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: 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
• 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/10589550
• 关键词: Affect; Affinity; Attention; Binding; Binding Proteins; Cell membrane; Complex; Defect; Disease; Drosophila genus; Exhibits; Family; Goals; Health; Homologous Gene; Knock-out; Link; Lipid Binding; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Metabolism; Modeling; Molecular; PH Domain; Pathway interactions; Phenotype; Phosphatidylinositols; Physiological; Protein Binding Domain; Proteins; RNA Interference; Regulation; Research; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Sum; System; Tertiary Protein Structure; Testing; Time; Ubiquitination; WNT Signaling Pathway; Work; fly; human disease; in vivo; link protein; novel; paralogous gene; parent grant; prevent; recruit; ubiquitin-protein ligase

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 父母补助金R 01 GM 131101 磷酸肌醇（PIP）和泛素化是调节空间信号转导的主要系统， 时间虽然它们的主要调节机制已得到很好的表征，但次级调节层， 特别是那些调节串扰的方法受到的关注较少。我们已经发现PIP之间存在一种新的联系， 和由PLEKHA 4介导的泛素化，PLEKHA 4是一种含有普列克底物蛋白同源（PH）结构域的蛋白。我们发现 这种多结构域蛋白在质膜的PI（4，5）P2富集区形成大的组装体， 脂质和蛋白质结合结构域的独特组合，并将E3泛素连接酶CUL 3 KLHL 12募集到这样的结构域中。 结构.令人惊讶的是，CUL 3 KLHL 12的这种重新定位伴随着E3连接酶活性的降低 向主要底物Dishevelled-3（DVL 3）的方向，导致DVL 3积累和Wnt信号传导增加， 其中DVL 3是关键中间体。PLEKHA 4如何在机制上调节 CUL 3 KLHL 12活性，在分子和功能水平。在这个提案中，我们将测试一部小说， 螯合模型来解释和理解这些结果。我们的长期研究目标是了解 PIP传感蛋白连接膜脂质组成以调节多种生理信号蛋白 contexts.本提案的目的是了解PLEKHA 4及其旁系同源物 PLEKHA 5/6/7影响CUL 3 KLHL 12 E3连接酶对DVL 3和Wnt信号传导的活性。核心假设 指导这项工作的是PLEKHA 4/5/6/7的寡聚簇介导CUL 3 KLHL 12在血浆中的螯合 膜处于失活状态，并通过阻止DVL 3遍在蛋白化，充当Wnt的正调节因子 发信号。我们提出了以下几个方面的研究目标：（1）阐明分子机制 控制PLEKHA 4对CUL 3 KLHL 12介导的DVL 3泛素化的调节。我们将测试隔离 通过用PLEKHA 4构建体进行RNAi诱导的表型的拯救的PLEKHA 4功能模型 缺乏不同的分子功能，包括膜结合和寡聚化。我们还将探索 PI（4，5）P2代谢的变化对PLEKHA 4功能的贡献。(2)确定机械基础 PLEKHA 4对果蝇模型中Wnt信号传导的生理作用。我们发现击倒对手 PLEKHA 4/5/6/7同源物的单一祖先果蝇表现出Wnt/Wingless信号转导缺陷。我们将执行 体内结构功能研究，以剖析这些表型的机制。(3)阐明 在PLEKHA 4/5/6/7中，在分子和细胞水平上的功能的专门化和保守性 家人我们将检验PLEKHA 4/5/6/7蛋白可以形成多功能PLEKHA的假设 复合物通过异源寡聚化和不同程度的膜和蛋白质亲和力。总的来说， 了解PLEKHA 4/5/6/7蛋白如何读取膜中动态变化的PIP含量 双分子层和双分子层的研究表明，调控E3连接酶活性的信息将定义一个新的机制框架， 重要信号通路的调节（例如，Wnt信号）在健康和疾病中的作用。

项目成果

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