ANALYSIS OF ADAPTER PROTEIN (LAT) IN TCR SIGNALING

描述（申请人的摘要）：T淋巴细胞是免疫调节的中心组分。 免疫系统和信号通过T细胞抗原受体（TCR）发挥作用， 在免疫应答中调节T细胞存活、增殖 分化和效应子功能。LAT（用于激活T细胞的接头） 是一种膜相关的衔接蛋白， activation.磷酸化LAT与Grb 2、Gads、PLC γ 1和其他蛋白结合。 信号分子来自LAT缺陷细胞和LAT敲除的当前研究 小鼠显示LAT对于TCR介导的信号转导是必需的， 胸腺细胞发育我们认为，在T细胞活化后，LAT偶联 Ras-MAPK激活和Ca 2+通量通过与TCR相互作用而与TCR接合 多种蛋白质LAT与其他信号蛋白的相互作用也是 在胸腺细胞发育的不同阶段所需。有三个具体的 旨在验证这一假设，并进一步了解LAT如何 在T细胞活化中起作用。在具体目标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 磷酸化位点是理解其他信号 蛋白质与LAT相互作用以及LAT功能如何调节。具体目标 2，我们将使用不同的LAT突变体来解剖LAT介导的蛋白复合物 并确定这些复合物在TCR信号传导中的重要性。在 具体目标3，我们将重建来自LAT缺陷小鼠的骨髓细胞 用表达不同LAT突变体的重组逆转录病毒， 将这些转导的细胞回输给LAT缺陷小鼠，以确定LAT在 胸腺细胞发育LAT函数的确定和进一步理解 TCR信号通路可能有助于设计合理的方法， 增强或抑制自身免疫、变态反应和组织中T细胞增殖 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.