Spatiotemporal control of ubiquitination by phosphoinositide-binding proteins

磷酸肌醇结合蛋白对泛素化的时空控制

基本信息

批准号：
10446171
负责人：
JEREMY BASKIN
金额：
$ 30.94万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10446171
关键词：
Affect Anaphase Behavior Binding Binding Proteins Biological Assay Biotinylation Cell Cycle Cell Cycle Checkpoint Cell Cycle Progression Cell Cycle Stage Cell membrane Cells Cellular Membrane Code Complex Cytoskeleton Data Defect Dimensions Disease Event Exhibits G2/M Transition Goals Health Immunofluorescence Immunologic In Vitro Interphase Link Lipids Location Malignant Neoplasms Mass Spectrum Analysis Mediating Membrane Membrane Lipids Metaphase Methods Microtubules Mitosis Mitotic Molecular PH Domain Pathway interactions Phenotype Phosphatidylinositols Phosphorylation Play Process Property Proteins Proteolysis Proteomics RNA Interference Regulation Research Role Signal Transduction Signaling Protein Small Interfering RNA Spatial Behavior Structure System Testing Ubiquitin Ubiquitination Work anaphase-promoting complex base biochemical tools human disease in vitro activity in vivo multicatalytic endopeptidase complex novel programs protein degradation recruit spatiotemporal tool ubiquitin-protein ligase

项目摘要

Project Summary/Abstract Progression through the cell cycle involves spatiotemporal coordination of cytoskeletal and membrane dynamics with controlled proteolysis events. The anaphase-promoting complex/cyclosome (APC/C) is the main E3 ubiquitin ligase regulating mitosis. Whereas the temporal control of APC/C-mediated ubiquitination is well established, the spatial organization of APC/C function is a key uncharacterized dimension to its activity, access to substrates, and effects on mitosis. We have identified a novel link between phosphoinositides (PIPs), which form a lipid-based code of membrane identity, the microtubule cytoskeleton, and the APC/C that is mediated by PLEKHA5, a pleckstrin homology (PH) domain-containing, PIP-binding protein. We discovered PLEKHA5 as a microtubule- and plasma membrane-localized protein interactor of the APC/C whose depletion by siRNA antagonizes mitotic progression, causing a buildup of APC/C substrates. We propose that PLEKHA5 regulates APC/C subcellular localization and thus controls access to key mitotic substrates. Yet, it is unknown which aspects of PLEKHA5’s molecular properties are required for its cell cycle functions. As well, the localization of the APC/C at different stages of the cell cycle still remains largely a mystery, as is the effect of PLEKHA5 on APC/C localization and function. Further, the role of PLEKHA5 in modulating the composition and E3 ligase activity of the APC/C is unknown. Our long-term research goal is to understand how PIP-sensing proteins read the dynamically changing lipid composition of membranes and transduce this information to regulate the localization and function of important proteins in cell signaling. The objective of this proposal is to understand the molecular events through which PLEKHA5 controls the localization and activity of the APC/C and thus regulates cell cycle progression. The central hypothesis guiding this work is that PLEKHA5 engages the plasma membrane and the microtubule cytoskeleton in a spatiotemporally controlled fashion and recruits the APC/C to these locations to access substrates whose ubiquitination is critical for progression through mitosis. In this proposal, we will first establish molecular mechanisms governing PLEKHA5 regulation of mitotic entry and progression by testing the hypothesis that PLEKHA5 localization and interactions with APC/C are important for its effects on mitosis. In addition, we will determine subcellular localizations of APC/C and the role of PLEKHA5 in controlling APC/C localization and function by developing and applying a suite of “in vivo biochemistry” tools to assess the localization of APC/C, evaluate its colocalization with PLEKHA5, elucidate the effects of PLEKHA5 depletion on APC/C localization, and ascertain how ectopic localization of PLEKHA5 affects its cell cycle functions. Finally, we will determine the direct effects of PLEKHA5 on the composition and in vitro E3 ubiquitin ligase activity of the APC/C. Our studies will establish a new mechanistic framework for understanding how spatial organization of the ubiquitination machinery affects cellular pathways important for health and disease.

项目概要/摘要细胞周期的进展涉及细胞骨架和膜的时空协调受控蛋白水解事件的动力学是主要的。 E3 泛素连接酶在 APC/C 介导的泛素化的时间控制中发挥着良好的调节作用。建立后，APC/C 功能的空间组织是其活动、访问的一个关键的未表征维度。我们发现了磷酸肌醇 (PIP) 之间的新联系。形成基于脂质的膜身份代码、微管细胞骨架和由以下介导的 APC/C PLEKHA5，一种含有 pleckstrin 同源 (PH) 结构域的 PIP 结合蛋白。 APC/C 的微管和质膜定位蛋白相互作用蛋白，其被 siRNA 耗尽拮抗有丝分裂进展，导致 APC/C 底物积聚。我们建议 PLEKHA5 进行调节。 APC/C 亚细胞定位，从而控制对关键有丝分裂底物的访问，但尚不清楚是哪个。 PLEKHA5 的分子特性也是其细胞周期功能所必需的。细胞周期不同阶段的 APC/C 在很大程度上仍然是一个谜，PLEKHA5 对细胞周期的影响也是如此。 APC/C 定位和功能此外，PLEKHA5 在调节组成和 E3 连接酶中的作用。 APC/C 的活性尚不清楚，我们的长期研究目标是了解 PIP 传感蛋白如何读取。膜的动态变化的脂质成分并转换该信息来调节该提案的目的是了解细胞信号传导中重要蛋白质的定位和功能。 PLEKHA5 通过其控制 APC/C 的定位和活性的分子事件，从而指导这项工作的中心假设是 PLEKHA5 参与血浆。膜和微管细胞骨架以时空控制的方式并招募 APC/C 这些位置访问底物，其泛素化对于有丝分裂的进展至关重要。根据建议，我们将首先建立控制 PLEKHA5 有丝分裂进入和调节的分子机制通过测试 PLEKHA5 定位和与 APC/C 的相互作用对于此外，我们将确定 APC/C 的亚细胞定位和 PLEKHA5 的作用。通过开发和应用一套“体内生物化学”工具来控制 APC/C 定位和功能评估 APC/C 的定位，评估其与 PLEKHA5 的共定位，阐明 PLEKHA5 的作用 APC/C 定位的耗尽，并确定 PLEKHA5 的异位定位如何影响其细胞周期最后，我们将确定 PLEKHA5 对组成和体外 E3 泛素的直接影响。我们的研究将建立一个新的机制框架来理解 APC/C 的连接酶活性。泛素化机制的空间组织影响对健康和疾病很重要的细胞通路。