CSK INHIBITION OF C-SRC ACTIVITY REQUIRES BOTH THE SH2 AND SH3 DOMAINS OF SRC

The protein tyrosine kinase c-Src is negatively regulated by phosphory lation of Tyr527 in its carboxy-terminal tail. A kinase that phosphory lates Tyr527, called Csk, has recently been identified. We expressed c -Src in yeast to test the role of the SH2 and SH3 domains of Src in th e negative regulation exerted by Tyr527 phosphorylation. Inducible exp ression of c-Src in Schizosaccharomyces pombe caused cell death. Co-ex pression of Csk counteracted this effect. Src proteins mutated in eith er the SH2 or SH3 domain were as lethal as wild type c-Src, but were i nsensitive to Csk, even though they were substrates for Csk in vivo. P eptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interac ting with the tail. These data support the model of an SH2 domain-phos phorylated tail interaction repressing c-Src activity, but expand it t o include a role for the SH3 domain. We propose that the SH3 domain co ntributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain - phosphorylated tail i nteraction. Moreover, the system we describe here allows for further s tudy of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.