Deletion of the SH3 domain of Src interferes with regulation by the phosphorylated carboxyl-terminal tyrosine

• Published in: The Journal of biological chemistry Vol. 268; no. 24; pp. 18070 - 18075
• Main Authors: Okada, M., Howell, B.W., Broome, M.A., Cooper, J.A.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25-08-1993; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Biological and medical sciences; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Chick Embryo; CSK Tyrosine-Protein Kinase; Fibroblasts; Fundamental and applied biological sciences. Psychology; Genes, src; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Molecular and cellular biology; Mutagenesis, Site-Directed; Oncogene Protein pp60(v-src) - genetics; Oncogene Protein pp60(v-src) - isolation & purification; Oncogene Protein pp60(v-src) - metabolism; Phosphotyrosine; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - isolation & purification; Protein-Tyrosine Kinases - metabolism; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Restriction Mapping; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sequence Deletion; src-Family Kinases; Transformation, Genetic; Tyrosine - analogs & derivatives; Tyrosine - metabolism; Phosphorylation; Enzyme; Transferases; Molecular interaction; SH3 Domain; In vitro; Structure function relationship; SH2 Domain; Regulation(control); Enzymatic activity; C-Onc gene; Deletion; Protein-tyrosine kinase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)46812-8

A current model for the regulation of the Src protein-tyrosine kinase proposes that the COOH-terminal phosphotyrosine, Tyr-527, binds to the Src homology 2 (SH2) region in an intramolecular interaction that represses the kinase domain. This model is consistent with the activation of Src by mutations in the SH2 domain or COOH terminus. Mutations in the SH3 domain also activate Src, although this region is not thought to bind phosphotyrosine. Seidel-Dugan et al. (Seidel-Dugan, C., Meyer, B. E., Thomas, S. M., and Brugge, J. S. (1992) Mol. Cell. Biol. 12, 1835-1845) have shown that Src mutants with deletions in the SH2 or SH3 domain transform chicken embryo fibroblasts and have increased kinase activity. These mutant proteins are underphosphorylated at Tyr-527, a change that could in itself activate the mutants. Therefore, it is not possible to distinguish whether the SH2 and SH3 domains are needed for phosphorylation of Tyr-527 or for Src to adopt or maintain the repressed state. We have artificially increased the level of Tyr-527 phosphorylation of SH2 and SH3 deletion mutants by coexpressing them with the Tyr-527 kinase, Csk, in yeast cells. We find that both the SH2 and SH3 domains are needed for inhibition of Src by Csk. The SH2 domain is needed for efficient phosphorylation by Csk, both in yeast cells and in vitro. The SH3 domain is needed for Src to be inhibited when Tyr-527 is phosphorylated by Csk. This suggests that the SH3 domain cooperates with the SH2 domain and phosphorylated Tyr-527 to inhibit the kinase domain. Dephosphorylation of SH3 domain mutants at Tyr-527 in fibroblasts could be a consequence of a failure of the proposed SH2/phosphotyrosine interaction.