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. 2001 Feb 20;40(7):2004-10.
doi: 10.1021/bi002342n.

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

D Wang  1 , X Y Huang, P A Cole
Affiliations

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

D Wang et al. Biochemistry. .

Abstract

Phosphorylation of a critical tail tyrosine residue in Src modulates its three-dimensional structure and protein tyrosine kinase activity. The protein tyrosine kinase Csk is responsible for catalyzing the phosphorylation of this key Src tyrosine residue, but the detailed molecular basis for Src recognition and catalysis is poorly understood. In this study, we investigate this phosphorylation event using purified recombinant Csk and Src proteins and mutants. It was shown that the apparent k(cat) and K(m) values for Csk phosphorylation of catalytically impaired Src (dSrc) are similar to the parameters for Csk-catalyzed phosphorylation of the Src family member Lck. The SH3 (Src homology 3) and SH2 (Src homology 2) domains of dSrc were fully dispensable with respect to rapid phosphorylation, indicating that the catalytic domain and tail of dSrc are sufficient for the high efficiency of dSrc as a substrate. Of the eight Src tail residues examined, only the fully conserved Glu (Y-3 position) and Gln (Y-1 position) investigated by alanine scanning mutagenesis caused large reductions (10--40-fold) in dSrc substrate efficiency. The Y-3 Glu requirement was stringent as conservative replacements with Asp or Gln were no better than Ala whereas replacement of the Y-1 Gln with Ile was readily tolerated. Interestingly, en bloc replacement of the tail with a seven amino acid consensus sequence derived from a peptide library analysis was no better than the wild-type sequence. Surprisingly, the dSrc Y527F protein, although not a Csk substrate, enhanced Csk-catalyzed phosphorylation of dSrc. These results and other data suggest that Src dimerization (or higher order oligomerization) is important for high-efficiency Csk-catalyzed phosphorylation of the Src tail.

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