DELETION OF THE SH3 DOMAIN OF SRC INTERFERES WITH REGULATION BY THE PHOSPHORYLATED CARBOXYL-TERMINAL TYROSINE

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 rep resses 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, 1 835-1845) 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 no t possible to distinguish whether the SH2 and SH3 domains are needed f or phosphorylation of Tyr-527 or for Src to adopt or maintain the repr essed state. We have artificially increased the level of Tyr-527 phosp horylation of SH2 and SH3 deletion mutants by coexpressing them with t he 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-52 7 is phosphorylated by Csk. This suggests that the SH3 domain cooperat es with the SH2 domain and phosphorylated Tyr-527 to inhibit the kinas e domain. Dephosphorylation of SH3 domain mutants at Tyr-527 in fibrob lasts could be a consequence of a failure of the proposed SH2/phosphot yrosine interaction.