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：
10589550
负责人：
JEREMY BASKIN
金额：
$ 1.07万
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

项目摘要

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.

项目摘要父母赠款R01 GM131101 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。从机械上讲，Plekha4如何调制它仍然未知 CUL3KLHL12活性在分子和功能水平上。在此提案中，我们将测试一本小说隔离模型解释和理解这些结果。我们的长期研究目标是了解如何管道感应蛋白将膜脂质组成链接以调节潜水员生理的信号蛋白上下文。该提案的目的是了解Plekha4及其旁系同源物的机制 Plekha5/6/7影响Cul3KlHl12 E3连接酶的活性对DVL3和WNT信号传导。中心假设指导这项工作的是等离子体的Plekha4/5/5/6/7的低聚群中cul3klhl12的中间群膜处于不活跃状态，通过防止DVL3泛素化，是Wnt的积极调节剂信号。我们提出以下目标以实现我们的目标：（1）阐明分子机制 Cul3KlHl12介导的DVL3泛素化的plekha4调节。我们将测试会话 Plekha4功能的模型通过使用Plekha4构建体进行RNAi诱导的表型来拯救不同分子功能的碎片环境，包括膜结合和寡聚。我们还将探索 PI（4,5）P2代谢变化对Plekha4功能的贡献。（2）确定机械基础对于果蝇模型中Plekha4对Wnt信号传导的物理影响。我们发现一个淘汰赛在单一祖先飞行中，Plekha4/5/6/7同源物在无翼/无翼信号中表现出缺陷。我们将表演体内结构功能研究旨在剖析促成这些表型的机制。（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 通过杂音寡聚和膜和蛋白质亲和力的差异程度进行复合物。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）。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Getting a Grip on Greasy Molecules

控制油腻分子

DOI：
10.1016/j.tibs.2019.03.006
发表时间：
2019
期刊：
Trends in Biochemical Sciences
影响因子：
13.8
作者：
Bumpus, Timothy W.;Baskin, Jeremy M.
通讯作者：
Baskin, Jeremy M.

For Wnt Signaling, Fucosylation of LRP6 Is a Bitter Pill

对于 Wnt 信号传导，LRP6 的岩藻糖基化是一剂苦药丸

DOI：
10.1016/j.chembiol.2020.08.003
发表时间：
2020
期刊：
Cell Chemical Biology
影响因子：
8.6
作者：
Shami Shah, Adnan;Sun, Hongyan;Baskin, Jeremy M.
通讯作者：
Baskin, Jeremy M.

Phosphoinositide phosphorylation sans kinase.

无激酶的磷酸肌醇磷酸化。

DOI：
10.1038/s41556-022-00885-0
发表时间：
2022
期刊：
Nature cell biology
影响因子：
21.3
作者：
Cao,Xiaofu;Baskin,JeremyM
通讯作者：
Baskin,JeremyM

The chemistry and biology of phosphatidylinositol 4-phosphate at the plasma membrane.

DOI：
10.1016/j.bmc.2021.116190
发表时间：
2021-06-15
期刊：
Bioorganic & medicinal chemistry
影响因子：
3.5
作者：
Batrouni AG;Baskin JM
通讯作者：
Baskin JM

ESCRT-III and ER-PM contacts maintain lipid homeostasis.

ESCRT-III 和 ER-PM 接触维持脂质稳态。

DOI：
10.1091/mbc.e20-01-0061
发表时间：
2020
期刊：
Molecular biology of the cell
影响因子：
3.3
作者：
Jorgensen,JeffR;Tei,Reika;Baskin,JeremyM;Michel,AgnèsH;Kornmann,Benoît;Emr,ScottD
通讯作者：
Emr,ScottD