SLAT: A novel GEF coordinating actin remodeling and Ca2+ signaling in T cells

描述(申请人提供)：这项建议旨在继续我们对一种新的TCR偶联信号蛋白的研究，SWAP-70样T细胞适配器(SLAT)，它主要在T细胞中表达。SLAT是Th1、Th2和Th17细胞介导的炎症反应所必需的CDC42/rac1蛋白，反映了其在TCR刺激的细胞内内质网钙释放以及NFAT(而不是NF-：B或AP-1)激活中的必需作用。我们的工作还揭示了抗原诱导的SLAT到免疫突触(IS)的移位是由LCK依赖的SLAT ITAM样基序的磷酸化所介导的，SLAT的钙/NFAT调节活性依赖于肌动蛋白聚合和活性的CDC42/rac1。这些研究的总体目标是进一步了解SLAT在T细胞生物学中的功能和调节，确定其在感染病原体免疫中的作用，并验证其作为一种有前景的T细胞特异性免疫抑制药物的靶点。我们的研究将致力于以下目标：目的1)我们将使用高分辨率成像方法来确定野生型SLAT和SLAT突变体相对于IS和TCR微簇(MC)的时空模式，并确定SLAT缺失如何影响IS动力学；目的2)结合SLAT的磷酸化ITAM基序的蛋白质的质谱分析和免疫共沉淀实验确定ERM蛋白Ezrin为SLAT相互作用蛋白。我们将使用生化和遗传学的方法来解决这一假设，即SLAT与Ezrin的相互作用在控制IS动力学中发挥重要作用，并将SLAT耦合到CDC42/rac1激活；目的3)基于我们初步发现TCR诱导的SLAT与三磷酸肌醇受体1(IP3R1)之间的关联，我们将与IP3R领域的专家合作，分析这种相互作用的结构决定因素及其与T细胞激活的功能相关性，并解决SLAT是T细胞正常的IP3R功能所必需的工作假说；目的：SLAT是针对非复制性抗原的免疫/炎症反应所必需的，但其在病原体清除中的作用尚不清楚，而CD8+T细胞在其中起关键作用。我们将基于我们的初步发现，记录SLAT在CD8+T细胞激活和扩增中的作用，以研究SLAT是否是清除病原体(单核细胞增生性李斯特菌和痘苗病毒)所必需的，以及钙信号缺陷是否是在SLAT-/-小鼠中观察到的CD8+T细胞反应受损的原因。拟议研究的结果将代表着基础/科学和临床领域的重大进步。首先，他们将阐明TCR诱导的钙/NFAT信号通路中一个关键的新角色的机制，该信号通路在T细胞生物学的各个方面发挥着关键作用。其次，他们可能会验证SLAT作为治疗自身免疫性疾病的新药靶点，并以此作为鉴定临床有用的T细胞和钙信号通路选择性药物的起点。

DESCRIPTION (provided by applicant): 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.