Evidence for one or more Raf-1 kinase kinase(s) activated by insulin and polypeptide growth factors.
The protein product of the Raf-1 proto-oncogene is a protein serine/threonine kinase that is activated after stimulation of cells with insulin and other mitogens. To investigate the mechanism of this activation, we used purified Raf-1 expressed in E. coli as a substrate for a putative Raf-1 protein kinase kinase. In three different insulin-sensitive cell types, insulin activated Raf-1 kinase kinase activity in crude cytosolic cellular fractions. The insulin stimulation of this activity was evident as early as 2 min after exposure to insulin, maximal at 5-8 min, and inapparent at 15 min. Phosphoamino acid analysis of phosphorylated Raf-1 revealed that serine was the primary phosphate acceptor for the insulin-activated kinase or kinases; small amounts of phosphothreonine were also detected. The insulin effect occurred in cells depleted of protein kinase C, and in extracts depleted of endogenous Raf-1 kinase by immunodepletion; these data argue against protein kinase C or Raf-1 kinase itself being the insulin-stimulated activity. The insulin-activated kinase or kinases phosphorylated the Raf-1 protein on multiple sites in vitro, as evidenced by tryptic mapping; at least some of these appeared to overlap with sites phosphorylated in response to serum in intact cells. Several other mitogens and growth factors stimulated Raf-1 kinase kinase activity, including epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, serum, and phorbol 12-myristate 13-acetate. This insulin- and mitogen-stimulated Raf-1 kinase kinase activity may play a role in mediating the phosphorylation and possibly the activation of the Raf-1 kinase by insulin and other growth factors.
Lee
R M
RM
Howard Hughes Medical Institute Laboratories, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Rapp
U R
UR
Blackshear
P J
PJ
D000818:Animals; D015153:Blotting, Western; D002413:Cations, Divalent; D002460:Cell Line; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008934:Mitogens / Q000494:pharmacology; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D014407:Tumor Cells, Cultured
D002413:Cations, Divalent; D006133:Growth Substances; D007328:Insulin; C000715470:MAS1 protein, human; D008934:Mitogens; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; D019908:Proto-Oncogene Proteins c-raf