Phosphoinositide-Binding Proteins as Regulators of Ubiquitination and Wnt Signaling

磷酸肌醇结合蛋白作为泛素化和 Wnt 信号转导的调节剂

• 批准号: 10458495
• 负责人: JEREMY BASKIN
• 金额: $ 30.77万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458495
• 关键词: 1-Phosphatidylinositol 4-Kinase; Adult; Affect; Affinity; Attention; Binding; Binding Proteins; Biological Assay; Cell membrane; Cells; Complex; Cytosol; Defect; Disease; Drosophila genus; Drosophila melanogaster; Enzymes; Eukaryota; Event; Exhibits; Family; Goals; Hair; Health; Homologous Gene; Human; In Vitro; Knock-out; Link; Lipid Bilayers; Lipid Binding; Lipids; Liposomes; Mammalian Cell; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Metabolism; Microscopic; Modeling; Molecular; Orthologous Gene; PH Domain; Pathway interactions; Pattern; Phenotype; Phosphatidylinositols; Phospholipase C; Phosphotransferases; Physiological; Play; Process; Property; Protein Binding Domain; Protein Family; Proteins; RNA Interference; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Sum; System; Tertiary Protein Structure; Testing; Time; Ubiquitination; Up-Regulation; WNT Signaling Pathway; Wing; Work; base; fly; human disease; in vivo; inositol-1,4,5-trisphosphate 5-phosphatase; link protein; novel; paralogous gene; prevent; recruit; trafficking; ubiquitin-protein ligase

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.

项目摘要/摘要 磷酸肌醇（PIPS）和泛素化是调节空间中信号转导的主要系统 时间。虽然它们的主要调节机制的特征是调节的次要层，但 尤其是那些注册串扰的人受到较少的关注。我们已经确定了PIP之间的新颖链接 和泛素化蛋白Plekha4介导的泛素化。我们发现了 该多域蛋白在PI（4,5）的P2富含P2 脂质和蛋白质结合结构域的独特组合，并募集E3泛素连接酶Cul3KlHl12到此类 结构。令人惊讶的是，Cul3KlHl12的这种重新定位是通过降低E3连接酶活性来实现的 向主要的底物，DISEVELLED-3（DVL3），导致DVL3积累并增加Wnt信号传导， 其中DVL3是一个关键中间体。在机械上，plekha4如何调节cul3klhl12仍然未知 活性，在分子和功能水平上。在此提案中，我们将测试一种新型的固存模型 解释并理解这些结果。我们的长期研究目标是了解如何感应蛋白 将膜脂质组成链接到在潜水员物理环境中调节信号蛋白。目的 该建议是了解plekha4及其旁系同源物plekha5/6/7如何影响cul3klhl12的机制 E3连接酶对DVL3和Wnt信号传导的活性。指导这项工作的中心假设是低聚 Plekha4/5/6/7的簇在质膜处的CUL3KLHL12的介导疗法在不活跃状态下 并且，通过防止DVL3泛素化，充当Wnt信号的正调节剂。我们提出以下内容 目的是实现我们的目标：（1）阐明有关Plekha4调节的分子机制 CUL3KLHL12介导的DVL3泛素化。我们将通过 用plekha4构建在不同的分子中挽救RNAi诱导的表型 功能，包括膜结合和低聚。我们还将探讨变更的贡献 PI（4,5）P2代谢为Plekha4功能。 （2）确定plekha4的机械基础 果蝇模型中WNT信号传导的生理影响。我们发现单曲的淘汰赛 祖先蝇plekha4/5/6/7同源物在无翼/无翼信号中表现出缺陷。我们将在体内表演 结构 - 功能研究，以剖析导致这些表型的机制。 （3）阐明 Plekha4/5/5/6/7内的分子和细胞水平的功能的专业化和守恒 家庭。我们将检验以下假设：plekha4/5/6/7蛋白可以形成多个功能性plekha 通过杂极化和膜和蛋白质亲和力的差异度通过差异。总而 了解plekha4/5/6/7蛋白如何读取膜的动态变化的PIP含量 双层并将这些信息转换为调节E3连接酶活动将定义一个新的机械框架 调节健康和疾病中重要的信号通路（例如WNT信号传导）。