Superti-Furga G, Fumagalli S, Koegl M, Courtneidge S A, Draetta G
European Molecular Biology Laboratory, Heidelberg, Germany.
EMBO J. 1993 Jul;12(7):2625-34. doi: 10.1002/j.1460-2075.1993.tb05923.x.
The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.
蛋白质酪氨酸激酶c-Src在其羧基末端尾巴的Tyr527磷酸化时受到负调控。最近鉴定出一种使Tyr527磷酸化的激酶，称为Csk。我们在酵母中表达c-Src，以测试Src的SH2和SH3结构域在Tyr527磷酸化所施加的负调控中的作用。粟酒裂殖酵母中c-Src的诱导型表达导致细胞死亡。Csk的共表达抵消了这种效应。在SH2或SH3结构域中发生突变的Src蛋白与野生型c-Src一样具有致死性，但对Csk不敏感，尽管它们在体内是Csk的底物。肽结合实验表明，具有突变SH3结构域的Src蛋白采用了一种SH2结构域不与尾巴相互作用的构象。这些数据支持SH2结构域-磷酸化尾巴相互作用抑制c-Src活性的模型，但将其扩展以包括SH3结构域的作用。我们提出，SH3结构域通过稳定SH2结构域-磷酸化尾巴的相互作用，有助于维持Src分子折叠的无活性构象。此外，我们在此描述的系统允许在中性背景和适合进行遗传分析的生物体中进一步研究酪氨酸激酶的调控。
