DIRECT INTERACTION OF V-SRC WITH THE FOCAL ADHESION KINASE MEDIATED BY THE SRC SH2 DOMAIN
DIRECT INTERACTION OF V-SRC WITH THE FOCAL ADHESION KINASE MEDIATED BY THE SRC SH2 DOMAIN
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DOI：
10.1091/mbc.5.4.413
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发表时间：
1994-04-01
影响因子：
3.3
通讯作者：
GUAN, JL
中科院分区：
文献类型：
--
作者：
XING, Z;CHEN, HC;GUAN, JL
最近描述的粘着斑激酶（FAK）已被牵连的细胞粘附受体整合素和神经肽生长因子启动的信号转导途径。为了研究FAK将信号从膜传递到细胞内部的机制，我们进行了一系列实验来检测FAK与含有Src同源2（SH 2）结构域的蛋白质的潜在相互作用，所述Src同源2（SH 2）结构域是重要的细胞内信号分子。使用v-Src转化的NIH 3 T3细胞，我们发现FAK存在于由抗Src抗体沉淀的免疫复合物中，表明FAK与v-Src在体内的潜在相互作用。我们还显示了潜在的直接相互作用的FAK与v-Src在体内使用酵母双杂交系统。使用在昆虫细胞中表达的重组FAK和含有Src SH 2结构域的细菌融合蛋白，我们在体外显示FAK与Src SH 2结构域直接结合，但不与SH 3结构域结合。一个激酶缺陷的突变体FAK，这是不是autophosphorylated，没有相互作用的Src SH 2结构域在相同的条件下，这表明参与的FAK autophosphorylation网站。用蛋白酪氨酸磷酸酶处理FAK降低了其与Src SH 2结构域的结合，而体外自磷酸化增加了其结合。这些结果证实了FAK自身磷酸化位点在其与含SH 2结构域的蛋白质相互作用中的重要性。两者合计，这些结果表明，FAK可能介导的信号转导事件启动的细胞表面上的激酶激活和自磷酸化，导致其结合到其他关键的细胞内信号分子。
The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.