Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.
The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.
Yaciuk
P
P
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Choi
J K
JK
Shalloway
D
D
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002471:Cell Transformation, Neoplastic; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src); D012694:Serine / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate
D000596:Amino Acids; D011518:Proto-Oncogene Proteins; D012694:Serine; D011494:Protein Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate