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.

项目概要家长补助金 R01 GM131101 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 如何进行机械调节。 CUL3KLHL12 在分子和功能水平上的活性在本提案中，我们将测试一种新的活性。我们的长期研究目标是了解如何利用封存模型来解释和理解这些结果。 PIP 传感蛋白将膜脂成分联系起来，调节多种生理学中的信号蛋白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 连接酶对 DVL3 和 Wnt 信号传导的活性。指导这项工作的是 PLEKHA4/5/6/7 的寡聚簇介导 CUL3KLHL12 在血浆中的隔离膜处于非活性状态，并通过阻止 DVL3 泛素化，充当 Wnt 的正调节因子我们提出以下目标来实现我们的目标：（1）阐明分子机制。我们将测试隔离作用。通过使用 PLEKHA4 构建体拯救 RNAi 诱导的表型来建立 PLEKHA4 功能模型我们还将探索不同分子功能的缺陷，包括膜结合和寡聚化。 PI(4,5)P2 代谢变化对 PLEKHA4 功能的贡献 (2) 确定机制基础。对于 PLEKHA4 对果蝇模型中 Wnt 信号传导的生理影响，我们发现了敲除。我们将进行单祖果蝇 PLEKHA4/5/6/7 同系物的 Wnt/Wingless 信号传导缺陷。 (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。

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