Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail

Phosphorylation of a critical tail tyrosine residue in Src modulates its th ree-dimensional structure and protein tyrosine kinase activity. The protein tyrosine kinase Csk is responsible for catalyzing the phosphorylation of t his key Src tyrosine residue, but the detailed molecular basis for Src reco gnition and catalysis is poorly understood. In this study, we investigate t his phosphorylation event using purified recombinant Csk and Src proteins a nd mutants. It was shown that the apparent k(cat) and K-m values for Csk ph osphorylation of catalytically impaired Src (dSrc) are similar to the param eters for Csk-catalyzed phosphorylation of the Src family member Lck. The S H3 (Src homology 3) and SH2 (Src homology 2) domains of dSrc were fully dis pensable with respect to rapid phosphorylation, indicating that the catalyt ic domain and tail of dSrc are sufficient for the high efficiency of dSrc a s a substrate. Of the eight Src tail residues examined, only the fully cons erved Glu (Y-3 position) and Gin (Y-1 position) investigated by alanine sca nning mutagenesis caused large reductions (10-40-fold) in dSrc substrate ef ficiency. The Y-3 Glu requirement was stringent as conservative replacement s with Asp or Gin were no better than Ala whereas replacement of the Y-1 Gi n with Ile was readily tolerated. Interestingly, en bloc replacement of the tail with a seven amino acid consensus sequence derived from a peptide lib rary analysis was no better than the wild-type sequence, Surprisingly, the dSrc Y527F protein, although not a Csk substrate, enhanced Csk-catalyzed ph osphorylation of dSrc. These results and other data suggest that Src dimeri zation (or higher order oligomerization) is important for high-efficiency C sk-catalyzed phosphorylation of the Src tail.