Hypertrophic agonists stimulate the activities of the protein kinases c-Raf and A-Raf in cultured ventricular myocytes.
We detected expression of two Raf isoforms, c-Raf and A-Raf, in neonatal rat heart. Both isoforms phosphorylated, activated, and formed complexes with mitogen-activated protein kinase kinase 1 in vitro. However, these isoforms were differentially activated by hypertrophic stimuli such as peptide growth factors, endothelin-1 (ET1), or 12-O-tetradecanoylphorbol-13-acetate (TPA) that activate the mitogen-activated protein kinase cascade. Exposure of cultured ventricular myocytes to acidic fibroblast growth factor activated c-Raf but not A-Raf. In contrast, TPA produced a sustained activation of A-Raf and only transiently activated c-Raf. ET1 transiently activated both isoforms. TPA and ET1 were the most potent activators of c-Raf and A-Raf. Both utilized protein kinase C-dependent pathways, but stimulation by ET1 was also partially sensitive to pertussis toxin pretreatment. cRaf was inhibited by activation of cAMP-dependent protein kinase although A-Raf was less affected. Fetal calf serum, phenylephrine, and carbachol were less potent activators of c-Raf and A-Raf. These results demonstrate that A-Raf and c-Raf are differentially regulated and that A-Raf may be an important mediator of mitogen-activated protein kinase cascade activation when cAMP is elevated.
Bogoyevitch
M A
MA
National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Marshall
C J
CJ
Sugden
P H
PH
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000201:enzymology; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D003470:Culture Media; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D016232:Endothelins / Q000494:pharmacology*; D004789:Enzyme Activation; D016220:Fibroblast Growth Factor 1 / Q000494:pharmacology; D015870:Gene Expression; D006133:Growth Substances / Q000494:pharmacology*; D006352:Heart Ventricles; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D037342:Pertussis Toxin; D010656:Phenylephrine / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D003470:Culture Media; D016232:Endothelins; D006133:Growth Substances; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D016220:Fibroblast Growth Factor 1; D010656:Phenylephrine; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D002217:Carbachol; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate