Differential regulation of Ras nucleotide exchange factors

描述(申请人提供)：RAS的激活是细胞外刺激和下游事件之间的关键环节，导致细胞增殖和分化。已知导致RAS不适当激活的突变会导致白血病和实体瘤，以及努南综合征等发育障碍。RAS循环在活跃的GTP结合和非活跃的GDP结合状态之间，受RAS鸟核苷酸交换因子(GEF)和鸟嘌呤核苷酸激活蛋白(GAP)控制，RAS对这些蛋白质不敏感，或GEF或GAP的不适当激活通常是这些疾病的基础。T细胞受体的激活可以通过两个GEF RASGRP1和SOS的联合作用引起不同程度的RAS刺激，从而导致细胞增殖或死亡，这取决于细胞外信号的强度和持续时间。这种对未成熟T细胞(胸腺细胞)进行阳性或阴性选择的过程是适应性免疫和预防自身免疫反应的关键途径。该项目的长期目标是在模型膜上重建体外T细胞受体信号通路，以了解阳性和阴性胸腺细胞选择的结构和生化基础。为了激活RAS，RASGRP1和/或SOS必须首先被上游信号激活，但Rasgef激活的许多方面的分子基础尚未确定。目前也不清楚为什么在这个信号级联中需要两个不同的RasGEF。这一建议将研究SOS和RASGRP1被允许协同激活RAS的独特信号激活的假设。以下具体目标将检验这一假设。1)激活受体复合体募集SOS膜对催化活性的影响将通过与膜锚定的RAS的荧光核苷酸交换实验来阐明。2)RASGRP1的调控机制可通过测定不同RASGRP1结构的核苷酸交换活性来阐明，这些RASGRP1结构只含有催化结构域或该结构域与C-末端EF-HANDS、C1结构域和螺旋卷曲基序。此外，将调查辅助RAS结合位点的存在，以确定RASGRP1是否含有负责正反馈环的变构结合口袋，如在SOS中发现的那样。最后，RASGRP1和SOS协同激活RAS的能力将通过在核苷酸交换实验中结合这两种蛋白质来测试。3)用X射线结晶学方法探讨了RASGRP1催化作用的结构基础和侧翼结构域效应。RASGRP1和SOS之间的这些比较研究将为RAS在T细胞中如何激活，以及两种生化活性相似的酶如何激活而不同地诱导不同的细胞命运提供结构和生化基础。

DESCRIPTION (provided by applicant): Activation of Ras is a key link between extracellular stimuli and downstream events that lead to cell proliferation and differentiation. Mutations leading to inappropriate Ras activation are known to cause leukemia and solid tumors, as well as developmental disorders such as Noonan Syndrome. Ras cycles between active GTP-bound and inactive GDP-bound states, which is controlled by Ras guanine nucleotide exchange factors (GEFs) and guanine nucleotide activating proteins (GAPs) and insensitivity of Ras to these proteins, or inappropriate activation of GEFs or GAPS often underlie these diseases. T-cell receptor activation can cause varying degrees of Ras stimulation through the combined action of two GEFs, RasGrp1 and SOS, which leads to cell proliferation or death, depending on the strength and duration of the extracellular signal. This process of positive or negative selection of immature T cells (thymocytes) is a key pathway for adaptive immunity and the prevention of autoimmune responses. The long-term goal of this project is to reconstitute the T-cell receptor signaling pathway in vitro on model membranes to understand the structural and biochemical basis for positive and negative thymocyte selection. For Ras activation to occur, RasGrp1 and/or SOS must first be activated by upstream signals, but the molecular basis for many aspects of RasGEF activation have not been determined. It is also unclear why two different RasGEFs are necessary in this signaling cascade. This proposal will investigate the hypothesis that SOS and RasGrp1 are activated by unique signals that allow for cooperative Ras activation. The following specific aims will test this hypothesis. 1) The impact of SOS membrane recruitment by activated receptor complexes on catalytic activity will be elucidated using a fluorescence nucleotide exchange assay with membrane anchored Ras. 2) Regulatory mechanisms of RasGrp1 will be elucidated by measuring nucleotide exchange activity of different RasGrp1 constructs that contain only the catalytic domain or this domain with the C-terminal EF-hands, C1 domain and coiled-coil motif. In addition, the presence of auxiliary Ras binding sites will be investigated to determine if RasGrp1 contains an allosteric binding pocket responsible for a positive feedback loop such as that found in SOS. Finally, the ability of RasGrp1 and SOS to cooperatively activate Ras will be tested by combining the two proteins in nucleotide exchange experiments. 3) The structural basis for RasGrp1 catalysis and flanking domain effects will be probed using X-ray crystallography. These comparative studies between RasGrp1 and SOS will provide a structural and biochemical basis for how Ras activation occurs in T-cells, and how activation of two enzymes with similar biochemical activities can differentially induce distinct cell fates.