Accessibility of the c-Src SH2-domain for binding is increased during mitosis

Abstract

The Src homology 2 (SH2) region is a noncatalytic domain of Src-family tyrosine kinases and other proteins which participants in inter- and intramolecular interactions of tyrosine-phosphorylated proteins. A synthetic peptide modeled on the c-Src carboxyl terminus, which contains phosphotyrosine at position 527, binds recombinant SH2 and the SH2-domain of c-Src which lacks phosphotyrosine 527. Unphosphorylated peptide does not bind detectably. Thus, the phosphorylated peptide is a specific probe for investigating SH2 accessibility. Since Src and other tyrosine kinases may participate in regulating events in mitosis, we used the SH2-binding probe to test the prediction that decreased tyrosine 527 phosphorylation would lead to increased accessibility of the c-Src SH2-domain during mitosis. Probe binding to overexpressed chicken c-Src was enhanced at least 6-fold during mitosis, indicating that the c-Src SH2-domain is more accessible in this part of the cell cycle. This suggests that there may be mitosis-specific interactions of the c-Src SH2-domain with cellular proteins in vivo.