Regulation of the cellular src protein tyrosine kinase: interactions of the carboxyl terminal sequences residing between the kinase domain and tyrosine-527.
Negative regulation of the cellular Src tyrosine kinase (pp60c-src) is mediated through the phosphorylation of a C-terminal tyrosine residue, Tyr-527. Current models predict that inhibition of c-Src kinase activity results from an interaction of phosphorylated Tyr-527 with the amino terminal SH2 domain. Tyr-527 is located 11 residues C-terminal from the end of the kinase domain. Insertion or deletion of residues within these 11 residues of pp60c-src activates kinase activity and induces morphological transformation. The resultant variant Src proteins also exhibit a reduced level of phosphorylation of Tyr-527. We have used antibodies to phosphotyrosine, susceptibility to tyrosine phosphatases and binding of mutant Src proteins to peptides mimicking the tyrosine phosphorylated C-terminus of pp60c-src to investigate the tyrosine phosphorylated and unphosphorylated forms of such insertion/deletion variants. The reactivity of variant proteins with phosphotyrosine antibodies and the susceptibility of phosphorylated Tyr-527 to tyrosine phosphatases were similar to that of wild type pp60c-src. In addition, the results of binding experiments performed with a C-terminal peptide containing phosphorylated Tyr-527 indicated that only dephosphorylated forms of variant Src proteins bound phospho-peptide. These data suggest that insertion or deletion mutations within the C-terminal region of pp60c-src do not substantially alter the interaction of phosphorylated Tyr-527 with the SH2 domain. Rather, the data are consistent with the hypothesis that the reduction of phosphorylation of Tyr-527 and the accompanying activation of these variants may be due to the action of a tyrosine phosphatase and the inefficient phosphorylation of Tyr-527 by a regulatory kinase.
Cobb
B S
BS
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.
Parsons
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism*
D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D017027:Protein Tyrosine Phosphatases