Inter-Domain Regulation of p120RasGAP

项目摘要 GAP（GT3活化蛋白）在GT3循环中起着重要作用，允许调节GT3的表达。 复杂的过程，如信号转导;因此，他们的功能障碍有显着的分歧。在 p120 RasGAP（RasGAP，p120; RASA 1）是第一个描述的GAP，功能障碍与 血管疾病，如毛细血管畸形-动静脉畸形综合征（CM-AVM）和静脉畸形， Galen畸形（VOGM）。该蛋白由N-末端Src同源2（SH 2）-SH 3-SH 2盒组成， 随后是普列克底物蛋白同源性（PH）、C2和GAP结构域。尽管它的重要性和相对悠久的历史 作为研究的目标，p120 RasGAP尚未在结构和生物化学水平上得到充分分析。 特别地，尽管全长p120 RasGAP已显示出比差距域具有更大的活性， PH和C2结构域的贡献还没有被特别分离出来。我假设 p120 RasGAP的GAP活性通过蛋白质的构象变化诱导的构象变化来控制。 调节结构域，其又受脂质头部基团和/或其他结合蛋白的影响。具体地说， 我的初步数据表明C2结构域可能在GAP调节中起作用。我会测试这些 两个目标的机制。在目标1中，我已经可以表达和纯化差距、C2和PH结构域的构建体 初步的酶分析表明，C2结构域加速了p120 RasGAP的酶促降解， 活动我将在体外进行详细的酶研究，使用GAP测定来评估这些结构域的作用。 然后，我将评估致病突变对酶活性的影响。此外，我将量化 PH和C2结构域对不同脂质头部基团的亲和力，并评估C2钙敏感性。这些 然后将数据用于使用囊泡结合的Ras进行体外GAP测定，以测试膜的作用。 PH和C2调节行为的关联。在目标2中，我已经获得并正在提炼第一颗水晶 p120 RasGAP的C2-GAP区域的结构。这种晶体结构表明，C2和GAP 结构域通过柔性接头连接，并说明C2结构域理想地定位成与 Ras的变构叶形成“对生拇指”结构。我也会得到C2- GAP区域与HRas复合以直接观察这种结合。基于对这些结构的分析， 通过定点诱变和GAP试验验证C2结构域的相互作用。采取 这些目标将为PH和C2领域在监管中的作用提供全面的分析 的p120 RasGAP和揭示的相互作用负责观察到的活性变化。这些研究将 阐明血管疾病包括CM-AVM和VOGM的分子基础。

PROJECT SUMMARY GAPs (GTPase activating proteins) play an essential role in the GTPase cycle, allowing for the regulation of complex processes such as signal transduction; consequently, their dysfunction has significant ramifications. In the case of p120RasGAP (RasGAP, p120; RASA1), the first GAP described, dysfunctions have been linked to vascular diseases such as capillary malformation-arteriovenous malformation syndromes (CM-AVM) and vein of Galen malformations (VOGM). The protein consists of an N-terminal Src homology 2 (SH2)-SH3-SH2 cassette, followed by pleckstrin homology (PH), C2, and GAP domains. Despite its importance and relatively long history as a target of study, p120RasGAP has not been adequately analyzed at the structural and biochemical levels. In particular, although full-length p120RasGAP has been shown to have greater activity than the GAP domain alone, the contributions of the PH and C2 domains have not specifically been isolated. I hypothesize that the GAP activity of p120RasGAP is controlled through conformational changes induced by the protein’s regulatory domains, which are in turn affected by lipid head groups and/or other binding proteins. Specifically, my preliminary data demonstrate that the C2 domain may play a role in GAP regulation. I will test these mechanisms in two aims. In Aim 1, I can already express and purify constructs of the GAP, C2, and PH domains of p120RasGAP, and preliminary enzymatic analysis suggests that the C2 domain accelerates enzymatic activity. I will conduct detailed enzymatic studies in vitro using GAP assays to assess the roles of these domains. I will then assess the effect of disease-causing mutations on enzyme activity. Additionally, I will quantify the affinities of the PH and C2 domains for different lipid head groups and assess C2 calcium sensitivity. These data will then be used to conduct in vitro GAP assays using vesicle-bound Ras to test the role of membrane association in PH and C2 regulatory behavior. In Aim 2, I have already obtained and am refining the first crystal structure of the C2-GAP region of p120RasGAP. This crystal structure demonstrates that the C2 and GAP domains are connected by a flexible linker and illustrates that the C2 domain is ideally positioned to interact with the allosteric lobes of Ras in an “opposable thumb” formation. I will also obtain a crystal structure of the C2- GAP region in complex with HRas to observe this binding directly. Based on analysis of these structures, I will validate interactions made by the C2 domain via site-directed point mutagenesis and GAP assays. Taken together, these aims will provide a comprehensive analysis of the roles of the PH and C2 domains in regulation of p120RasGAP and reveal interactions responsible for the observed changes in activity. These studies will elucidate molecular bases for vascular diseases including CM-AVM and VOGM.