Phosphoinositide-Binding Proteins as Regulators of Ubiquitination and Wnt Signaling

项目总结 家长资助金R01 GM131101 磷脂酰肌醇(PIP)和泛素化是调节太空和太空中信号转导的主要系统。 时间到了。虽然它们的主要监管机制有很好的特点，但第二层监管， 尤其是那些监管串音的，受到的关注较少。我们已经确定了PIP之间的一种新的联系 以及由PLEKHA4介导的泛素化，PLEKHA4是一种含有Pleckstrin同源(PH)结构域的蛋白。我们发现 这种多结构域蛋白在质膜富含PI(4，5)P2的区域通过一种 脂蛋白结合域的独特组合，并招募E3泛素连接酶CUL3KLHL12 结构。令人惊讶的是，CUL3KLHL12的这种重新定位伴随着E3连接酶活性的下降 朝向主要底物杂乱-3(DVL3)，导致DVL3积累和Wnt信号增加， 其中DVL3是关键的中间体。目前尚不清楚PLEKHA4是如何在机制上调节 CUL3KLHL12活性，分子水平和功能水平。在这个提案中，我们将测试一部小说 隔离模型来解释和理解这些结果。我们的长期研究目标是了解 PIP敏感蛋白连接膜脂组成，调节不同生理状态下的信号蛋白 上下文。这项建议的目的是了解PLEKHA4及其类似物如何 PLEKHA5/6/7影响CUL3KLHL12 E3连接酶对DVL3和Wnt信号的活性。中心假说 指导这项工作的是PLEKHA4/5/6/7的寡聚体簇介导CUL3KLHL12在血浆中的隔离 膜处于非活性状态，并通过阻止DVL3泛素化，发挥Wnt的正调节作用 发信号。我们提出以下目标来实现我们的目标：(1)阐明分子机制 控制PLEKHA4调节CUL3KLHL12介导的DVL3泛素化。我们将测试自动减支 用PLEKHA4构建拯救RNAi诱导的表型的PLEKHA4功能模型 缺乏不同的分子功能，包括膜结合和齐聚。我们还将探索 PI(4，5)P2代谢变化对PLEKHA4功能的影响(2)确定机制基础 关于PLEKHA4‘S在果蝇模型中对Wnt信号的生理影响。我们发现了一种击倒 PLEKHA4/5/6/7同源基因在Wnt/Wingless信号通路中存在缺陷。我们将表演 体内结构功能研究，以剖析导致这些表型的机制。(3)澄清 PLEKHA4/5/6/7在分子和细胞水平上的功能特化和保守 一家人。我们将检验PLEKHA4/5/6/7蛋白可以形成多功能PLEKHA的假设 通过异位齐聚和不同程度的膜和蛋白质亲和力形成的复合体。总而言之， 了解PLEKHA4/5/6/7蛋白如何读取膜上动态变化的PIP含量 双分子层和转导该信息来调节E3连接酶活性将定义一个新的机制框架 在健康和疾病中调节重要的信号通路(例如，Wnt信号)。

PROJECT SUMMARY Parent Grant R01 GM131101 Phosphoinositides (PIPs) and ubiquitination are major systems that modulate signal transduction in space and time. While their primary regulatory mechanisms are well characterized, secondary layers of regulation, particularly those regulating crosstalk, have received less attention. We have identified a novel link between PIPs and ubiquitination mediated by PLEKHA4, a pleckstrin homology (PH) domain-containing protein. We discovered that this multi-domain protein forms large assemblies at PI(4,5)P2-rich regions of the plasma membrane, via a unique combination of lipid- and protein-binding domains, and recruits the E3 ubiquitin ligase CUL3KLHL12 to such structures. Surprisingly, this relocalization of CUL3KLHL12 is accompanied by a decrease in E3 ligase activity toward a major substrate, Dishevelled-3 (DVL3), leading to DVL3 accumulation and increases in Wnt signaling, in which DVL3 is a key intermediate. It remains unknown how, mechanistically, PLEKHA4 modulates CUL3KLHL12 activity, at both the molecular and functional levels. In this proposal we will test a novel sequestration model to explain and understand these results. Our long-term research goal is to understand how PIP-sensing proteins link membrane lipid composition to regulate signaling proteins in diverse physiological contexts. The objective of this proposal is to understand mechanisms of how PLEKHA4 and its paralogs PLEKHA5/6/7 affect CUL3KLHL12 E3 ligase activity toward DVL3 and Wnt signaling. The central hypothesis guiding this work is that oligomeric clusters of PLEKHA4/5/6/7 mediate sequestration of CUL3KLHL12 at the plasma membrane in an inactive state and, via preventing DVL3 ubiquitination, act as positive regulators of Wnt signaling. We propose the following aims to achieve our goals: (1) Elucidate molecular mechanisms governing PLEKHA4 regulation of CUL3KLHL12-mediated DVL3 ubiquitination. We will test the sequestration model for PLEKHA4 function by performing rescue of RNAi-induced phenotypes with PLEKHA4 constructs deficient in different molecular functions, including membrane binding and oligomerization. We will also explore contributions of changes in PI(4,5)P2 metabolism to PLEKHA4 function. (2) Determine the mechanistic basis for PLEKHA4’s physiological effects on Wnt signaling in the Drosophila model. We found that a knockout of the single ancestral fly PLEKHA4/5/6/7 homolog exhibits defects in Wnt/Wingless signaling. We will perform in vivo structure function studies to dissect the mechanisms contributing to these phenotypes. (3) Elucidate specialization and conservation of function at the molecular and cellular levels within the PLEKHA4/5/6/7 family. We will test the hypothesis that the PLEKHA4/5/6/7 proteins can form multiple functional PLEKHA complexes via hetero oligomerization and differential degrees of membrane and protein affinities. In sum, understanding how the PLEKHA4/5/6/7 proteins can read the dynamically changing PIP content of membrane bilayers and transduce that information to regulate E3 ligase activity will define a new mechanistic framework for regulation of important signaling pathways (e.g., Wnt signaling) in health and disease.