Abstract:
:The C-terminal src kinase (CSK) is a ubiquitously expressed, cytosolic enzyme capable of phosphorylating and inactivating several plasma membrane-bound src-family protein tyrosine kinases in vitro [Nada, S., Okada, 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 the various src-family kinases as its substrates. A novel assay method was developed for assaying the ability of CSK to inactivate src-family tyrosine kinases. With this assay method, we demonstrated that CSK inactivated p56lyn with a significantly higher efficiency than pp60c-src. Phosphopeptide mapping of CSK-phosphorylated p56lyn and pp60c-src shows that the consensus tyrosine residue (also termed tail tyrosine) in the C-terminal regulatory domain of p56lyn was phosphorylated by CSK with an efficiency much higher than that of pp60c-src. Thus, the higher efficiency of inactivation of p56lyn by CSK is a result of the ability of p56lyn to serve as a better substrate of CSK. The synthetic peptides derived from the C-terminal portion of p56lyn and pp60c-src were much poorer substrates than the intact src-family kinases for CSK, indicating that the local structure around the tail tyrosine is not sufficient to direct efficient phosphorylation of p56lyn by CSK. Nevertheless, the slightly higher efficiency displayed by CSK in phosphorylating the peptide derived from the C-terminal portion of p56lyn than that from pp60c-src suggests that the structural differences between the C-terminal portions of p56lyn and pp60c-src contribute to the differential efficiencies displayed by CSK in phosphorylating the two kinases. Determination of the CSK-phosphorylation site in the src-C-terminal peptide by phosphopeptide mapping reveals that the whole C-terminal regulatory domain and an adjacent part of the protein kinase domain contain some of the structural determinants directing CSK to phosphorylate the consensus tail tyrosine of the src-family kinases.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Cheng HC,Bjorge JD,Aebersold R,Fujita DJ,Wang JHdoi
10.1021/bi9603940subject
Has Abstractpub_date
1996-09-10 00:00:00pages
11874-87issue
36eissn
0006-2960issn
1520-4995pii
bi9603940journal_volume
35pub_type
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