Structural Investigation of p120RasGAP's regulation by phosphorylated binding partners

磷酸化结合伴侣对 p120RasGAP 调节的结构研究

• 批准号: 10843716
• 负责人: Kimberly Jean Vish
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10843716
• 关键词: Affect; Affinity; Area; Arteries; Arteriovenous malformation; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Blood; Blood Circulation; Blood Vessels; Blood capillaries; Brain; Calorimetry; Cell Extracts; Cells; Complex; Data; Development; Disease; Family; Fluorescence; GRLF1 gene; GTPase-Activating Proteins; Galen Vein; Guanosine Triphosphate; In Vitro; Investigation; Knowledge; Length; Maps; Measures; Molecular; Molecular Conformation; Mutation; N-terminal; Newborn Infant; Outcome; Pathogenicity; Pathway interactions; Patients; Peptides; Phosphopeptides; Phosphorylation; Phosphotyrosine; Proteins; RAS genes; Receptor Protein-Tyrosine Kinases; Regulation; Roentgen Rays; Signal Transduction; Structure; Syndrome; Tertiary Protein Structure; Testing; Titrations; Tyrosine Phosphorylation; Vascular Diseases; Work; X-Ray Crystallography; base; disease phenotype; malformation; mutant; p120 GTPase Activating Protein; protein purification; ras GTPase-Activating Proteins; recruit; rho; rho GTPase-activating protein; src Homology Region 2 Domain

Project Summary/Abstract The EphB4-p120RasGAP signaling axis is critical for vascular development and mutations in both EphB4 and p120RasGAP are associated with vascular disorders including Vein of Galen Malformation (VOGM) and Capillary Malformation-Arteriovenous Malformation (CM-AVM) Syndrome. One key protein along this pathway, p120RasGAP (RasGAP), is particularly under studied. Despite RasGAP being the first GTPase Activating Protein (GAP) discovered it is still unclear how RasGAP is regulated. RasGAP contains two SH2 domains, allowing it to signal downstream of phosphotyrosine-containing proteins, such as the receptor tyrosine kinase EphB4 and the RhoGAP p190RhoGAP (p190). Multiple tyrosine phosphorylated binding partners of RasGAP elicit different signaling outcomes, and previous studies suggest these binding partners may influence RasGAP activity. However, the molecular basis for how this occurs is unknown. Uncovering how these binding partners regulate RasGAP’s activity paves the way to understand how mutations associated with VOGM and CM-AVM might disrupt this regulation. In this proposal I will conduct structural, biophysical, and enzymatic studies to test the hypothesis phosphotyrosine-containing binding partners regulate RasGAP’s signaling activity. Aim 1: To determine the effects of non-catalytic domains, binding partner interactions, and mutations on GAP regulation. Due to studies in cell extract and immunoprecipated protein, phosphorylated binding partners are hypothesized to influence RasGAP’s GAP activity. However, there have been no studies performed using purified proteins to assess this hypothesis. In Aim 1 I propose in purified protein fluorescent GAP assays to determine how RasGAP’s non-catalytic domains, binding partners, and disease mutations influence GAP activity. Aim 2: To determine conformational changes in RasGAP upon different binding partner interactions. Despite the importance of the EphB4-RasGAP interaction it is still unknown how these proteins interact. A structure determined in the Boggon Lab of RasGAP’s N-terminal domains with a doubly phosphorylated p190 peptide show it is impossible for EphB4’s phosphotyrosine residues to engage RasGAP the same way. Therefore, in Aim 2 I will determine how EphB4 engages RasGAP and determine the conformational effects on RasGAP. I propose X-ray crystallography, Small Angle X-ray Scattering, and Isothermal Titration Calorimetry to probe the conformational changes induced in RasGAP by p190 and EphB4 binding. I will then assess the impact of disease-associated mutations in the context of these biophysical data and probe the impact of disease- associated mutations on conformation, structure, and activity.

项目总结/摘要 EphB4-p120RasGAP信号传导轴对于血管发育和EphB4和p120RasGAP中的突变都是至关重要的。 p120RasGAP与血管疾病相关，包括Galen静脉畸形（VOGM）和 毛细血管畸形-动静脉畸形（CM-AVM）综合征。沿着这条通路的一个关键蛋白， p120RasGAP（RasGAP）是一种特殊的基因。尽管RasGAP是第一个激活GTcycle的 目前还不清楚RasGAP是如何被调控的。RasGAP含有两个SH2结构域， 使其向含磷酸酪氨酸的蛋白质如受体酪氨酸激酶的下游发出信号 EphB4和RhoGAP p190 RhoGAP（p190）。RasGAP的多个酪氨酸磷酸化结合配偶体 引发不同的信号传导结果，先前的研究表明这些结合伴侣可能会影响RasGAP 活动然而，这种情况如何发生的分子基础是未知的。揭示了这些绑定 合作伙伴调节RasGAP的活动铺平了道路，以了解如何与VOGM相关的突变， CM-AVM可能会破坏这种调节。在这个建议中，我将进行结构，生物物理，和酶 一项旨在验证含有磷酸酪氨酸的结合伴侣调节RasGAP信号传导的假设的研究 活动目的1：确定非催化结构域、结合伴侣相互作用和突变的影响 关于GAP监管由于对细胞提取物和免疫沉淀蛋白的研究，磷酸化结合伴侣 被假设为影响RasGAP的GAP活性。然而，还没有研究使用 纯化的蛋白质来评估这一假设。在目的1中，我提出在纯化蛋白荧光GAP测定中， 确定RasGAP的非催化结构域、结合伴侣和疾病突变如何影响GAP 活动目的2：确定RasGAP在不同结合配偶体相互作用后的构象变化。 尽管EphB4-RasGAP相互作用的重要性，但这些蛋白质如何相互作用仍然是未知的。一 在Boggon实验室中确定的RasGAP的N-末端结构域具有双重磷酸化的p190 肽显示EphB4的磷酸酪氨酸残基不可能以相同的方式接合RasGAP。 因此，在目标2中，我将确定EphB4如何接合RasGAP并确定对RasGAP的构象影响。 RasGAP。我建议用X射线晶体学、小角X射线散射和等温滴定量热法来 探测由p190和EphB4结合在RasGAP中诱导的构象变化。然后我会评估 在这些生物物理数据的背景下，研究疾病相关突变，并探索疾病的影响- 在构象、结构和活性上的相关突变。