Structural Biology of Tyrosine Kinase Regulation

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DESCRIPTION (provided by applicant): The Src-family of nonreceptor kinases includes eight distinct proteins identified in humans. These proteins play wide-ranging roles in signal transduction in a variety of cell types, and are implicated in numerous diseases including cancer, HIV infection, rheumatoid arthritis, and Type I diabetes (DM1). Inhibition of Hck prevents onset of diabetes in animal models, suggesting this protein as a target for anti- DM1 drug development. The plethora of human tyrosine kinases makes design of high-specificity inhibitors difficult. The long term objective of this project is to develop a comprehensive, detailed understanding of the structural basis of Hck regulation which will be of use in the design of highly specific Hck inhibitors. Such drugs should have broad applicability in the treatment of rheumatoid arthritis and DM1. The specific aims of this pilot project are (1) to establish a bacterial expression system to produce Hck suitable for analysis by solution-state NMR spectroscopy and (2) to initiate NMR studies using these molecules. Specific aim 1 entails stable-isotope labeling of recombinant Hck and in vitro phosphorylation. Specific aim 2 entails assignment of 1H, 13C, and 15N resonance of Hck and collection and analysis of backbone 15N relaxation data. These studies will establish the feasibility of comprehensive analysis of tyrosine kinase structure and dynamics using NMR.