T lymphocytes are the central components of immune system and signaling via the T-cell antigen receptor (TCR) plays an important role in immune responses to regulate T-cell survival, proliferation, differentiation, and effector function. LAT (linker for activation of T-cells) is a membrane-associated adaptor protein that is phosphorylated upon T-cell activation. Phosphorylated LAT associates with Grb2, Gads, PLCyl, and other signaling molecules. Current studies from LAT-deficient cells and LAT knock-out mice show that LAT is essential for TCR-mediated signal transduction and thymocyte development. We propose that, upon T-cell activation, LAT couples Ras-MAPK activation and Ca2+ flux to TCR engagement by interacting with multiple proteins. LAT interaction with other signaling proteins is also required in different stages of thymocyte development. There are three specific aims designed to test this hypothesis and to further understand how LAT functions in T-cell activation.
In specific aim 1, we will map the in vivo and in vitro tyrosine and serine/threonine phosphorylation sites of LAT. Mapping the phosphorylation sites is required to understand how other signaling proteins interact with LAT and how LAT function is regulated.
In specific aim 2, we will use different LAT mutants to dissect LAT-mediated protein complexes and to determine the importance of these complexes in TCR signaling.
In specific aim 3, we will reconstitute bone marrow cells from LAT-deficient mice with recombinant retroviruses expressing different LAT mutants and transfer these transduced cells back to LAT-deficient mice to determine LAT function in thymocyte development. Determination of LAT function and further understanding TCR signaling pathway could facilitate the design of a rational approach to augment or inhibit T-cell proliferation in autoimmunity, allergy, and tissue and organ transplantation.
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