The protein-tyrosine kinase p56(lck) is a member of the src-family of oncogene encoded proteins. The proximity of these family members to the plasma membrane coupled with their proven ability to transform cells has suggested a role for these proteins in transducing extracellular activation signals. This suggestion has been strengthened by the recent findings that several of these traditionally non-receptor tyrosine kinases are indeed receptor associated. This proposal focuses on p56(lck), a lymphocyte- specific tyrosine kinase found associated with the transmembrane receptors CD4 and CD8. This proposal addresses the hypothesis that p56(lck) is a component of a signaling complex whose localization to the plasma membrane is necessary for its proper functioning. The testing of this hypothesis will be approached by 1) identifying and characterizing members of the complex which may serve as substrates for p56(lck) (p56(lck)-associated proteins); 2) characterizing potential complex-members that modify p56(lck) (protein kinases for which p56(lck) serves as a substrate); and 3) defining structural modifications of p56(lck) which direct complex formation. This proposal offers evidence for two potential substrates for p56(lck), for two potential kinases which phosphorylate p56(lck) and for the fatty acylation of p56(lck) by palmitoylation. Strategies are presented for understanding how each of these factors contributes to the functioning of the p56(lck) signaling complex. The methodologies used in this proposal include: 1) protein purification, 2) lymphocyte activation studies using normal mouse lymphocytes and transformed lymphocyte cell lines, 3) biosynthetic labeling and peptide mapping, 4) immunoprecipitations and immune complex kinase assays, 5) immunoblot analysis, 6) myristoylation inhibition studies, 7) cellular distribution and localization studies, and 8) screening of protein expression libraries and DNA cloning. An appreciation of the normal functioning of p56(lck) is critical to understanding how its inappropriate regulation can cause cell transformation.
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