The function of MEKK3 interaction with CCM2

 描述（由申请方提供）：脑海绵状血管畸形（CCM）是一种脑血管病变，估计影响高达0.5%的人群。CCM患者可能患有慢性头痛、癫痫、癫痫发作、中风和局灶性神经功能缺损。已鉴定出三种疾病相关基因，分别编码CCM 1/KRIT 1、CCM 2/malcavernin/OSM和CCM 3/PDCD 10。已知这些CCM蛋白形成多蛋白复合物（CCM复合物信号传导平台），并且这些蛋白中的任何一种的功能丧失导致CCM病理。尽管我们在小鼠模型中对CCM蛋白的遗传学和分子功能的理解取得了重大进展，但CCM基因突变患者中CCM是如何发展的仍然很不清楚。CCM复合物的已知结合配偶体之一是促分裂原活化蛋白激酶MEKK 3。重要的是，MEKK 3在血管系统中的作用似乎与CCM蛋白，特别是CCM 2的作用重叠。因此，本项目的目标是了解MEKK 3：CCM 2相互作用的结构要求和功能后果以及下游信号传导的相关变化，这些变化可能影响与CCM疾病相关的关键血管表型。为此，我们的研究旨在全面解决我们的中心假设，即CCM 2募集MEKK 3对血管完整性至关重要。我们将在三个目标中讨论这个假设。在目标1中，我们将定义MEKK 3募集到CCM 2的结构和功能机制。该目标将使用结构、生物化学和生物物理工具来提供基本的分子水平框架，以了解MEKK 3如何通过与CCM 2的相互作用被募集到CCM复合物中。在目的2中，我们将通过使用生物化学和基于细胞的测定来探测CCM 2对MEKK 3的调节，从而研究CCM 2在MEKK 3信号传导调节中的作用。我们还将研究MEKK 3：CCM 2复合物的特异性磷酸化靶点，并研究复合物的靶基因。在目标3中，我们将发现MEKK 3募集到CCM 2的体内功能作用。我们利用结构导向的体内研究，其中我们特异性地破坏MEKK 3：CCM 2相互作用而不损害MEKK 3的总体活性，以测试与CCM疾病相关的关键脉管系统表型以及下游信号传导的相关变化是否由MEKK 3：CCM 2相互作用的丧失引起。总体而言，我们预计我们提出的研究将确定MEKK 3与CCM 2相互作用的功能重要性，并解决与CCM疾病相关的血管病理是否由MEKK 3：CCM 2相互作用的丧失和下游信号传导的相关变化引起。

 DESCRIPTION (provided by applicant): Cerebral Cavernous Malformations (CCMs) are brain vascular lesions estimated to affect up to 0.5% of the population. CCM patients can suffer chronic headaches, epilepsy, seizures, stroke and focal neurological deficits. Three disease-associated genes, which encode CCM1/KRIT1, CCM2/malcavernin/OSM, and CCM3/PDCD10, respectively, have been identified. These CCM proteins are known to form a multi-protein complex (the CCM complex signaling platform) and a loss of function of any one of these proteins leads to CCM pathology. Despite major progress in our understanding of the genetics and molecular functions of CCM proteins in mouse models, precisely how CCM is developed in patients with mutations in CCM genes remains largely unclear. One of the known binding partners of the CCM complex is a mitogen-activated protein kinase kinase kinase, MEKK3. Importantly, the role of MEKK3 in the vascular system appears to be overlapping with that of CCM proteins, especially that of CCM2. Therefore the goal of this project is to understand the structural requirement and functional consequences of MEKK3:CCM2 interaction and the associated changes in downstream signaling that may impact the critical vasculature phenotypes associated with CCM disease. Towards this end, our studies are designed to comprehensively address our central hypothesis that recruitment of MEKK3 by CCM2 is critical for vascular integrity. We will address this hypothesis in three Aims. In Aim 1 we will define the structural and functional mechanisms for MEKK3 recruitment to CCM2. This aim will use structural, biochemical and biophysical tools to provide the basic molecular level framework for how MEKK3 is recruited to the CCM complex by its interaction with CCM2. In Aim 2 we will investigate the role of CCM2 in regulation of MEKK3 signaling by probing MEKK3 regulation by CCM2 using biochemical and cell based assays. We will also investigate the specific phosphorylation targets of the MEKK3:CCM2 complex and investigate the target genes of the complex. In Aim 3 we will discover the in vivo functional role of MEKK3 recruitment to CCM2. We utilize structure-directed in vivo studies, in which we specifically disrupt the MEKK3:CCM2 interaction without compromising the overall activity of MEKK3, to test whether critical vasculature phenotypes associated with CCM disease, and associated changes in downstream signaling, result from loss of the MEKK3:CCM2 interaction. Overall, we expect that the studies we propose will define the functional importance of MEKK3 interaction with CCM2 and resolve whether the vascular pathology associated with CCM disease result from loss of the MEKK3:CCM2 interaction and associated changes in downstream signaling.