Mayer B J, Hirai H, Sakai R
Howard Hughes Medical Institute, Children's Hospital, Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Curr Biol. 1995 Mar 1;5(3):296-305. doi: 10.1016/s0960-9822(95)00060-1.
Non-receptor protein-tyrosine kinases often contain at least one Src homology 2 (SH2) domain, a protein module that binds with high affinity to tyrosine-phosphorylated peptides. Because SH2 domains would be predicted to bind with high affinity to proteins phosphorylated by the kinase, but not to the unphosphorylated substrate, their presence in tyrosine kinases has been puzzling. An important role for the SH2 domain of the Abl tyrosine kinase was suggested by work showing that Abl requires an intact SH2 domain in order to malignantly transform cells, and that replacement of the Abl SH2 domain with heterologous SH2 domains alters the spectrum of proteins phosphorylated detectably by Abl in vivo.
We have used purified wild-type and mutant Abl kinases to examine the roles of the Abl's SH2 and catalytic domains in phosphorylation of p130CAS, a model substrate that has multiple potential phosphorylation sites. We find that an SH2 domain is required for efficient hyperphosphorylation of p130 in vitro. We use chimeric mutants with heterologous SH2 domains to demonstrate that the SH2 domain of the oncogenically transforming adaptor protein Crk, which is the SH2 domain predicted to bind with highest affinity (of those tested) to potential phosphorylation sites in p130, is best able to facilitate hyperphosphorylation. This is the case whether the catalytic domain of the kinase is derived from Abl or from its distant relative, Src. These studies also reveal a role for binding of Crk to Abl in mediating phosphorylation by the kinase. Using purified proteins, we demonstrate that association with Crk strikingly enhances the ability of Abl to hyperphosphorylate p130. There is an excellent correlation between the ability of mutant Crk proteins to promote hyperphosphorylation of p130 by Abl and their ability to transform rodent fibroblasts.
Our data suggest that, ultimately, the substrate specificity of a non-receptor tyrosine kinase is dependent on the binding specificity of its associated SH2 domain. The SH2 domain binds tightly to a subset of proteins phosphorylated by the catalytic domain, leading to processive phosphorylation of those proteins. Substrate specificity can be broadened by an association between the kinase and proteins, such as Crk, that contain additional SH2 domains; this may play a role in malignant transformation by Crk.
非受体蛋白酪氨酸激酶通常至少包含一个Src同源2（SH2）结构域，这是一种能与酪氨酸磷酸化肽段高亲和力结合的蛋白质模块。由于预计SH2结构域会与激酶磷酸化的蛋白质高亲和力结合，而不与未磷酸化的底物结合，因此它们在酪氨酸激酶中的存在一直令人费解。Abl酪氨酸激酶的SH2结构域的重要作用已被相关研究表明，这些研究显示Abl需要完整的SH2结构域才能恶性转化细胞，并且用异源SH2结构域替换Abl的SH2结构域会改变Abl在体内可检测到的磷酸化蛋白质谱。
我们使用纯化的野生型和突变型Abl激酶来研究Abl的SH2和催化结构域在p130CAS磷酸化中的作用，p130CAS是一种具有多个潜在磷酸化位点的模型底物。我们发现在体外高效过度磷酸化p130需要SH2结构域。我们使用带有异源SH2结构域的嵌合突变体来证明致癌转化衔接蛋白Crk的SH2结构域最能促进过度磷酸化，在测试的SH2结构域中，Crk的SH2结构域预计与p130中的潜在磷酸化位点具有最高亲和力。无论激酶的催化结构域源自Abl还是其远亲Src，都是如此。这些研究还揭示了Crk与Abl结合在介导激酶磷酸化中的作用。使用纯化的蛋白质，我们证明与Crk结合显著增强了Abl过度磷酸化p130的能力。突变型Crk蛋白促进Abl对p130过度磷酸化的能力与其转化啮齿动物成纤维细胞的能力之间存在极好的相关性。
我们的数据表明，最终，非受体酪氨酸激酶的底物特异性取决于其相关SH2结构域的结合特异性。SH2结构域紧密结合催化结构域磷酸化的一部分蛋白质，导致这些蛋白质的持续性磷酸化。激酶与含有额外SH2结构域的蛋白质（如Crk）之间的结合可拓宽底物特异性；这可能在Crk的恶性转化中起作用。
