Protein-tyrosine kinases regulate the growth properties of cells in response to extracellular mitogens. The long-term objective of this project has been to understand, on a molecular basis, how cytoplasmic protein-tyrosine kinases function to couple receptors that lack enzymatic activity to the activation of intracellular pathways that lead to cell proliferation or differentiation. To accomplish this goal, we have focused our studies on the tyrosine kinases involved in the activation of B lymphocytes through the engagement of cell surface antigen receptors. Our major focus is on the tyrosine kinase, Syk, which is a required component of the signaling machinery in B cells. We hypothesize that the covalent modification of Syk on amino acid residues located with the linker region that separates the N-terminal SH2 domains from the C-terminal catalytic domain regulate multiple aspects of Syk's function in B cells. Preliminary studies indicate that these covalent modifications of both serine and tyrosine residues regulate the ability of Syk to couple the BCR to different intracellular pathways, the export of Syk from the nucleus, and the ability of Syk to function in general following receptor cross-linking. Consequently, we hypothesize that the effects of inhibitory or stimulatory co- receptors that regulate B cell responsiveness arise, in part, through alterations in the state of phosphorylation of the Syk linker region. To explore these hypotheses, we plan to accomplish three specific aims: 1) to investigate the role of the tyrosine-phosphorylation of Syk in the physiological outcome of BCR-stimulated signaling in B lymphocytes, 2) to investigate the role of the serine-phosphorylation of Syk in the regulation of Syk function and nuclear export and 3) to characterize proteins that interact with Syk as defined by yeast two-hybrid analyses. Methodologies to be used include 1) generation of transgenic mice, 2) structure determination of protein-protein interactions by high resolution NMR, 3) analyses of protein phosphorylation and signal transduction in cellular model systems, and 4) biochemical and genetic measurements of protein-protein interactions.
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