Hauck C R, Hunter T, Schlaepfer D D
Department of Immunology, The Scripps Research Institute and The Salk Institute, La Jolla, California 92037, USA.
J Biol Chem. 2001 May 25;276(21):17653-62. doi: 10.1074/jbc.M009329200. Epub 2001 Mar 1.
Integrins facilitate cell attachment to the extracellular matrix, and these interactions generate cell survival, proliferation, and motility signals. Integrin signals are relayed in part by focal adhesion kinase (FAK) activation and the formation of a transient signaling complex initiated by Src homology 2 (SH2)-dependent binding of Src family protein-tyrosine kinases to the FAK Tyr-397 autophosphorylation site. Here we show that in viral Src (v-Src)-transformed NIH3T3 fibroblasts, an adhesion-independent FAK-Src signaling complex occurs. Co-expression studies in human 293T cells showed that v-Src could associate with and phosphorylate a Phe-397 FAK mutant at Tyr-925 promoting Grb2 binding to FAK in suspended cells. In vitro, glutathione S-transferase fusion proteins of the v-Src SH3 but not c-Src SH3 domain bound to FAK in lysates of NIH3T3 fibroblasts. The v-Src SH3-binding sites were mapped to known proline-X-X-proline (PXXP) SH3-binding motifs in the FAK N- (residues 371-377) and C-terminal domains (residues 712-718 and 871-882) by in vitro pull-down assays, and these sites are composed of a PXXPXXPhi (where Phi is a hydrophobic residue) v-Src SH3 binding consensus. Sequence comparisons show that residues in the RT loop region of the c-Src and v-Src SH3 domains differ. Substitution of c-Src RT loop residues (Arg-97 and Thr-98) for those found in the v-Src SH3 domain (Trp-97 and Ile-98) enhanced the binding of distinct NIH3T3 cellular proteins to a glutathione S-transferase fusion protein of the c-Src (Trp-97 + Ile-98) SH3 domain. FAK was identified as a c-Src (Trp-97 + Ile-98) SH3 domain target in fibroblasts, and co-expression studies in 293T cells showed that full-length c-Src (Trp-97 + Ile-98) could associate in vivo with Phe-397 FAK in an SH2-independent manner. These studies establish a functional role for the v-Src SH3 domain in stabilizing an adhesion-independent signaling complex with FAK.
整合素促进细胞与细胞外基质的附着，这些相互作用产生细胞存活、增殖和迁移信号。整合素信号部分通过粘着斑激酶（FAK）激活以及由Src家族蛋白酪氨酸激酶依赖Src同源2（SH2）结构域与FAK Tyr-397自磷酸化位点结合而启动的瞬时信号复合物的形成来传递。在这里，我们表明在病毒Src（v-Src）转化的NIH3T3成纤维细胞中，发生了一种不依赖黏附的FAK-Src信号复合物。在人293T细胞中的共表达研究表明，v-Src可以与Phe-397 FAK突变体结合并在Tyr-925位点使其磷酸化，从而促进Grb2在悬浮细胞中与FAK结合。在体外，v-Src SH3结构域的谷胱甘肽S-转移酶融合蛋白而非c-Src SH3结构域的融合蛋白与NIH3T3成纤维细胞裂解物中的FAK结合。通过体外下拉实验，v-Src SH3结合位点被定位到FAK N端（第371-377位氨基酸残基）和C端结构域（第712-718位和第871-882位氨基酸残基）中已知的脯氨酸-X-X-脯氨酸（PXXP）SH3结合基序，这些位点由PXXPXXPhi（其中Phi是一个疏水残基）的v-Src SH3结合共有序列组成。序列比较表明，c-Src和v-Src SH3结构域的RT环区域中的氨基酸残基不同。将c-Src RT环区域的氨基酸残基（Arg-97和Thr-98）替换为v-Src SH3结构域中的氨基酸残基（Trp-97和Ile-98），增强了不同的NIH3T3细胞蛋白与c-Src（Trp-97 + Ile-98）SH3结构域的谷胱甘肽S-转移酶融合蛋白的结合。FAK被鉴定为成纤维细胞中c-Src（Trp-97 + Ile-98）SH3结构域的靶标，在293T细胞中的共表达研究表明，全长c-Src（Trp-97 + Ile-98）可以在体内以不依赖SH2的方式与Phe-397 FAK结合。这些研究确定了v-Src SH3结构域在稳定与FAK的不依赖黏附的信号复合物中的功能作用。
