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

基本信息

批准号：
10220078
负责人：
JEREMY BASKIN
金额：
$ 30.83万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
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/10220078
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

项目摘要

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.

项目概要/摘要磷酸肌醇 (PIP) 和泛素化是调节空间和信号转导的主要系统。虽然它们的主要监管机制已得到很好的描述，但二级监管机制，特别是那些调节串扰的方法，受到的关注较少。我们发现了 PIP 之间的一种新联系。我们发现了由 PLEKHA4 介导的泛素化，PLEKHA4 是一种含有 pleckstrin 同源 (PH) 结构域的蛋白质。这种多结构域蛋白质通过质膜的 PI(4,5)P2 丰富区域形成大型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 CUL3KLHL12 招募到此类令人惊讶的是，CUL3KLHL12 的这种重新定位伴随着 E3 连接酶活性的降低。朝向主要底物 Dishevelled-3 (DVL3)，导致 DVL3 积累并增加 Wnt 信号传导，其中 DVL3 是关键中间体，目前尚不清楚 PLEKHA4 如何机械地调节 CUL3KLHL12。在本提案中，我们将在分子和功能水平上测试一种新颖的隔离模型。解释和理解这些结果是我们的长期研究目标是了解 PIP 传感蛋白是如何工作的。连接膜脂质成分以调节不同生理环境中的信号蛋白。该提案旨在了解 PLEKHA4 及其旁系同源物如何影响 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寡聚体。 PLEKHA4/5/6/7 簇在非活性状态下介导 CUL3KLHL12 在质膜上的隔离并且，通过阻止 DVL3 泛素化，充当 Wnt 信号传导的正调节因子。旨在实现我们的目标：（1）阐明 PLEKHA4 调节的分子机制我们将通过 CUL3KLHL12 介导的 DVL3 泛素化来测试 PLEKHA4 功能的隔离模型。使用缺乏不同分子的 PLEKHA4 构建体来拯救 RNAi 诱导的表型我们还将探讨变化的贡献。 PI(4,5)P2 代谢至 PLEKHA4 功能 (2) 确定 PLEKHA4 的机制基础。在果蝇模型中对 Wnt 信号传导的生理影响我们发现单个基因敲除。祖先果蝇 PLEKHA4/5/6/7 同源物在 Wnt/Wingless 信号传导中表现出缺陷，我们将在体内进行。 (3) 阐明 PLEKHA4/5/6/7 内分子和细胞水平的功能专业化和保守我们将测试 PLEKHA4/5/6/7 蛋白可以形成多功能 PLEKHA 的假设。通过异源寡聚化和不同程度的膜和蛋白质亲和力形成复合物。了解 PLEKHA4/5/6/7 蛋白如何读取膜上动态变化的 PIP 含量双层并转导调节 E3 连接酶活性的信息将定义一个新的机制框架健康和疾病中重要信号通路（例如 Wnt 信号传导）的调节。