Identification of p130cas as an in vivo substrate of receptor protein-tyrosine phosphatase α
Identification of p130cas as an in vivo substrate of receptor protein-tyrosine phosphatase α
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DOI：
10.1074/jbc.m001626200
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发表时间：
2000-07-07
影响因子：
4.8
通讯作者：
den Hertog, J
中科院分区：
文献类型：
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作者：
Buist, A;Blanchetot, C;den Hertog, J
我们采用底物捕捉策略来确定受体蛋白-酪氨酸磷酸酶α(RPTPα)的生理底物。在这里，我们报告了RPTPα膜近端催化结构域(D1)的底物捕获突变体RPTPα-D1C433S，它特异性地结合到过钒酸处理细胞的酪氨酸磷酸化蛋白上。RPTPα(D2)及其突变体的膜远端催化结构域不与酪氨酸磷酸化蛋白结合。与RPTPα-D1-C433S结合的酪氨酸磷酸化蛋白的模式在不同的细胞系中有所不同，但从每个细胞系中都提取了大约130 kDa的蛋白质。该蛋白被鉴定为p130(Cas)。纤维连接蛋白刺激的NIH3T3细胞的酪氨酸磷酸化p130(Cas)与RPTPα-D1-C433S以网络形式结合，提示p130(Cas)是RPTPα的生理底物。RPTPα在体外使p130(CaS)去磷酸化，并与p130(CaS)亚群共定位于质膜。与激活的SrcY529F、p130(CAS)和RPTPα或无活性的突变体RPTPα的共转染实验表明，RPTPα在体内可使p130(CAS)去磷酸化。在此条件下，RPTPα不能使酪氨酸磷酸化的表皮生长因子受体去磷酸化，提示p130(Cas)是活细胞中RPTPα的特异性底物。综上所述，我们的结果为p130(Cas)是RPTPα在体内的生理底物提供了证据。
We have employed a substrate trapping strategy to identify physiological substrates of the receptor protein-tyrosine phosphatase alpha (RPTP alpha). Here we report that a substrate-trapping mutant of the RPTP alpha membrane proximal catalytic domain (D1), RPTP alpha-D1C433S, specifically bound to tyrosine-phosphorylated proteins from pervanadate-treated cells. The membrane distal catalytic domain of RPTP alpha (D2) and mutants thereof did not bind to tyrosine-phosphorylated proteins. The pattern of tyrosine-phosphorylated proteins that bound to RPTP alpha-D1-C433S varied between cell lines, but a protein of approximately 130 kDa was pulled down from every cell line. This protein was identified as p130(cas). Tyrosine-phosphorylated p130(cas) from fibronectin-stimulated NIH3T3 cells bound to RPTP alpha-D1-C433S as web, suggesting that p130(cas) is a physiological substrate of RPTP alpha. RPTP alpha dephosphorylated p130(cas) in vitro, and RPTP alpha co-localized with a subpopulation of p130(cas) to the plasma membrane. Co-transfection experiments with activated SrcY529F, p130(cas), and RPTP alpha or inactive, mutant RPTP alpha indicated that RPTP alpha dephosphorylated p130(cas) in vivo. Tyrosine-phosphorylated epidermal growth factor receptor was not dephosphorylated by RPTP alpha under these conditions, suggesting that p130(cas) is a specific substrate of RPTP alpha in living cells. In conclusion, our results provide evidence that p130(cas) is a physiological substrate of RPTP alpha in vivo.