Structural Investigation of p120RasGAP's regulation by phosphorylated binding partners

项目摘要/摘要 EphB4-p120RasGAP信号轴对血管发育和EphB4和EphB4基因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。这条途径上的一个关键蛋白质， P120RasGAP(RasGAP)，目前正在研究中。尽管RasGAP是第一个激活的GTP酶 蛋白质(GAP)发现目前仍不清楚RasGAP是如何受到调控的。RasGAP包含两个SH2结构域， 允许它向含有磷酸酪氨酸的蛋白下游发出信号，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 引发不同的信号结果，以前的研究表明，这些结合伙伴可能会影响RasGAP 活动。然而，这种情况发生的分子基础尚不清楚。揭示这些绑定是如何 合作伙伴规范RasGAP的活动为了解与VOGM和VOGM相关的突变铺平了道路 CM-AVM可能会破坏这一调节。在这个提案中，我将进行结构、生物物理和酶 含磷酸酪氨酸结合伙伴调控RasGAP信号转导的研究 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 关于缺口监管。由于对细胞提取液和免疫沉淀蛋白的研究，磷酸化结合伙伴 假设会影响RasGAP的GAP活动。然而，目前还没有使用 纯化的蛋白质来评估这一假说。在目标1中，我建议在纯化的蛋白质荧光间隙分析中 确定RasGAP的非催化结构域、结合伙伴和疾病突变如何影响GAP 活动。目的2：确定RasGAP在不同结合伙伴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。一个 RasGAP N-末端结构域的Boggon实验室确定的双磷酸化p190结构 多肽表明，EphB4‘S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如何与RasGAP结合，并确定其构象效应 RasGAP。我建议用X射线结晶学、小角X射线散射和等温滴定量热法来 探讨p190和EphB4结合对RasGAP细胞构象的影响。然后我会评估影响 在这些生物物理数据的背景下研究与疾病相关的突变，并探索疾病- 构象、结构和活性上的相关突变。

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.