A potential SH3 domain-binding site in the Crk SH2 domain
A potential SH3 domain-binding site in the Crk SH2 domain
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DOI：
10.1074/jbc.271.35.21365
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发表时间：
1996-08-30
影响因子：
4.8
通讯作者：
Pawson, T
中科院分区：
文献类型：
--
作者：
Anafi, M;Rosen, MK;Pawson, T
哺乳动物接头蛋白Crk-II的Src同源2（SH 2）结构域含有富含脯氨酸的插入物，预测位于延伸的DE环内，其被磷酸肽结合所限制。使用酵母双杂交系统，发现Crk-II SH 2结构域的该区域与SH 3结构域的子集，特别是Abl SH 3结构域相互作用。此外，发现这种富含脯氨酸的插入物改变了Crk-II被p140（c-abl）酪氨酸激酶磷酸化的效率。在体外，全长非磷酸化的Crk-II与谷胱甘肽S-转移酶Abl SH 3结构域融合蛋白的相互作用非常弱，然而，Crk-II在Tyr-221上的磷酸化诱导了与SH 2结构域的分子内缔合，或添加对应于Crk-II Tyr-221磷酸化位点的磷酸肽，刺激Crk-II与Abl SH 3结构域的结合，NMR光谱分析显示Tyr-221磷酸肽与Crk SH 2结构域的结合诱导位于富含脯氨酸的插入物中的瓦尔-71的化学位移变化，这些结果表明，Crk SH 2结构域中的富含脯氨酸的插入物构成了一个SH 3结构域结合位点，该位点可以通过磷酸肽配体与Crk SH 2结构域的结合来调节。
The Src homology 2 (SH2) domain of the mammalian adaptor protein Crk-II contains a proline-rich insert, predicted to lie within an extended DE loop, which is dispensable for phosphopeptide binding, Using the yeast two-hybrid system, this region of the Crk-II SH2 domain was found to interact with a subset of SH3 domains, notably the Abl SH3 domain. Furthermore, this proline-rich insert was found to modify the efficiency with which Crk-II was phosphorylated by the p140(c-abl) tyrosine kinase, In vitro, the interaction of full length non-phosphorylated Crk-II with a glutathione S-transferase Abl SH3 domain fusion protein was very weak, However, phosphorylation of Crk-II on Tyr-221 which induces an intramolecular association with the SH2 domain, or addition of a phosphopeptide corresponding to the Crk-II Tyr-221 phosphorylation site, stimulated association of Crk-II with the Abl SH3 domain, NMR spectroscopic analysis showed that binding of the Tyr-221 phosphopeptide to the Crk SH2 domain induced a chemical shift change in Val-71, located in the proline-rich insert, indicative of a change in the structure of the proline-rich loop in response of Crk SH2 pTyr-221 interaction, These results suggest that the proline-rich insert in the Crk SH2 domain constitutes an SH3 domain-binding site that can be regulated by binding of a phosphopeptide ligand to the Crk SH2 domain.