Functional analysis of a regulator for Src-family kinases

src家族激酶是位于质膜外围的非受体型蛋白酪氨酸激酶。有人认为，它们通过与缺乏酪氨酸激酶活性的某些类型的受体的物理关联，作为细胞外信号的转导器起作用，但它们的真正功能仍不清楚。已知src家族激酶的激酶活性受c末端调节酪氨酸残基的磷酸化调节。为了阐明src家族激酶的功能，有必要弄清楚其调控机制。最近，我们在体外发现了一种新的激酶，可以磷酸化调节酪氨酸（称为Csk）。在体外获得的一系列证据表明，Csk是最有可能参与调控系统的候选者，但关于Csk是否确实在体内作为src家族激酶的调节剂，还有更多的问题有待回答。为了解决这个问题，我们计划制造在特定组织中过度表达Csk或缺乏Csk的突变小鼠，并检查Csk功能的获得或丧失对src家族激酶的影响。在华盛顿大学（Roger Perlmutter实验室），我们试图产生高水平表达Csk蛋白的转基因小鼠，特别是在淋巴系统。然而，没有小鼠产生足够量的Csk蛋白。我们还观察到，即使在培养的细胞中，也很难获得过表达Csk蛋白的细胞系。因此，预测过表达Csk可能会影响动物或细胞的生存能力。在Fred Hutchinson Cancer Research Center (Jonathan Cooper Lab)，我们分析了Csk介导Src家族激酶调控的详细机制，发现Src的SH3结构域对调控也很重要。在日本（蛋白质研究所和物理化学研究所），我们通过胚胎干细胞的基因靶向产生了csk缺陷小鼠胚胎。这些胚胎在10 ~ 12岁发育受阻，表现出生长迟缓和神经组织坏死。Src、Fyn和Lyn在这些胚胎中的激酶活性明显增强，这是特异性活性增强的结果。激酶活性的增加与几种细胞蛋白酪氨酸磷酸化的增加有关。这些发现表明，在体内，Csk确实是src家族激酶不可缺少的负调节因子。少

Functional analysis of a regulator for Src-family kinasesSrc-family kinases are non-receptor type of protein-tyrosine kinases which are localized at the periphery of plasma membrane. It has been suggested that they function as transducers of extracellular signals through physical association with certain type of receptors that lack tyrosine kinase activity, but their genuine functions still remain unknown. The kinase activities of the Src-family kinases are known to be regulated by the phosphorylation of C-terminal regulatory tyrosine residues. In order to shed light on the functions of the Src-family kinases, it seems necessary to figure out the regulation mechanism. Recently we found a novel kinase that can phosphorylate the regulatory tyrosines (termed as Csk) in vitro. A line of evidence obtained in vitro have shown that Csk is the most probable candidate involved in the regulation system, but a question of whether Csk is indeed acting as a regulator for Src-family kinases in vivo … More remains to be answered. To address this, we planed to make mutant mice that overexpress Csk in particular tissues or lack Csk, and examine the effects of gain or loss of Csk function on the Src-family kinases.In University of Washington (Roger Perlmutter lab), we have attempted to generate transgenic mice expressing high level of Csk protein particularly in lymphoid system. However, no mouse produced sufficient amount of Csk protein. We also observed even in cultured cells that it was difficult to gain a cell line overexpressing Csk protein. Thus it was predicted that overexpression of Csk may affect the viability of animals or cells. In Fred Hutchinson Cancer Research Center (Jonathan Cooper Lab), we analyzed the detailed mechanism of the regulation of Src-family kinases mediated by Csk, and found that SH3 domain of Src is also important for the regulation. In Japan (Institute for Protein Research and The Institute of Physical and Chemical Research), we generated Csk-deficient mouse embryos by gene targeting in embryonic stem cells. These embryos were developmentally arrested at the 10 to 12 somite stage and exhibited growth retardation and necrosis in the neural tissues. The kinase activity of Src, Fyn and Lyn in these embryos was greatly enhanced as an apparent consequence of enhanced specific activity. The increase in kinase activity was associated with an increase in tyrosine phosphorylation of several cellular proteins. These findings suggested that Csk indeed act as an indispensable negative regulator of Src-family kinases in vivo. Less