Tyrosine Protein Kinases and Lymphocyte Activation

Purdue University, West Lafayette IN

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Abstract

DESCRIPTION (provided by applicant): This application is submitted in response to Notice Number NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. In this application, additional funds are sought to expand the scope of research currently underway on grant number 5RO1CA037372-26 entitled "Tyrosine Protein Kinases and Lymphocyte Activation", awarded by the National Cancer Institute. The long-term goal of our research has been to understand, on a molecular level, how cytoplasmic protein-tyrosine kinases regulate the growth properties of immune cells in response to extracellular stimuli. We are focused on the tyrosine kinases involved in the activation of B lymphocytes with a special emphasis on Syk, which was discovered as part of this work. The specific aims of the parent proposal are: 1) to investigate the role of the multi-functional docking site on Syk on coupling the B cell receptor for antigen to downstream signal transduction pathways, 2) to investigate the role of Syk in regulating signal transduction pathways downstream of TNF-family receptors, and 3) to investigate the role of Syk in the modulation of cellular responses to stress stimuli through the identification and characterization of its binding partners and substrates. In this supplemental application, an expansion of this project is sought to add a fourth specific aim: 4) to develop a knock-in mouse expressing an analog-sensitive form of Syk as a tool for investigating Syk's role in immune cell signaling. In this aim, a knock-in mouse will be generated in which the gene encoding for endogenous Syk is replaced with one coding for an "analog-sensitive" form of the kinase in which mutations within the ATP-binding site have been made to enable the enzyme to bind selectively orthogonal inhibitors or ATP-analogs modified to contain bulky substituents. Thus, the activity of Syk in cells derived from this animal will be uniquely sensitive to the application of the orthogonal inhibitor making it possible to evaluate unambiguously of Syk's role in a number of diverse signaling pathways. The unique sensitivity of the engineered kinase to ATP-analogs also allows for the evaluation of novel kinase substrates. The availability of this animal will greatly enhance and accelerate our own research efforts as well as provide a valuable tool to the larger research community. PUBLIC HEALTH RELEVANCE: The Syk protein-tyrosine kinase is an enzyme that is absolutely essential for the ability of B lymphocytes in the immune system to develop properly and to respond to foreign antigens and generate antibodies as part of the adaptive immune response. However, Syk also is required for regulating the proliferation of certain types of leukemia and lymphoma cells;acting as a promoter of cancer cell growth in some examples and as a suppressor of tumor growth in others. Studies of how Syk functions within a cell are essential for gaining an understanding of how it regulates cell proliferation and how its activity might be controlled.