Functional analysis of a regulator for Src-family kinases

Src家族激酶是一种非受体型的蛋白酪氨酸激酶，定位于细胞质膜的外周。已经表明，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缔合而作为细胞外信号的转导子起作用，但它们的真正功能仍然未知。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，似乎有必要弄清楚其调控机制。最近，我们发现了一种新的激酶，可以磷酸化的调节酪氨酸（称为Csk）在体外。一系列体外研究表明，Csk是最有可能参与调控系统的候选者，但Csk是否真的在体内作为Src家族激酶的调节剂仍是一个问题 ...更多信息 仍有待解答。为了解决这一问题，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，并研究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），我们试图制造特别是在淋巴系统中表达高水平Csk蛋白的转基因小鼠。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，也难以获得过表达Csk蛋白的细胞系。因此，预测Csk的过表达可能影响动物或细胞的活力。在Fred哈钦森癌症研究中心（Jonathan库珀实验室），我们分析了Csk介导的Src家族激酶调控的详细机制，发现Src的SH 3结构域在调控中也很重要。在日本（蛋白质研究所和理化研究所），我们通过胚胎干细胞中的基因靶向产生了缺乏Csk的小鼠胚胎。这些胚胎发育停滞在10至12体节阶段，并表现出生长迟缓和神经组织坏死。Src、Fyn和林恩在这些胚胎中的激酶活性作为增强的比活性的明显结果而大大增强。激酶活性的增加与几种细胞蛋白的酪氨酸磷酸化的增加有关。这些结果表明，Csk确实作为一个不可或缺的负调控Src家族激酶在体内。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