Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms.
We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12-myristate 13-acetate (PMA) to activate Raf-1. Specifically, we determined the role of Ras binding and the phosphorylation of serines 338/339, tyrosines 340/341 and the activation loop (491-508) in response to these stimuli in COS-7 cells. An intact Ras binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA. Brief treatment of COS-7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340/341 phosphorylation sites also blocked Raf-1 activation by all stimuli tested. Loss of the serine 497 and serine 499 PKCalpha phosphorylation sites failed to significantly reduce Raf-1 activation by any stimulus including PMA. Alanine substitution of all other potential phosphorylation sites within the Raf-1 activation loop had little or no effect on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of different stimuli through a common mechanism involving association with Ras-GTP and multiple phosphorylations of the amino-terminal region of the catalytic domain. Phosphorylation of the activation loop does not appear to be a significant mechanism of Raf-1 kinase regulation in COS-7 cells.
Barnard
D
D
Department of Medicine, Walther Oncology Center, Indiana University School of Medicine, Indianapolis 46202, USA.
Diaz
B
B
Clawson
D
D
Marshall
M
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004815:Epidermal Growth Factor / Q000502:physiology*; D005786:Gene Expression Regulation; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009857:Oncogenes; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism; D018631:ras Proteins / Q000502:physiology
D014443:Tyrosine; D012694:Serine; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate