Acidic residues at the carboxyl terminus of p60c-src are required for regulation of tyrosine kinase activity and transformation.
Protein phosphorylation sites act to transduce signals into changes in enzymatic activity, representing a point of interaction within a regulatory pathway. The amino acid sequence surrounding a phosphorylation site may well have several functions, including recognition by an appropriate kinase. By generating random mutations in its immediate vicinity, we have examined the sequence requirements of a regulatory tyrosine phosphorylation site, Tyr527, in the proto-oncogene product, p60c-src. The transforming and kinase activities of p60c-src are repressed by phosphorylation of Tyr527. Mutations were made around Tyr527 without changing Tyr527 or the kinase domain. Twenty-nine mutants were sequenced and classified as transforming or nontransforming for Rat-2 cells. Nontransforming mutants contained a surprising variety of COOH-terminal mutations, although acidic residues were present at positions 518 and 524 in all nontransforming mutants. Transforming mutants that contained single-residue changes at Asp518 and Ser522 demonstrated the importance of these residues. Other transforming mutants contained two or more substitutions, but the results are most simply explained if residues Glu524 and Thr523 are also important for normal regulation. Transforming mutations reduced the phosphorylation of Tyr527. We conclude that only a few of the residues in the COOH terminus other than Tyr527 are required to ensure normal phosphorylation and repression of activity in fibroblasts. Other residues may have been conserved during evolution to permit normal function and regulation in other cell types.
MacAuley
A
A
Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Cooper
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D004247:DNA; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)