Engagement of multicomponent immunoreceptors such as the T cell antigen receptor (TCR) results in rapid activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate a number of enzymes and adapter molecules involved in a complex signaling cascade. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells). LAT is a 36-38kD integral membrane protein. We have been able to purify this molecule, and have cloned the cDNA that encodes it. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing these adaptor molecules and enzymes to the plasma membrane in the vicinity of the activated TCR. Two independently derived mutants of the Jurkat human T cell line have been shown to be deficient in LAT. TCR crosslinking in these cells fails to result in any indication of T cell activation. However, reconstitution of these cells with LAT completely restores TCR-mediated activation, thus demonstrating the importance of this molecule for TCR signaling. LAT-deficient mice have also been generated using standard gene targeting procedures. T cell development in these mice is blocked at an early stage within the thymus. Thus this complex developmental pathway is also dependent on LAT. Mast cells also express LAT, and engagement of the mast cell immunoreceptor, FceRI, fails to induce optimal degranulation or cytokine production. Current studies on LAT function focus on the role of individual tyrosine residues. Tyrosine to phenylalanine mutations have been introduced, and these mutant molecules have been re-expressed into LAT-deficient cells and mice. In cell lines we have mapped the residues involved in the binding of phospholipase C, grb2 and gads to LAT. We are using genetic techniques to introduce these mutations directly into the germline of mice to evaluate how these sites of LAT tyrosine phosphorylation regulate T cell development. Additional studies are directed at defining how LAT-coupled pathways regulate cytoskeletal reorganization in T cells, and how LAT is involved in cytotoxic T cell function.
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