ANALYSIS OF ADAPTER PROTEIN (LAT) IN TCR SIGNALING

描述(申请人摘要)：T淋巴细胞是 免疫系统和信号通过T细胞抗原受体(TCR)发挥作用 在免疫反应中的重要作用，调节T细胞的存活，增殖， 分化，和效应器功能。Lat(激活T细胞的连接物) 是一种在T细胞上被磷酸化的膜相关接头蛋白 激活。磷酸化的LAT与Grb2、Gads、PLCyl和其他 信号分子。LAT缺陷细胞和LAT基因敲除的研究现状 小鼠表明LAT对TCR介导的信号转导和 胸腺细胞发育。我们认为，在T细胞激活后，LAT偶联 Ras-MAPK激活和钙离子通量与TCR相互作用 多种蛋白质。后来与其他信号蛋白相互作用也是 胸腺细胞发育的不同阶段所需的。有三个具体的 旨在检验这一假设并进一步了解LAT如何 在T细胞激活中起作用。在特定的目标1中，我们将绘制体内和 LAT的体外酪氨酸和丝氨酸/苏氨酸磷酸化位点。映射 需要这些磷酸化位点来理解其他信号如何 蛋白质与LAT相互作用，以及LAT功能是如何被调节的。以特定的目标 2、我们将使用不同的LAT突变体来解剖LAT介导的蛋白质复合体 并确定这些复合体在TCR信号转导中的重要性。在……里面 特定目标3，我们将重建LAT缺陷小鼠的骨髓细胞 表达不同LAT突变体的重组逆转录病毒及其转移 这些转导细胞返回LAT缺陷小鼠以确定LAT功能 胸腺细胞发育。LAT功能的测定及进一步认识 TCR信号通路有助于设计一种合理的途径 增强或抑制自身免疫、过敏和组织中T细胞的增殖 和器官移植。

DESCRIPTION (Applicant's Abstract): T lymphocytes are the central components of immune system and signaling via the T-cell antigen receptor (TCR) plays an important role in immune responses to regulate T-cell survival, proliferation, differentiation, and effector function. LAT (linker for activation of T-cells) is a membrane-associated adaptor protein that is phosphorylated upon T-cell activation. Phosphorylated LAT associates with Grb2, Gads, PLCyl, and other signaling molecules. Current studies from LAT-deficient cells and LAT knock-out mice show that LAT is essential for TCR-mediated signal transduction and thymocyte development. We propose that, upon T-cell activation, LAT couples Ras-MAPK activation and Ca2+ flux to TCR engagement by interacting with multiple proteins. LAT interaction with other signaling proteins is also required in different stages of thymocyte development. There are three specific aims designed to test this hypothesis and to further understand how LAT functions in T-cell activation. In specific aim 1, we will map the in vivo and in vitro tyrosine and serine/threonine phosphorylation sites of LAT. Mapping the phosphorylation sites is required to understand how other signaling proteins interact with LAT and how LAT function is regulated. In specific aim 2, we will use different LAT mutants to dissect LAT-mediated protein complexes and to determine the importance of these complexes in TCR signaling. In specific aim 3, we will reconstitute bone marrow cells from LAT-deficient mice with recombinant retroviruses expressing different LAT mutants and transfer these transduced cells back to LAT-deficient mice to determine LAT function in thymocyte development. Determination of LAT function and further understanding TCR signaling pathway could facilitate the design of a rational approach to augment or inhibit T-cell proliferation in autoimmunity, allergy, and tissue and organ transplantation.