ET-induced FAK activation. Y-27632 (10 micromol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKC epsilon-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 micromol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKC epsilon-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 micromol/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 PKC epsilon, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.
Heidkamp
Maria C
MC
The Cardiovascular Institute, Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153, USA. mheidka@lumc.edu
Bayer
Allison L
AL
Scully
Brian T
BT
Eble
Diane M
DM
Samarel
Allen M
AM
D008841:Actin Cytoskeleton / Q000378:metabolism; D051339:Actin Depolymerizing Factors; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D016232:Endothelins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005799:Genes, Dominant; D006352:Heart Ventricles; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins / Q000378:metabolism; D009154:Mutation; D032383:Myocytes, Cardiac / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D011108:Polymers / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051744:Protein Kinase C-epsilon; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution; D054460:rho-Associated Kinases; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
D051339:Actin Depolymerizing Factors; D016232:Endothelins; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D011108:Polymers; C497257:Prkce protein, rat; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; C496054:Ptk2b protein, rat; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon