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

• 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
• 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

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.

项目摘要/摘要 通过细胞周期的进展涉及细胞骨架和膜的时空配位 具有控制蛋白水解事件的动力学。后期促进复合体/循环体（APC/C）是主要的 E3泛素连接酶调节有丝分裂。 APC/C介导的泛素化的临时控制很好 建立的APC/C功能的空间组织是其活动的关键未表征的维度，访问 对底物以及对有丝分裂的影响。我们已经确定了磷酸肌醇（PIP）之间的新联系， 形成膜身份的基于脂质的代码，微管细胞骨架和由由APC/C介导的APC/C Plekha5，一种含有域的Pleckstrin同源性（pH）结合蛋白。我们发现Plekha5是 APC/C的微管和质膜 - 定位的蛋白质相互作用，其siRNA耗尽 拮抗有丝分裂的进展，导致APC/C底物的积累。我们建议Plekha5调节 APC/C亚细胞定位，因此控制对关键有丝分裂底物的访问。但是，未知是 Plekha5分子特性的各个方面是其细胞周期功能所必需的。同样，本地化 在细胞周期的不同阶段的APC/C仍然很神秘，Plekha5对 APC/C的本地化和功能。此外，plekha5在调节组成和E3连接酶中的作用 APC/C的活动尚不清楚。我们的长期研究目标是了解管道感应蛋白如何读取 膜的动态变化脂质组成，并翻译此信息以调节 重要蛋白在细胞信号传导中的定位和功能。该提议的目的是了解 Plekha5控制APC/C的定位和活性的分子事件，从而 调节细胞周期进程。指导这项工作的中心假设是Plekha5参与等离子 膜和微管细胞骨架以空间控制的方式招募APC/C 这些位置访问泛素化对于通过有丝分裂进展至关重要的底物。在这个 提案，我们将首先建立有关Plekha5有丝分裂进入和的分子机制 通过测试plekha5定位和与APC/C相互作用的假设来进展对于 它对有丝分裂的影响。此外，我们将确定APC/C的亚细胞位置和Plekha5的作用 通过开发和应用“体内生物化学”工具来控制APC/C的本地化和功能 为了评估APC/C的定位，评估其与Plekha5的共定位，阐明了Plekha5的效果 APC/C定位的耗竭，并确定Plekha5的教会定位如何影响其细胞周期 功能。最后，我们将确定plekha5对组成和体外E3泛素的直接影响 APC/C的连接酶活性。我们的研究将建立一个新的机械框架，以了解如何 泛素化机制的空间组织会影响对健康和疾病重要的细胞途径。