Non-linear transduction of TCR signals leading to Ras activation

有效的胸腺细胞选择对于产生具有非常广谱的特异性T细胞受体（TCR）的T淋巴细胞是至关重要的，所述特异性T细胞受体（TCR）识别外来抗原，同时避免自身免疫的发展。胸腺细胞阳性选择与阴性选择的确切分子机制尚不清楚，但长期以来一直假设涉及不同的Ras-MAP激酶（MAPK）激活模式。MAPK是位于活性Ras下游并调节基因表达的可调信号分子。我们假设Ras鸟嘌呤核苷酸交换因子的选择，即RasGRP或SOS +， RasGRP，支配不同的Ras活化特征和胸腺细胞选择。该项目将侧重于了解Ras-MAPK信号的RasGRP 1和SOS 1的特点，以及如何影响胸腺细胞的命运决定结合Kuriyan（UCB），格罗夫斯（UCB），Chakraborty（MIT）和鲁塞（UCSF）实验室的生物物理，计算和细胞生化专业知识。以下的组合：1）纯化的重组蛋白; 2）含有定义的脂质的模型二维脂质双层系统 大量的重组蛋白; 3）计算建模;和4）Jurkat T细胞、DT 40 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没有采用的独特方法。项目#2的总体目标是了解通过LAT下游的RasGRP 1或SOS 1激活Ras-MAPK的独特特征，以及这些信号事件如何影响胸腺细胞选择。在目标1中，我们将利用三个科学的方法来表征RasGRP 1 RasGEF函数。 迭代方法中的纪律。在目标2中，我们将定义RasGEF SOS 1的变构调节的要求。目标3.我们将定义RasGRP 1-SOS 1协同作用的分子基础。在目标4中，我们将表征LAT在数字Ras-MAPK信号传导中的作用。目标5。我们将明确SOS介导的Ras活化在胸腺细胞选择中的作用。了解Ras激活的特定性质以及ERK、P38和JNK MAPK的下游激活将是本项目的终点目标; 具体地说，我们将关注RasGRP 1和SOS 1的RasGEF特征的作用以及磷酸-LAT，其可以作为模拟和数字Ras信号传导之间的分叉点。我们期望从这三个学科之间的竞合和矛盾中得到启示。通过逐步建立越来越复杂的模型，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.