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

基本信息

批准号：
10535097
负责人：
Kimberly Jean Vish
金额：
$ 4.68万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10535097
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 Crystallization Data Development Disease Family Fluorescence GRLF1 gene GTPase-Activating Proteins Galen Vein Guanosine Triphosphate In Vitro Investigation Knowledge Lead Length Maps Measures Molecular Molecular Conformation Mutation N-terminal Newborn Infant Outcome Pathogenicity Pathway interactions Patients Peptides Phosphopeptides Phosphotyrosine Proteins RAS genes Receptor Protein-Tyrosine Kinases Regulation Roentgen Rays Signal Transduction Structure Syndrome Tertiary Protein Structure Testing Titrations Tyrosine Vascular Diseases Work X-Ray Crystallography base disease phenotype malformation mutant p120 GTPase Activating Protein recruit rho rho GTPase-activating protein src Homology Region 2 Domain

项目摘要

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.

项目概要/摘要 EphB4-p120RasGAP 信号轴对于 EphB4 和 EphB4 的血管发育和突变至关重要。 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。这一途径的一个关键蛋白，尽管 RasGAP 是第一个 GTP 酶激活剂，但 p120RasGAP (RasGAP) 仍处于研究之中。 RasGAP 蛋白 (GAP) 的发现目前尚不清楚 RasGAP 是如何调控的，RasGAP 包含两个 SH2 结构域。允许其向含磷酸酪氨酸的蛋白质下游发出信号，例如受体酪氨酸激酶 EphB4 和 RhoGAP p190RhoGAP (p190) RasGAP 的多个酪氨酸磷酸化结合伴侣。引发不同的信号传导结果，之前的研究表明这些结合伙伴可能会影响 RasGAP 然而，这种现象如何发生的分子基础尚不清楚。合作伙伴调节 RasGAP 的活性为了解突变如何与 VOGM 和相关性相关铺平了道路 CM-AVM 可能会破坏这种调节，在这个提案中我将进行结构、生物物理和酶学研究。研究检验含磷酸酪氨酸的结合配偶体调节 RasGAP 信号传导的假设目标 1：确定非催化结构域、结合伙伴相互作用和突变的影响。由于对细胞提取物和免疫沉淀蛋白、磷酸化结合伴侣的研究。被突袭以影响 RasGAP 的 GAP 活性，但尚未进行任何研究。纯化蛋白质来评估这一假设在目标 1 中，我建议通过纯化蛋白质荧光 GAP 测定来评估这一假设。确定 RasGAP 的非催化结构域、结合伙伴和疾病突变如何影响 GAP 目标 2：确定 RasGAP 在不同结合伴侣相互作用时的构象变化。尽管 EphB4-RasGAP 相互作用很重要，但这些蛋白质如何相互作用仍不清楚。 Boggon 实验室确定的 RasGAP N 端结构域的双磷酸化 p190 结构肽表明 EphB4 的磷酸酪氨酸残基不可能以同样的方式与 RasGAP 结合。因此，在目标 2 中，我将确定 EphB4 如何参与 RasGAP 并确定其构象效应我建议使用 X 射线晶体学、小角 X 射线散射和等温滴定量热法来探究 p190 和 EphB4 结合在 RasGAP 中诱导的构象变化，然后我将评估其影响。在这些生物物理数据的背景下分析与疾病相关的突变，并探讨疾病的影响- 构象、结构和活性的相关突变。