Spatiotemporal control of ubiquitination by phosphoinositide-binding proteins

项目摘要/摘要 细胞周期的进展涉及细胞骨架和膜的时空协调 具有受控蛋白分解事件的动力学。后期促进复合体/环体(APC/C)是主要的 E3泛素连接酶调节有丝分裂。而APC/C介导的泛素化的时间控制性很好 APC/C职能的空间组织已经确立，是其活动的一个关键的未确定的维度，即访问 对底物的影响以及对有丝分裂的影响。我们已经确定了磷脂酰肌醇(PIP)之间的一种新的联系，它 形成基于脂质的膜识别密码、微管细胞骨架和APC/C，后者由 PLEKHA5，一种含有Pleckstrin同源(PH)结构域的PIP结合蛋白。我们发现PLEKHA5是一种 APC/C的微管和质膜定位的蛋白相互作用，其被siRNA耗尽 抑制有丝分裂进程，导致APC/C底物堆积。我们建议PLEKHA5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。然而，目前还不清楚是谁 PLEKHA5的S分子特性是其细胞周期功能所必需的。同样，本地化也是 细胞周期不同阶段的APC/C仍然是一个谜，PLEKHA5对细胞周期的影响也是如此。 APC/C的本地化和功能。此外，PLEKHA5在调节成分和E3连接酶中的作用 APC/C的活动未知。我们的长期研究目标是了解PIP感应蛋白是如何阅读的 动态变化的膜脂成分，并将这一信息转导到调节 细胞信号转导中重要蛋白质的定位和功能。这项建议的目标是理解 PLEKHA5调控APC/C定位和活性的分子事件 调节细胞周期进程。指导这项工作的中心假设是PLEKHA5与等离子体接触 膜和微管细胞骨架以时空控制的方式并招募APC/C来 这些位置可以获得其泛素化对有丝分裂的进展至关重要的底物。在这 提议，我们将首先建立PLEKHA5调节有丝分裂进入和 通过检验PLEKHA5的定位和与APC/C的相互作用对 它对有丝分裂的影响。此外，我们还将确定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的定位和功能 评估APC/C的定位，评估其与PLEKHA5的共定位，阐明PLEKHA5的作用 去除APC/C定位，并确定PLEKHA5的异位定位如何影响其细胞周期 功能。最后，我们将确定PLEKHA5对成分和体外E3泛素的直接影响 连接酶活性的APC/C我们的研究将建立一个新的机制框架，以了解如何 泛素化机制的空间组织影响对健康和疾病至关重要的细胞通路。

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.