权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：
9642167
负责人：
JEREMY BASKIN
金额：
$ 31万
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/9642167
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.

项目总结/文摘