Insulin and platelet-derived growth factor stimulate phosphorylation of the c-raf product at serine and threonine residues in intact cells.
We have examined the phosphorylation of the serine threonine kinase, the product of c-raf proto-oncogene in response to insulin or platelet-derived growth factor in intact cells. Both insulin and platelet-derived growth factor stimulated phosphorylation of the c-raf protein about 2- to 3-fold. The phosphorylation occurred exclusively on serine and threonine residues; phosphotyrosine was not detected. In immune-complex kinase assays, treatment with insulin, and platelet-derived growth factor increased autophosphorylation of the c-raf kinase, suggesting activation of its kinase activity. To investigate whether the phosphorylation of the c-raf protein in intact cells results from an autophosphorylation event or from the phosphorylation by other cellular kinase(s), we replaced lysine 375 in the putative ATP-binding domain of the c-raf protein with alanine using oligonucleotide site-directed mutagenesis and expressed the mutated protein in NIH3T3 cells. The substitution resulted in the inactivation of the serine/threonine-specific autophosphorylation in immune-complex kinase assays. In intact cells, however, although phosphorylation of the mutant protein in response to insulin and platelet-derived growth factor occurred to a lesser extent than that of the wild-type protein, the phosphopeptide maps were indistinguishable. These results suggest that serine threonine phosphorylation might be responsible for the activation of c-raf kinase upon treatment of cells with insulin and platelet-derived growth factor, and most of the phosphate associated with the c-raf protein results from its phosphorylation by as yet uncharacterized cellular serine/threonine kinase(s).
Izumi
T
T
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Tamemoto
H
H
Nagao
M
M
Kadowaki
T
T
Takaku
F
F
Kasuga
M
M
D001483:Base Sequence; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D027384:Oncogene Proteins v-raf; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
D007328:Insulin; D010982:Platelet-Derived Growth Factor; D015682:Retroviridae Proteins, Oncogenic; D013912:Threonine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D027384:Oncogene Proteins v-raf