Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity.
We have examined the phosphorylation and the serine/threonine-specific kinase activity of the protooncogene product Raf-1 (formerly c-raf) in response to oncogenic transformation or growth-factor treatment of mouse 3T3 cells. Expression of the membrane-bound oncogene products encoded by v-fms, v-src, v-sis, polyoma virus middle-sized tumor antigen, and Ha-ras increased the apparent molecular weight and phosphorylation of the Raf-1 protein, while expression of the nuclear oncogene and protooncogene products encoded by v-fos and c-myc did not. Changes in electrophoretic mobility and phosphorylation occurred rapidly in response to treatment of cells with platelet-derived growth factor, acidic fibroblast growth factor, epidermal growth factor, and the protein kinase C activator phorbol 12-myristate 13-acetate, but not insulin. The phosphorylation of the Raf-1 protein occurred primarily on serine and threonine residues. However, a subpopulation of Raf-1 molecules was phosphorylated on tyrosine residues in cells transformed by v-src or stimulated with platelet-derived growth factor. Transformation by v-src, or treatment with platelet-derived growth factor or phorbol 12-myristate 13-acetate, activated the Raf-1-associated serine/kinase activity as measured in immune-complex kinase assays. These findings suggest that proliferative signals generated at the membrane result in the phosphorylation of the Raf-1 protein and the activation of its serine/threonine kinase activity. Raf-1 activation may thus serve to transduce signals from the membrane to the cytoplasm and perhaps on to the nucleus.
Morrison
D K
DK
Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Kaplan
D R
DR
Rapp
U
U
Roberts
T M
TM
D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D010449:Peptide Mapping; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011519:Proto-Oncogenes; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007328:Insulin; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D019908:Proto-Oncogene Proteins c-raf; D013755:Tetradecanoylphorbol Acetate