The function of MEKK3 interaction with CCM2

 描述(申请人提供)：脑海绵状畸形(CCM)是一种脑血管病变，估计影响高达0.5%的人口。CCM患者可能患有慢性头痛、癫痫、癫痫、中风和局灶性神经功能障碍。目前已鉴定出3个疾病相关基因，分别编码CCM1/KRIT1、CCM2/Malcavernin/OSM和CCM3/PDCD10。已知这些CCM蛋白形成一个多蛋白复合体(CCM复合体信号平台)，其中任何一个蛋白的功能丧失都会导致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基因突变患者的CCM究竟是如何发生的仍然很大程度上尚不清楚。CCM复合体的已知结合伙伴之一是丝裂原激活的蛋白激酶激酶，MEKK3。重要的是，MEKK3在血管系统中的作用似乎与CCM蛋白，特别是CCM2的作用重叠。因此，本项目的目标是了解MEKK3：CCM2相互作用的结构要求和功能结果，以及可能影响与CCM疾病相关的关键血管表型的下游信号的相关变化。为此，我们的研究旨在全面解决我们的核心假设，即CCM2招募MEKK3对血管完整性至关重要。我们将从三个方面阐述这一假说。在目标1中，我们将确定MEKK3招募到CCM2的结构和功能机制。这一目标将使用结构、生化和生物物理工具来提供基本的分子水平框架，说明MEKK3如何通过与CCM2的相互作用被招募到CCM复合体中。在目标2中，我们将通过生化和基于细胞的分析来探讨CCM2对MEKK3信号的调控作用。我们还将研究MEKK3：CCM2复合体的特定磷酸化靶点和该复合体的靶基因。在目标3中，我们将发现MEKK3募集到CCM2的体内功能作用。我们利用结构导向的体内研究，在不损害MEKK3的整体活性的情况下特异性地干扰MEKK3：CCM2的相互作用，以测试与CCM疾病相关的关键血管表型以及下游信号的相关变化是否源于MEKK3：CCM2相互作用的丧失。总体而言，我们预计我们提出的研究将确定MEKK3与CCM2相互作用的功能重要性，并确定与CCM疾病相关的血管病理是否源于MEKK3：CCM2相互作用的丧失和下游信号的相关变化。

 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.