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

描述（由申请人提供）：该提案旨在继续我们对一种主要在T细胞中表达的新型tcr偶联信号蛋白SWAP-70-like adapter of T cells （SLAT）的研究。SLAT是一种Cdc42/Rac1 GEF，是由Th1、Th2和Th17细胞介导的炎症反应所必需的，反映了它在tcr刺激的细胞内ER储存的Ca2+释放中所起的必要作用，从而在NFAT（但不包括NF-:B或AP-1）激活中起作用。我们的工作还揭示了抗原诱导的SLAT向免疫突触（IS）的易位是由SLAT itam样基元的Lck激酶依赖性磷酸化介导的，SLAT的Ca2+/NFAT调节活性依赖于肌动蛋白聚合和活性Cdc42/Rac1。本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控，确定其在感染性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靶点。目的1)利用高分辨率成像方法确定野生型SLAT和SLAT突变体对IS和TCR微簇（MCs）的时空格局，并确定SLAT缺失对IS动态的影响；目的2)结合SLAT磷酸化- itam基序蛋白的质谱分析和共免疫沉淀实验证实ERM蛋白ezrin是SLAT相互作用蛋白。我们将利用生物化学和遗传学的方法来解决SLAT与ezrin的相互作用在控制IS动力学中起重要作用的假设，并将SLAT与Cdc42/Rac1激活耦合；目标3)基于我们对tcr诱导的SLAT与肌醇1,4,5-三磷酸受体1 （IP3R1）之间关联的初步发现，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细胞中IP3R功能所需的工作假设；目的4)SLAT是针对非复制抗原的免疫/炎症反应所必需的，但其在病原体清除中的作用尚不清楚，其中CD8+ T细胞至关重要。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，研究SLAT是否需要病原体（单核增生李斯特菌和牛痘病毒）清除，以及Ca2+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。拟议的研究结果将代表在基础/科学和临床领域的重大进展。首先，他们将阐明在tcr诱导的Ca2+/NFAT信号通路中一个关键的新参与者的机制方面，这在T细胞生物学的各个方面起着关键作用。其次，他们可能会验证SLAT作为自身免疫性疾病的新药物靶点，并以此作为鉴定临床有用的T细胞和Ca2+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.