Identification of protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines
Identification of protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines
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DOI：
10.1074/jbc.m004852200
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发表时间：
2000-12-29
影响因子：
4.8
通讯作者：
Fujita, DJ
中科院分区：
文献类型：
--
作者：
Bjorge, JD;Pang, A;Fujita, DJ
c-Src 酪氨酸激酶活性在几种类型的人类癌症中升高，这归因于 c-Src 表达水平升高、c-Src 比活性增加以及 c-Src 中的激活突变。我们发现许多 c-Src 比活性升高的人类乳腺癌细胞系也具有针对 Src 家族激酶羧基末端负调节结构域的磷酸酶活性升高。为了鉴定这种磷酸酶，我们对 MDA-MB-435S 细胞的细胞提取物进行了鉴定。进行色谱分析并测定级分的磷酸酶活性。检测到针对非特异性底物聚（Glu/Tyr）的磷酸酶活性的四个峰。一个峰还使针对 c-Src 羧基末端负调节结构域和完整人 c-Src 建模的肽去磷酸化。免疫印迹和免疫耗竭实验将磷酸酶鉴定为蛋白酪氨酸磷酸酶 1B (PTP1B)。对几种 c-Src 活性增加的人乳腺癌细胞系的检查显示，相对于正常对照乳腺细胞，PTP1B 蛋白水平升高。体外c-Src再激活实验证实了PTP1B去磷酸化和激活c-Src的能力，293细胞中PTP1B的体内过表达导致内源性c-Src激酶活性增加2倍。我们的研究结果表明，PTP1B 是能够在几种人乳腺癌细胞系中使 c-Src 去磷酸化的主要蛋白酪氨酸磷酸酶，并表明 PTP1B 在控制 c-Src 激酶活性中具有调节作用。
c-Src tyrosine kinase activity is elevated in several types of human cancer, and this has been attributed to elevated c-Src expression levels, increased c-Src specific activity, and activating mutations in c-Src, We have found a number of human breast cancer cell lines with elevated c-Src specific activity that also possess elevated phosphatase activity directed against the carboxyl-terminal negative regulatory domain of Src family kinases, To identify this phosphatase, cell extracts from MDA-MB-435S cells were chromatographed and the fractions were assayed for phosphatase activity. Four peaks of phosphatase activity directed against the nonspecific substrate poly(Glu/Tyr) were detected. One peak also dephosphorylated a peptide modeled against the c-Src carboxyl-terminal negative regulatory domain and intact human c-Src. Immunoblotting and immunodepletion experiments identified the phosphatase as protein-tyrosine phosphatase 1B (PTP1B), Examination of several human breast cancer cell lines with increased c-Src activity showed elevated levels of PTP1B protein relative to normal control breast cells. In vitro c-Src reactivation experiments confirmed the ability of PTP1B to dephosphorylate and activate c-Src, In vivo overexpression of PTP1B in 293 cells caused a 2-fold increase of endogenous c-Src kinase activity. Our findings indicate that PTP1B is the primary protein-tyrosine phosphatase capable of dephosphorylating c-Src in several human breast cancer cell lines and suggests a regulatory role for PTP1B in the control of c-Src kinase activity.