Immune regulation by SLAT, a Novel Th2-Expressed Protein

描述（由申请人提供）：我们最近分离了一种新的蛋白，SLAT，它是rac特异性激活因子的一个新亚家族的成员。SLAT在Th2细胞中上调，抗原刺激诱导其免疫突触定位和ZAP-70关联，导致TCR募集减少和ZAP-70激活。我们的二，非排他性。工作假设是：1)SLAT通过其关联的ZAP-70抑制激酶的TCR募集和/或激活，从而以促进Th2分化、扩张和/或激活的方式调节TCR信号的质量；2) SLAT和/或其替代产品SLAT2在Th2细胞中作为Cdc42/ rac特异性GEF起作用；这种活性在决定Th2细胞中IS和脂质筏聚集的独特质量以及下游信号方面起着非冗余和独特的作用。我们将通过探索以下四个目标来解决这些假设并分析SLAT的表达，功能和调控的其他方面：1)利用原代小鼠T细胞，成像和生化技术，不同的刺激和受体阻断抗体，我们将分析SLAT/SLAT2 mRNA或蛋白的表达，研究它们在细胞内的定位，绘制膜/SLAT2定位所需的残基，并评估这种定位的意义。2)我们将对SLAT1”小鼠进行详细的分析，包括Th1/Th2分化，近端和下游信号事件，抗体和T细胞反应，以及对与Th1或Th2表型优势相关的实验疾病的易感性。3)我们将通过绘制相互作用位点来探索SLAT和ZAP-70（或Syk）之间关联的生物学相关性，并评估相互作用缺陷的SLAT或ZAP-70/Syk突变体或显性阴性ZAP-70/Syk对Th1/Th1发育和激活的影响。4)我们将使用生化和遗传学方法，包括SLAT1小鼠或重组SLAT突变体的T细胞，绘制负责GEF活性的SLAT残基，研究调节SLAT GEF活性的机制，并确定这种活性对SLAT的Th2偏态效应和Th2细胞中IS的不同组织的意义。这些研究将描述一种新的、多功能的tcr -近端蛋白。

DESCRIPTION (provided by applicant): 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.