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.

项目摘要/摘要 EPHB4-P120RASGAP信号轴对EPHB4和EPHB4和突变至关重要 P120RASGAP与包括Galen畸形（VOGM）和 毛细血管畸形 - 解剖畸形（CM-AVM）综合征。沿该路径的一种关键蛋白质， P120RASGAP（RASGAP）尤其在研究下。尽管RasGap是第一个激活的GTPase 蛋白质（GAP）发现仍不清楚如何调节RasGap。 RasGap包含两个SH2域， 允许它在含磷酪氨酸的蛋白（例如受体酪氨酸激酶）下游发出信号 EPHB4和RHOGAP P190RHOGAP（P190）。 Rasgap的多个酪氨酸磷酸化结合伴侣 引起不同的信号传导结果，以前的研究表明这些结合伴侣可能会影响RasGap 活动。但是，未知的分子基础是未知的。揭示这些绑定如何 合作伙伴规范拉斯加普的活动为了解与VOGM和VOGM相关的突变如何铺平了道路 CM-AVM可能会破坏这一法规。在此提案中，我将进行结构，生物物理和酶促 测试假设含磷酸酪氨酸的结合伙伴的研究调节RasGap的信号传导 活动。目标1：确定非催化域，结合伴侣相互作用和突变的影响 关于间隙调节。由于细胞提取物和免疫沉淀蛋白的研究，磷酸化结合伴侣 假设会影响RasGap的间隙活动。但是，没有使用 纯化的蛋白质以评估这一假设。在AIM 1中，我建议在纯化的蛋白质荧光间隙测定中提出 确定RasGap的非催化域，结合伴侣和疾病突变如何影响差距 活动。目标2：确定不同结合伴侣相互作用的RasGAP的构象变化。 尽管EPHB4-RASGAP相互作用很重要，但这些蛋白质如何相互作用仍然未知。一个 在Rasgap的N末端域的Boggon实验室中确定的结构，并具有双倍磷酸化的P190 肽表明，EPHB4的磷酸酪氨酸不可能以相同的方式参与RasGAP。 因此，在AIM 2中，我将确定EPHB4如何与Rasgap接合并确定对 rasgap。我提出X射线晶体学，小角度X射线散射和等温滴定量热法 探测P190和EPHB4结合在RasGAP中诱导的构象变化。然后我将评估影响 在这些生物物理数据的背景下与疾病相关的突变，并探究疾病的影响 会议，结构和活动的相关突变。