Non-linear transduction of TCR signals leading to Ras activation

有效的胸腺细胞选择是产生具有非常广泛的特异性T细胞受体(TCR)的T淋巴细胞的关键，这些T细胞受体可以识别外来抗原，同时避免发生自身免疫。胸腺细胞阳性选择和阴性选择的确切分子机制尚不清楚，但长期以来一直认为与不同的RAS-MAPK激活模式有关。MAPK是一种可调节的信号分子，位于活性RAS下游，调节基因表达。我们假设RAS鸟嘌呤核苷酸交换因子的选择用途，即RasGRP或SOS RasGRP控制着不同的RAS激活特征和胸腺细胞的选择。该项目将重点了解RASGRP1和SOS1的RAS-MAPK信号特征，以及这如何通过结合Kuriyan(UCB)、Groves(UCB)、Chakraborty(MIT)和Roose(UCSF)实验室的生物物理、计算和细胞生化专业知识影响胸腺细胞的命运决定。结合：1)纯化的重组蛋白；2)模型二维脂双层系统，包含 大量的重组蛋白质；3)计算模型；4)Jurkat T细胞、DT40 B细胞和突变细胞系的模型系统和小鼠模型研究提供了一种以前从未采用过的独特方法。项目2的总体目标是了解通过LAT下游的RASGRP1或SOS1激活RAS-MAPK的明显特征，以及这些信号事件如何影响胸腺细胞的选择。在目标1中，我们将利用三个科学的 迭代方法中的学科。在目标2中，我们将定义Rasgef SOS1的变构调节要求。在目标3中，我们将定义RASGRP1-SOS1协同作用的分子基础。在目标4中，我们将描述LAT在数字RAS-MAPK信号中的作用。在目标5中，我们将确定SOS介导的RAS激活在胸腺细胞选择中的作用。了解RAS激活的具体性质以及ERK、P38和JNK MAPK的下游激活将是该项目的终点目标； 具体地说，我们将重点讨论RASGRP1和SOS1的Rasgenf特性以及磷酸化LAT的作用，这可能是模拟和数字RAS信号的分叉点。我们希望从这三个学科之间的一致和矛盾中获得洞察力。通过逐步建立到日益复杂的模型，我们的目标是了解选择性Ras-MAPK激活模式的分子机制，以及这些模式如何影响胸腺细胞的选择。

Efficient thymocyte selection is critical to generate T lymphocytes with a very broad spectrum of specific T cell receptors (TCR) that recognize foreign antigens, while avoiding development of autoimmunity. The exact molecular mechanisms underlying thymocyte positive versus negative selection are not know, but different patterns of Ras-MAP kinase (MAPK) activation have long been postulated to be involved. MAPK are tunable signaling molecules that lie downstream of active Ras and that regulate gene expression. We hypothesize that the choice of Ras Guanine nucleotide Exchange Factors usage, namely RasGRP or SOS + RasGRP, governs distinct Ras activation characteristics and thymocyte selection. This project will focus on understanding the characteristics of Ras-MAPK signaling by RasGRP1 and SOS1 and how this affects thymocyte fate decisions by combining the biophysical, computational, and cellular-biochemical expertise of the Kuriyan (UCB), Groves (UCB), Chakraborty (MIT), and Roose (UCSF) labs. Combination of: 1) purified recombinant proteins; 2) a model two dimensional lipid bilayer system containing defined quantities of recombinant proteins; 3) computational modeling; and 4) model systems of the Jurkat T cell, DT40 B cell, and mutant cell lines and mouse model studies provide an unique approach that has not been taken previously. The overall goal of project #2 is to understand the distinct characteristics of Ras-MAPK activation via RasGRP1 or SOS1, downstream of LAT, and how these signaling events impact thymocyte selection. In Aim 1, we will characterize RasGRP1 RasGEF function utilizing the three scientific disciplines in iterative approaches. In Aim 2, we will define the requirement of allosteric regulation of the RasGEF SOS1. In Aim 3. we will define the molecular basis of RasGRP1-SOS1 synergy. In Aim 4, we will Characterize the role of LAT in digital Ras-MAPK signaling. In Aim 5. we will define the role of SOS-mediated Ras activation in thymocyte selection. Understanding the specific nature of Ras activation and the downstream activation of the ERK, P38, and JNK MAPK will be the endpoint goals for this project; specifically we will focus on the role of the RasGEF characteristics of RasGRP1 and SOS1 as well as on phospho-LAT, which could serve as a point of bifurcation between analog and digital Ras signaling. We expect that we will gain insights from both concurrence and contradiction between the three disciplines. By building step-wise to increasingly complex models we aim to understand the molecular mechanism of selective Ras-MAPK activation patterns and how these impact thymocyte selection.