Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product.
The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.
D000302:Adrenal Cortex / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004075:Diglycerides; D010718:Phosphatidylserines; D011518:Proto-Oncogene Proteins; D006160:Guanosine Triphosphate; D011493:Protein Kinase C; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate; C013965:diolein