A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity.
We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002451:Cell Compartmentation; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D009154:Mutation; D009374:Neoplasms, Experimental / Q000473:pathology; D015688:Oncogene Protein pp60(v-src); D009857:Oncogenes; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D051381:Rats; D013329:Structure-Activity Relationship; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)