Spatiotemporal control of ubiquitination by phosphoinositide-binding proteins

项目总结/摘要 通过细胞周期的进展涉及时空协调的细胞骨架和膜 控制蛋白水解事件的动力学。后期促进复合物/细胞周期体（APC/C）是主要的 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是很好的， 建立后，APC/C功能的空间组织是其活动的一个关键的非特征化维度， 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， 形成一个基于脂质的代码膜身份，微管细胞骨架，和APC/C，这是介导的 PLEKHA 5，一种含有普列克底物蛋白同源（PH）结构域的PIP结合蛋白。我们发现PLEKHA 5是一种 APC/C的微管和质膜定位的蛋白质相互作用物，其被siRNA耗尽 拮抗有丝分裂进程，导致APC/C底物的积累。我们认为PLEKHA 5调节 APC/C亚细胞定位，从而控制关键有丝分裂底物的进入。然而，我们不知道， PLEKHA 5分子特性的某些方面是其细胞周期功能所必需的。同时， APC/C在细胞周期的不同阶段仍然是一个谜，PLEKHA 5对细胞周期的影响也是如此。 APC/C定位和功能。此外，PLEKHA 5在调节组合物和E3连接酶中的作用也被证实。 APC/C的活性未知。我们的长期研究目标是了解PIP传感蛋白如何读取 动态变化的脂质组成的膜，并收集这一信息，以调节 细胞信号传导中重要蛋白质的定位和功能。本提案的目的是了解 PLEKHA 5通过其控制APC/C的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PLEKHA 5与等离子体结合， 细胞膜和微管细胞骨架在时空控制的方式，并招募APC/C， 这些位置来访问底物，其泛素化对于有丝分裂的进展至关重要。在这 建议，我们将首先建立PLEKHA 5调节有丝分裂进入的分子机制， 通过检验PLEKHA 5定位和与APC/C的相互作用对于PLEKHA 5的表达和进展是重要的这一假设， 对有丝分裂的影响。此外，我们将确定APC/C的亚细胞定位和PLEKHA 5的作用。 通过开发和应用一套“体内生物化学”工具， 评估APC/C的定位，评估其与PLEKHA 5的共定位，阐明PLEKHA 5的作用， 耗尽APC/C定位，并确定PLEKHA 5的异位定位如何影响其细胞周期 功能协调发展的最后，我们将确定PLEKHA 5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制框架， 泛素化机制的空间组织影响对健康和疾病重要的细胞途径。

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.