Okamura H, Resh M D
Cell Biology and Genetics Program, Sloan-Kettering Institute, New York, New York 10021, USA.
J Biol Chem. 1995 Nov 3;270(44):26613-8. doi: 10.1074/jbc.270.44.26613.
Expression of oncogenic variants of pp60src leads to dramatic changes in cytoskeletal organization characteristic of transformation. Activated Src associates with the cytoskeletal matrix, resulting in tyrosine phosphorylation of specific cytoskeletal substrates. We have previously shown that stable association of Src with the cytoskeletal matrix is mediated by the Src SH2 domain in a phosphotyrosine-dependent interaction. In this report, we demonstrate that one of the cytoskeletal binding partners of Src is p80/85 cortactin. The association was observed in lysates of transformed cells but was not seen in normal fibroblasts. The interaction could be reconstituted in vitro using transformed cell extracts and a glutathione S-transferase (GST) fusion protein containing the Src SH2 domain but not with GST-Src SH3 or with GST-Src SH2 containing a point mutation in the FLVRES sequence. Confocal microscopy revealed that cortactin redistributed and colocalized with v-Src and a Src SH3 deletion mutant in transformed cells. However, in cells expressing a Src SH2 deletion mutant, the redistribution of cortactin and colocalization with Src did not occur. Furthermore, biochemical fractionation of transformed cells indicated that a significant increase in cortactin distribution to the cytoskeletal fraction occurred, which correlated with a shift in the tyrosine-phosphorylated form of the protein. Cortactin fractionated from cells expressing kinase-defective or myristylation-defective Src mutants did not exhibit this shift. These data suggest a molecular mechanism by which tyrosine phosphorylation of cortactin and association with the Src SH2 domain influence the cytoskeletal reorganization induced in Src-transformed cells.
pp60src致癌变体的表达会导致细胞骨架组织发生显著变化，这是转化的特征。激活的Src与细胞骨架基质结合，导致特定细胞骨架底物的酪氨酸磷酸化。我们之前已经表明，Src与细胞骨架基质的稳定结合是由Src SH2结构域通过磷酸酪氨酸依赖性相互作用介导的。在本报告中，我们证明Src的细胞骨架结合伴侣之一是p80/85皮层肌动蛋白。在转化细胞的裂解物中观察到了这种结合，但在正常成纤维细胞中未观察到。使用转化细胞提取物和含有Src SH2结构域的谷胱甘肽S-转移酶（GST）融合蛋白可以在体外重建这种相互作用，但与GST-Src SH3或含有FLVRES序列点突变的GST-Src SH2则不能。共聚焦显微镜显示，在转化细胞中，皮层肌动蛋白重新分布并与v-Src和Src SH3缺失突变体共定位。然而，在表达Src SH2缺失突变体的细胞中，皮层肌动蛋白的重新分布以及与Src的共定位并未发生。此外，对转化细胞进行生化分级分离表明，皮层肌动蛋白向细胞骨架部分的分布显著增加，这与该蛋白酪氨酸磷酸化形式（的变化）相关。从表达激酶缺陷或肉豆蔻酰化缺陷的Src突变体的细胞中分级分离得到的皮层肌动蛋白并未表现出这种变化。这些数据提示了一种分子机制，通过该机制皮层肌动蛋白的酪氨酸磷酸化以及与Src SH2结构域的结合影响了Src转化细胞中诱导的细胞骨架重组。
