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新型TCR偶联信号蛋白SWAP-70样T细胞衔接子（SLAT）的研究，该蛋白主要在T细胞中表达。SLAT是由Th 1、Th 2和Th 17细胞介导的炎症反应所需的Cdc 42/Rac 1 GEF，反映了其在TCR刺激的Ca 2+从细胞内ER储存释放中的强制性作用，因此，在NFAT（但不是NF-：B或AP-1）活化中的强制性作用。我们的工作还表明，抗原诱导的移位SLAT的免疫突触（IS）是介导的Lck激酶依赖性磷酸化的SLAT ITAM样基序，和Ca 2 +/NFAT的调节活性的SLAT依赖于肌动蛋白聚合和活性Cdc 42/Rac 1。拟议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，确定其在感染性病原体免疫中的作用，并验证其作为有前途的T细胞特异性免疫抑制药物靶标。我们的研究将达到以下目的：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突变体维斯维斯IS和TCR微簇（MCs）的时空模式，并确定Slat缺失如何影响IS动力学;目的2）质谱（MS）分析结合SLAT的磷酸-ITAM基序的蛋白质和共-ITAM基序。免疫沉淀实验将ERM蛋白ezrin鉴定为SLAT相互作用蛋白。我们将使用生物化学和遗传学方法来解决这一假设，即SLAT与ezrin的相互作用在控制IS动力学中起着重要作用，并将SLAT与Cdc 42/Rac 1激活偶联;目的3）基于我们对TCR诱导的SLAT与1，4，5-三磷酸肌醇受体1（IP 3R 1）之间的关联的初步发现，我们将与IP 3R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细胞活化的功能相关性，并解决SLAT是T细胞中适当的IP 3R功能所必需的工作假设;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CD 8 + T细胞是关键的。我们将建立在我们的初步研究结果记录的作用，SLAT在CD 8 + T细胞活化和扩增，研究是否SLAT是所需的病原体（单核细胞增生李斯特菌和牛痘病毒）清除，以及是否Ca 2+信号转导缺陷是受损的CD 8 + T细胞反应的原因观察Slat-/-小鼠。拟议研究的结果将代表基础/科学和临床领域的重大进步。首先，他们将阐明TCR诱导的Ca 2 +/NFAT信号通路中关键新参与者的机制方面，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作为自身免疫性疾病的新型药物靶点，并在这样做时作为识别临床有用的T细胞和Ca 2+信号通路选择性药物的启动点。

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.