Analysis of protein-tyrosine phosphatases involved in positive regulation of Src family protein-tyrosine kinases

蛋白酪氨酸激酶在细胞外刺激引起的细胞内通路中发挥着重要的信号转导作用。Src家族蛋白酪氨酸激酶属于非受体类型的蛋白酪氨酸激酶，也被认为具有信号转导的功能，但其功能的分子机制尚未阐明，因为Src家族激酶在细胞受到细胞外信号刺激时是如何被激活的尚不完全清楚。研究表明，Src家族蛋白酪氨酸动力学的活性受到位于羧基末端区域的酪氨酸去磷酸化的调节位点的正调控。为了阐明它们的激活机制，我们试图确定一种酪氨酸磷酸酶，它能特异性作用于Src家族蛋白酪氨酸激酶的调控位点。因此，1)我们成功克隆了三种高度集中于神经系统的酪氨酸磷酸酶受体类型，并发现其中一种（称为BEM-1）具有体外脱磷酸化Src家族蛋白酪氨酸激酶调控位点的潜力。其体内功能目前正在研究中。2)以磷酸化的Src蛋白为底物，纯化了酪氨酸磷酸酶并确定了其部分氨基酸序列。序列显示纯化的酶与SH-PTP2相同，SH-PTP2是一种具有两个SH2结构域的胞质酪氨酸磷酸酶。SH-PTP2异位过表达成纤维细胞，当细胞与底物分离时，增强了Src家族蛋白酪氨酸激酶的激活，抑制了p130^<cas>蛋白的去磷酸化，这是Src家族激酶在体内的最佳靶点之一。这些结果表明SH-PTP2在体内可以作为Src家族蛋白酪氨酸激酶的正调节因子。

Protein-tyrosine kinases play important roles as signal transducers in the intracellular pathway evoked by extracellular stimuli. Src family protein-tyrosine kinases which belong to non-receptor type of protein-tyrosine kinases are also believed to function as signal transducers but the molecular mechanisms of their functions are yet to be elucidated because it is completely unknown how the Src family kinases are activated when the cells are stimulated by extracellular signals. It has been shown that the activities of Src family protein-tyrosine kinascs are positively regulated by tyrosine dephosphorylation at a regulatory site located in the carboxyl terminal region. To clarify their activation mechanism, we have attempted to identify a tyrosine phosphatase (s) that specifically acts on the regulatory site of the Src family protein-tyrosine kinases. Consequently, 1) we have succeeded in cDNA cloning of three receptor type of tyrosine phosphatases which are highly concentrated in the nervous system, and found that one of them (termed as BEM-1) had potential to dephosphorylate the regulatory site of Src family protein-tyrosine kinases in vitro. Its in vivo function is now under investigation. 2) Using phosphorylated Src protein as a substrate, we have purified a tyrosine phosphatase and determined its partial amino acid sequence. The sequence revealed that the purified enzyme was identical to SH-PTP2, a cytosolic tyrosine phosphatase having two SH2 domains. Ectopic overexpression of SH-PTP2 into fibroblast cells augmented the activation of Src family protein-tyrosine kinases when the cells were detached from the substrate and suppressed the dephosphorylation of p130^<cas> protein, which is known as one of the best targets of Src family kinases in vivo. These results suggested that SH-PTP2 can serve as a positive regulator for Src family protein-tyrosine kinases in vivo.