Differential regulation of Ras nucleotide exchange factors

描述（由申请人提供）：Ras的激活是细胞外刺激和导致细胞增殖和分化的下游事件之间的关键环节。已知导致不适当Ras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在活性gtp结合状态和非活性gdp结合状态之间循环，这是由Ras鸟嘌呤核苷酸交换因子（GEFs）和鸟嘌呤核苷酸激活蛋白（GAPs）控制的，Ras对这些蛋白不敏感，或GEFs或GAPs的不适当激活通常是这些疾病的基础。t细胞受体激活可通过RasGrp1和SOS两种gef的联合作用引起不同程度的Ras刺激，并根据细胞外信号的强度和持续时间导致细胞增殖或死亡。这种未成熟T细胞（胸腺细胞）的阳性或阴性选择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体外重建模型膜上的t细胞受体信号通路，以了解胸腺细胞阳性和阴性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。目前还不清楚为什么在这个信号级联中需要两个不同的rasgef。该提案将研究SOS和RasGrp1是由允许Ras协同激活的独特信号激活的假设。以下具体目标将检验这一假设。1)激活受体复合物募集SOS膜对催化活性的影响将通过膜锚定Ras的荧光核苷酸交换实验来阐明。2)通过测量只含有催化结构域或含有c端EF-hands、C1结构域和线圈基板的不同RasGrp1结构体的核苷酸交换活性来阐明RasGrp1的调控机制。此外，将研究辅助Ras结合位点的存在，以确定RasGrp1是否包含一个负责正反馈回路的变构结合口袋，如在SOS中发现的。最后，RasGrp1和SOS将在核苷酸交换实验中通过结合两种蛋白来测试它们协同激活Ras的能力。3)利用x射线晶体学研究RasGrp1催化和侧翼结构域效应的结构基础。这些RasGrp1和SOS的对比研究将为了解Ras在t细胞中的活化机制，以及两种生化活性相似的酶的活化如何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. PUBLIC HEALTH RELEVANCE: Ras is a key protein switch that is activated in signaling cascades important for cell growth, development and death. Activating Ras mutations are found in ~30% of cancers and improper Ras regulation can also result in developmental disorders including Noonan Syndrome. Because of the importance of Ras activity in human disease, it is important to understand the molecular mechanisms responsible for precisely tuning its functions at the membrane.