PURIFICATION OF BOVINE THYMUS CYTOSOLIC C-TERMINAL SRC KINASE (CSK) AND DEMONSTRATION OF DIFFERENTIAL EFFICIENCIES OF PHOSPHORYLATION AND INACTIVATION OF P56(LYN) AND PP60(C-SRC) BY CSK

The C-terminal src kinase (CSK) is a ubiquitously expressed, cytosolic enzyme capable of phosphorylating and inactivating several plasma mem brane-bound src-family protein tyrosine kinases bl vitro [Nada, S., Ok ada, M., MacAuley, A., Cooper, J. A., & Nakagawa, H. (1990) Nature 351 , 69-72; Bergman, M., Mustelin, T., Oetken, C., Partanen, J., Flint, N . A., Amrein, K. E., Autero, M., Burn, P., & Alitalo, K. (1992) EMBO J . 11, 2919-2924]. We purified CSK to apparent homogeneity from bovine thymus cytosol to study in vitro how the purified enzyme recognizes th e various src-family kinases as its substrates. A novel assay method w as developed for assaying the ability of CSK to inactivate src-family tyrosine kinases. With this assay method, we demonstrated that CSK ina ctivated p56(lyn) with a significantly higher efficiency than gp60(c-s rc). Phosphopeptide mapping of CSK-phosphorylated p56(lyn) and pp60(c- src) shows that the consensus tyrosine residue (also termed tail tyros ine) in the C-terminal regulatory domain of p56(lyn) was phosphorylate d by CSK with an efficiency much higher than that of pp60(c-src). Thus , the higher efficiency of inactivation of p56(lyn) by CSK is a result of the ability of p56(lyn) to serve as a better substrate of CSK. The synthetic peptides derived from the C-terminal portion of p56(lyn) an d pp60(c-src) were much poorer substrates than the intact are-family k inases for CSK, indicating that the local structure around the tail ty rosine is not sufficient to direct efficient phosphorylation of p56(ly n) by CSK. Nevertheless, the slightly higher efficiency displayed by C SK in phosphorylating the peptide derived from the C-terminal portion of p56(lyn) than that from pp60(c-src) suggests that the structural di fferences between the C-terminal portions of p56(lyn) and pp60(c-src) contribute to the differential efficiencies displayed by CSK in phosph orylating the two kinases. Determination of the CSK-phosphorylation si te in the src-C-terminal peptide by phosphopeptide mapping reveals tha t the whole C-terminal regulatory domain and an adjacent part of the p rotein kinase domain contain some of the structural determinants direc ting CSK to phosphorylate the consensus tail tyrosine of the src-famil y kinases.