We recently isolated a novel protein, SLAT, member of a novel subfamily of Rac-specific activators. SLAT is upregulated in Th2 cells and antigen stimulation induces its immune synapse localization and ZAP- 70 association, leading to reduced TCR recruitment and activation of ZAP-70. Our two, non-exclusive. working hypotheses are:1) SLAT, via its association ZAP-70, inhibits the kinase's TCR recruitment and/or activation, thereby modulating the quality of TCR signaling in a manner that promotes Th2 differentiation, expansion and/or activation; and 2) SLAT and/or its alternative product, SLAT2, functions in Th2 cells as a Cdc42/Rac-specific GEF; this activity plays a non-redundant and unique role in determining the distinct quality of the IS and lipid raft clustering and, consequently, downstream signals, in Th2 cells. We will address these hypotheses and analyze other aspects of the expression, function and regulation of SLAT by exploring four aims: 1) Using primary mouse T cells, imaging and biochemical techniques, different stimuli, and receptor-blocking antibodies, we will analyze the expression of SLAT/SLAT2 mRNA or protein, study their intracellular localization, map residues essential for the membrane/IS localization, and assess the significance of this localization. 2) We will conduct a detailed analysis of SLAT1"""""""" mice, including Th1/Th2 differentiation, proximal and downstream signaling events, antibody and T cell responses, and susceptibility to experimental diseases that are associated with predominance of the Th1 or Th2 phenotype. 3) We will explore the biological relevance of the association between SLAT and ZAP-70 (or Syk) by mapping the interaction sites, and assessing the effects of interaction-deficient SLAT or ZAP-70/Syk mutants, or dominant negative ZAP-70/Syk, on Th1/Th1 development and activation. 4) We will use biochemical and genetic approaches, including SLAT1' mice or T cells reconstituted with SLAT mutants, to map the SLAT residues responsible for GEF activity, study mechanisms that regulate the GEF activity of SLAT, and determine the significance of this activity for the Th2-skewing effect of SLAT and for distinct organization of the IS in Th2 cells. These studies will characterize a novel, multifunctional TCR-proximal protein.
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