DETECTION OF A PHYSICAL AND FUNCTIONAL INTERACTION BETWEEN CSK AND LCK WHICH INVOLVES THE SH2 DOMAIN OF CSK AND IS MEDIATED BY AUTOPHOSPHORYLATION OF LCK ON TYROSINE-394

The COOH-terminal Src kinase (Csk) is responsible for the phosphorylat ion of the conserved, negative regulatory, carboxyl-terminal tyrosine of most of the Src family protein tyrosine kinases. Up to now, no stab le binding of Csk to Src kinases has been detected. We therefore decid ed to analyze this interaction using two systems which allow detection of transient interaction. We produced and purified recombinant protei ns in the glutathione S transferase prokaryotic expression system. Fir st, using real-time biospecific interaction analysis (BIAcore(TM)), we detected in vitro a specific interaction between Csk and one of its s ubstrates Lck, a lymphocyte-specific member of the Src family. This in teraction requires the autophosphorylation of Lck: on tyrosine 394 (th e phosphorylation of which is correlated with an increase of the kinas e activity) and involves a functional Csk SH2 domain, Second, using th e yeast two-hybrid system, we confirmed in vivo the physical interacti on between Csk and Lck. Furthermore, in vitro we showed that autophosp horylation of Lck on tyrosine 394 enhances the phosphorylation of Lck by Csk on the negative regulatory site, tyrosine 505, suggesting that activated Lck serves preferentially as substrate for Csk. These findin gs might explain the mechanism(s) by which Csk interacts with most of Src kinases to downregulate their kinase activity.