Xu B, Miller W T
Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook 11794, USA.
Mol Cell Biochem. 1996 May 10;158(1):57-63. doi: 10.1007/BF00225883.
To examine the interactions between Src homology domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.
为了研究v-Src的Src同源结构域与酪氨酸激酶催化结构域之间的相互作用，已在细菌中作为融合蛋白表达了各种结构域组合。含有分离的催化结构域、SH2+催化结构域和SH3+SH2+催化结构域的构建体在自磷酸化测定中具有活性。对于v-Src的催化结构域，而不是v-Abl的催化结构域，添加外源Src SH3-SH2结构域可刺激自磷酸化活性。与自磷酸化结果相反，含有Src同源结构域的构建体对合成肽底物的活性高于分离的催化结构域。v-Src的SH2和SH3结构域稳定活性酶构象的能力也通过在盐酸胍中变性后复性得到证实。总体而言，数据表明，除了在分子间蛋白质-蛋白质相互作用中的作用外，v-Src的Src同源区域可能通过维持催化结构域的活性构象对酪氨酸激酶功能产生积极影响。
