Okada M, Howell B W, Broome M A, Cooper J A
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
J Biol Chem. 1993 Aug 25;268(24):18070-5.
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.
目前关于Src蛋白酪氨酸激酶调节的模型提出，COOH末端的磷酸酪氨酸Tyr-527通过分子内相互作用与Src同源2（SH2）区域结合，从而抑制激酶结构域。该模型与SH2结构域或COOH末端的突变激活Src的现象一致。SH3结构域的突变也能激活Src，尽管该区域被认为不与磷酸酪氨酸结合。Seidel-Dugan等人（Seidel-Dugan, C., Meyer, B. E., Thomas, S. M., and Brugge, J. S. (1992) Mol. Cell. Biol. 12, 1835 - 1845）已经表明，SH2或SH3结构域缺失的Src突变体可转化鸡胚成纤维细胞并具有增强的激酶活性。这些突变蛋白在Tyr-527处磷酸化不足，这种变化本身可能激活突变体。因此，无法区分SH2和SH3结构域是Tyr-527磷酸化所必需的，还是Src采用或维持抑制状态所必需的。我们通过在酵母细胞中与Tyr-527激酶Csk共表达，人为提高了SH2和SH3缺失突变体的Tyr-527磷酸化水平。我们发现，SH2和SH3结构域都是Csk抑制Src所必需的。在酵母细胞和体外，SH2结构域是Csk高效磷酸化所必需的。当Tyr-527被Csk磷酸化时，SH3结构域是Src被抑制所必需的。这表明SH3结构域与SH2结构域以及磷酸化的Tyr-527协同作用以抑制激酶结构域。成纤维细胞中SH3结构域突变体在Tyr-527处的去磷酸化可能是所提出的SH2/磷酸酪氨酸相互作用失败的结果。
