Differential regulation of Ras nucleotide exchange factors

Ras 核苷酸交换因子的差异调节

• 批准号: 8137067
• 负责人: Jeffrey Scott Iwig
• 金额: $ 4.84万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8137067
• 关键词: 1,2-diacylglycerol; Adaptor Signaling Protein; Affect; Allosteric Site; Apoptosis; Autoimmune Responses; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; Catalysis; Catalytic Domain; Cell Death; Cell Differentiation process; Cell Proliferation; Cells; Cessation of life; Comparative Study; Complex; DAG/PE-Binding Domain; Defect; Development; Diglycerides; Disease; EF Hand Motifs; Elements; Enzymes; Epidermal Growth Factor Receptor; Equilibrium; Event; Feedback; Fluorescence; Fluorescence Anisotropy; GTP Binding; GTPase-Activating Proteins; Goals; Growth and Development function; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Triphosphate; Human; Immune; In Vitro; Lead; Learning; Length; Link; Lipids; Malignant Neoplasms; Measures; Membrane; Membrane Proteins; Molecular; Mus; Mutagenesis; Mutation; Noise; Noonan Syndrome; Nucleotides; Pathway interactions; Phosphotyrosine; Prevention; Process; Proteins; Receptor Activation; Regulation; Role; SOS Response; Signal Transduction; Site; Solid Neoplasm; Son of Sevenless Proteins; Source; Stimulus; Structural Biochemistry; Structure; T Cell Receptor Signaling Pathway; T cell differentiation; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Tail; Testing; Thymocyte Selection; Training; Vesicle; X-Ray Crystallography; adaptive immunity; analog; base; cell growth; computer studies; developmental disease; digital; extracellular; human disease; in vivo; insight; leukemia; membrane model; protein function; ras Guanine Nucleotide Exchange Factors; ras Proteins; receptor; reconstitution; research study; response; structural biology; thymocyte

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.

描述（由申请人提供）：Ras的激活是细胞外刺激与导致细胞增殖和分化的下游事件之间的关键环节。已知导致不适当的Ras激活的突变会导致白血病和实体瘤，以及发育障碍，如努南综合征。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（GEF）和鸟嘌呤核苷酸激活蛋白（GAPS）控制的，Ras对这些蛋白的不敏感性，或GEF或GAPS的不适当激活通常是这些疾病的基础。T细胞受体激活可以通过两种GEF RasGrp 1和SOS的联合作用引起不同程度的Ras刺激，这取决于细胞外信号的强度和持续时间，导致细胞增殖或死亡。未成熟T细胞（胸腺细胞）的阳性或阴性选择过程是获得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模型膜上重建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。对于Ras激活发生，RasGrp 1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两种不同的RasGEF在这种信号级联中是必要的。该提案将调查SOS和RasGrp 1被允许合作Ras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)SOS膜招聘激活受体复合物对催化活性的影响将阐明使用荧光核苷酸交换测定与膜锚定的Ras。2)RasGrp 1的调控机制将通过测量不同的RasGrp 1结构的核苷酸交换活性来阐明，所述RasGrp 1结构仅包含催化结构域或具有C-末端EF-手、C1结构域和卷曲螺旋基序的该结构域。此外，将研究辅助Ras结合位点的存在，以确定RasGrp 1是否含有负责正反馈环（如SOS中发现的）的变构结合口袋。最后，RasGrp 1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)RasGrp 1催化和侧翼域效应的结构基础将使用X射线晶体学进行探讨。RasGrp 1和SOS之间的这些比较研究将为Ras激活如何在T细胞中发生以及具有相似生化活性的两种酶的激活如何差异诱导不同的细胞命运提供结构和生化基础。