Functional analysis of a regulator for Src-family kinases

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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