Protein-tyrosine kinases regulate the growth properties of cells in response to extracellular mitogens. The long-term objective of this study has been to understand how cytoplasmic protein-tyrosine kinases regulate the responses of cells to extracellular promoters of cell growth using lymphocyte activation as a model system. These studies are important for understanding the role of protein-tyrosine kinases in human malignancies and in diseases of the immune system. In many hematopoietic cells, the actions of activating antigens are mediated by a cytoplasmic protein-tyrosine kinase known as Syk (p72-syk) that is functionally and physically coupled to antigen receptors. How Syk, once activated by receptor ligation, interacts with the components of the downstream biochemical pathways that are stimulated by receptor ligation is the question that will be pursed in this proposal.
Three specific aims are to be investigated: (1) to determine the role of phosphorylation on the interactions of Syk with downstream signaling molecules in B cells; (2) to characterize the interactions of Syk with tubulin and to explore the effects of the Syk-catalyzed phosphorylation of tubulin on its participation in protein/protein interactions; and (3) to identify by genetic screens novel Syk interacting proteins. Methodologies to be used in achieving these objectives include (1) analysis of sites of protein phosphorylation, mutagenesis of tyrosine phosphorylation sites and reconstitution of signal transduction in Syk knockout cells, (2) analysis of the effects of phosphorylation on tubulin assembly, (3) structure determination by NMR of protein/protein interaction sties and (4) cloning of cDNAs for Syk-interacting proteins by expression screening and yeast two-hybrid screening. An analysis of the role of Syk in B cell receptor signaling will serve as a general model for understanding receptor-mediated signaling pathways in a variety of cell types.
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