The src gene is a proto-oncogene whose product, the Src protein, is representative of a sub-group of intracellular protein tyrosine kinases (the Src family). Mutations in the src gene that deregulate the tyrosine kinase activity of Src are oncogenic. Src family kinases are negatively regulated by a number of mechanisms, including an inhibitory C-terminal tyrosine phosphorylation. Normally, the stoichiometry of this phosphorylation is high because the rate of phosphate transfer to Src exceeds the rate of dephosphorylation. Clearly, it is important to identify and analyze the regulation of the kinases and phosphatases involved in these reactions in order to understand how Src family kinases are regulated. We have been characterizing a kinase, Csk, that specifically phosphorylates Src at the inhibitory site. We have now developed antibodies, in vitro kinase assays, and DNA constructs for expressing Csk. We have found that the intracellular localization of Csk is regulated by Src. One goal of this proposal is to study the mechanism of this localization, and to determine the significance of Csk translocation for its ability to repress Src. Another goal is to determine the functions of individual domains within Csk for regulation of Src. We will search for homologs of Csk. Finally, we are interested in the role of the Src homology 3 (SH3) domain of Src in Src regulation and transformation. We are screening for cell proteins that interact with the Src SH3 domain and propose experiments to analyze these interactions.
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