This proposal is aimed at continuing our studies of a novel TCR-coupled signaling protein, SWAP-70-like adapter of T cells (SLAT), which is predominantly expressed in T cells. SLAT is a Cdc42/Rac1 GEF that is required for inflammatory responses mediated by Th1, Th2 and Th17 cells, reflecting its obligatory role in TCR-stimulated Ca2+ release from intracellular ER stores and, consequently, in NFAT (but not NF-:B or AP-1) activation. Our work also revealed that antigen-induced translocation of SLAT to the immunological synapse (IS) is mediated by Lck kinase-dependent phosphorylation of a SLAT ITAM-like motif, and that the Ca2+/NFAT regulatory activity of SLAT depends on actin polymerization and active Cdc42/Rac1. The overall objective of the proposed studies is to further understand the function and regulation of SLAT in T cell biology, to determine its role in immunity to infectious pathogens, and to validate it as a promising, T cell-specific immunosuppressive drug target. Our studies will address the following Aims:
Aim 1) We will use high-resolution imaging approaches to determine the spatiotemporal pattern of wild-type SLAT and SLAT mutants vis-?-vis the IS and TCR microclusters (MCs), and determine how Slat deletion affects IS dynamics;
Aim 2) Mass spectrometry (MS) analysis of proteins binding the phospho-ITAM motif of SLAT and co-immunoprecipitation experiments identified the ERM protein ezrin as a SLAT-interacting protein. We will use biochemical and genetic approaches to address the hypothesis that the interaction of SLAT with ezrin plays an important role in controlling IS dynamics and couples SLAT to Cdc42/Rac1 activation;
Aim 3) Based on our preliminary finding of a TCR-induced association between SLAT and the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), we will collaborate with experts in the IP3R field to analyze the structural determinants of this interaction and its functional relevance for T cell activation, and address the working hypothesis that SLAT is required for proper IP3R function in T cells;
Aim 4) SLAT is required for immune/inflammatory responses against non-replicating antigens, but its role in pathogen clearance, in which CD8+ T cells are critical, is unknown. We will build upon our preliminary findings documenting a role for SLAT in CD8+ T cell activation and expansion to study whether SLAT is required for pathogen (Listeria Monocytogenes and Vaccinia virus) clearance, and whether the Ca2+ signaling defect is the cause of impaired CD8+ T cell responses observed in Slat-/- mice. Results of the proposed research will represent significant advancement in both the basic/scientific and clinical arenas. First, they will shed light on the mechanistic aspects of a key novel player in the TCR-induced Ca2+/NFAT signaling pathway, which plays a critical role in various aspects of T cell biology. Second, they are likely to validate SLAT as a novel drug target for autoimmune diseases and in doing so serve as a launching point for identification of clinically useful, T cell- and Ca2+ signaling pathway-selective drugs.
This project focuses on a novel signaling protein called SLAT, which we discovered in 2003, and which is critical for T cell-mediated inflammatory responses by virtue of its role as an essential regulator of calcium signals required for T cell activation. We will study the mechanisms that regulate the function and intracellular localization of SLAT, determine how it controls intracellular calcium release, and assess its role in immune protection against infectious pathogens. The proposed studies are likely to validate SLAT as a T cell-selective drug target for suppression of autoimmune and inflammatory diseases.
|CÃ´te, Marjorie; Fos, Camille; Canonigo-Balancio, Ann J et al. (2015) SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1. J Cell Sci 128:4341-52|
|Fos, Camille; Becart, Stephane; Canonigo Balancio, Ann J et al. (2014) Association of the EF-hand and PH domains of the guanine nucleotide exchange factor SLAT with IPâ‚ƒ receptor 1 promotes CaÂ²âº signaling in T cells. Sci Signal 7:ra93|
|Feau, Sonia; Schoenberger, Stephen P; Altman, Amnon et al. (2013) SLAT regulates CD8+ T cell clonal expansion in a Cdc42- and NFAT1-dependent manner. J Immunol 190:174-83|
|Mehta, Harshini; Glogauer, Michael; Becart, Stephane et al. (2009) Adaptor protein SLAT modulates Fcgamma receptor-mediated phagocytosis in murine macrophages. J Biol Chem 284:11882-91|
|Canonigo-Balancio, Ann J; Fos, Camille; Prod'homme, Thomas et al. (2009) SLAT/Def6 plays a critical role in the development of Th17 cell-mediated experimental autoimmune encephalomyelitis. J Immunol 183:7259-67|
|Becart, Stephane; Altman, Amnon (2009) SWAP-70-like adapter of T cells: a novel Lck-regulated guanine nucleotide exchange factor coordinating actin cytoskeleton reorganization and Ca2+ signaling in T cells. Immunol Rev 232:319-33|
|Becart, Stephane; Balancio, Ann J Canonigo; Charvet, Celine et al. (2008) Tyrosine-phosphorylation-dependent translocation of the SLAT protein to the immunological synapse is required for NFAT transcription factor activation. Immunity 29:704-19|
|Becart, Stephane; Charvet, Celine; Canonigo Balancio, Ann J et al. (2007) SLAT regulates Th1 and Th2 inflammatory responses by controlling Ca2+/NFAT signaling. J Clin Invest 117:2164-75|