The generation of immune responses against most foreign pathogens or host-derived cancer cells is critically dependent on an initial phase of T-cell activation and proliferation. Activation of T cells occurs because the TCR is able to bind antigenic peptides complexed with major histocompatibility complex-encoded molecules, and to relay this extracellular stimulus to the intracellular signaling machinery. Signal initiation from the ligand-bound TCR involves the sequential activation of Src family (Lck,Fyn) and Syk family (ZAP-70) protein tyrosine kinases (PTKs). These TCR-regulation PTKs, in tum, catalyze phosphorylation events leading to the activation of phospholipase PLC-gamma-l, Ras, and phosphatidylinositol-3 kinase (PI-3K). In spite of the impressive advances in our understanding of TCR function in recent years, the mechanism by which this receptor couples to downstream signaling proteins, and the relative contributions of these proteins to the T-cell activation program, are only partially understood. To further define the process of TCR-mediated signal transduction, Dr. Abraham's group has created a novel set of cellular and molecular reagents that will allow the application of powerful genetic approaches to analyses of several key components of the post-receptor signaling machinery. The human T-leukemic cell line, Jurkat, was chosen as a well-established base model system for the proposed studies. The principal readout for TCR signaling function will be a prototypical nuclear response to TCR engagement, i.e., interleukin-2 (IL-2) gene expression.
The specific aims of the proposed project are: (1) To study the mechanism of ZAP-70 activation and the roles of this PTK in TCR signaling using a ZAP-70-negative Jurkat mutant as the cellular model system. (2) To define the mechanism of PLC-gamma-l activation by the TCR, and to determine the contributions of various PLC-gamma-l subdomains to the signaling functions of this enzyme during T-cell activation. (3) To examine the roles of PLC-gamma-l,Ras,and PI-3K in IL-2 gene transcription using a virus-derived oncoprotein as a polyfunctional signal transducer in T cells.
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