Phosphoinositide-Binding Proteins as Regulators of Ubiquitination and Wnt Signaling

项目总结/摘要 磷酸肌醇（PIP）和泛素化是调节空间信号转导的主要系统， 时间虽然它们的主要调节机制已得到很好的表征，但次级调节层， 特别是那些调节串扰的方法受到的关注较少。我们已经发现PIP之间存在一种新的联系， 和由PLEKHA 4介导的泛素化，PLEKHA 4是一种含有普列克底物蛋白同源（PH）结构域的蛋白。我们发现 这种多结构域蛋白在质膜的PI（4，5）P2富集区形成大的组装体， 脂质和蛋白质结合结构域的独特组合，并将E3泛素连接酶CUL 3 KLHL 12募集到这样的结构域中。 结构.令人惊讶的是，CUL 3 KLHL 12的这种重新定位伴随着E3连接酶活性的降低 向主要底物Dishevelled-3（DVL 3）的方向，导致DVL 3积累和Wnt信号传导增加， 其中DVL 3是关键中间体。PLEKHA 4如何在机制上调节CUL 3 KLHL 12仍然是未知的 活性，在分子和功能水平上。在本提案中，我们将测试一种新的封存模型， 解释并理解这些结果。我们的长期研究目标是了解PIP传感蛋白如何 连接膜脂质组成，以调节不同生理环境中的信号蛋白。的目标 本研究旨在了解PLEKHA 4及其旁系同源物PLEKHA 5/6/7如何影响CUL 3 KLHL 12的机制 E3连接酶对DVL 3和Wnt信号传导的活性。指导这项工作的中心假设是， PLEKHA 4/5/6/7簇介导CUL 3 KLHL 12在质膜上以非活性状态的隔离 并且通过阻止DVL 3泛素化，作为Wnt信号传导的正调节剂。我们提议下列 目的是实现我们的目标：（1）阐明PLEKHA 4调控的分子机制， CUL 3 KLHL 12介导的DVL 3泛素化。我们将通过以下方式测试PLEKHA 4功能的螯合模型： 用不同分子缺陷的PLEKHA 4构建体进行RNAi诱导的表型的拯救 功能，包括膜结合和寡聚化。我们还将探讨变化的贡献， PI（4，5）P2代谢对PLEKHA 4功能的影响。(2)确定PLEKHA 4的机制基础 在果蝇模型中对Wnt信号传导的生理作用。我们发现一首淘汰赛的单曲 祖先果蝇PLEKHA 4/5/6/7同系物在Wnt/Wingless信号传导中表现出缺陷。我们将在体内进行 结构-功能研究来剖析促成这些表型的机制。(3)阐明 在PLEKHA 4/5/6/7中，在分子和细胞水平上的功能的专门化和保守性 家人我们将检验PLEKHA 4/5/6/7蛋白可以形成多功能PLEKHA的假设 复合物通过异源寡聚化和不同程度的膜和蛋白质亲和力。总之， 了解PLEKHA 4/5/6/7蛋白如何读取膜中动态变化的PIP含量 双分子层和双分子层的研究表明，调控E3连接酶活性的信息将定义一个新的机制框架， 重要信号传导途径的调节（例如，Wnt信号）在健康和疾病中的作用。

Project Summary/Abstract 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.