Regulation of the Anaphase-Promoting Complex by the Spindle Checkpoint

描述(由申请人提供)：细胞分裂周期的有序进行需要泛素-蛋白酶体系统周期性地降解关键的细胞周期调节蛋白。后期促进复合体或环体(APC/C)是一种大的多亚基泛素连接酶，它控制着包括中期到后期在内的几个细胞周期的转变。当所有姐妹染色单体都实现了与有丝分裂纺锤体的两极连接后，APC/C与其激活剂CDC20泛素化分离酶抑制物Securin。Securin的降解激活了分离酶，然后分离酶裂解粘附素的一个亚单位，触发姐妹染色单体分离和后期的开始。APC/C-CDC20的活性受到多种机制的严密调控，以防止姐妹染色单体过早分离。主轴检查点就是这样一种APC/C调节机制。我们的长期目标是了解纺锤体检查点如何抑制APC/C-CDC20的活性，并延迟后期对有丝分裂细胞中单个独立的动粒的反应。本提案重点研究了APC/C的重要检查点抑制因子有丝分裂检查点复合体(MCC)的组装和拆卸机制。MCC包含BubR1、Bub3、Cdc20和Mad2。BubR1和Bub3在整个细胞周期中以结构性方式相互结合，而BubR1-CDC20和MAD2-CDC20的相互作用在有丝分裂过程中得到加强。这项建议的具体目的是：(1)确定MAD1辅助的MAD2构象变化在MCC组装中的作用；(2)确定Bub1对CDc20的磷酸化在MCC组装中的作用；以及(3)确定CDc20的自素化和降解在MCC拆解中的作用。纺锤体检查点的故障被认为与人类癌症有关。抗有丝分裂类药物，如紫杉醇，通过利用这一途径中的缺陷来杀死癌细胞。这项提议中的实验将阐明纺锤体检查点的分子基础，这最终可能导致治疗人类癌症的新策略。

DESCRIPTION (provided by applicant): The orderly progression through the cell division cycle requires periodic degradation of key cell cycle regulatory proteins by the ubiquitin-proteasome system. The anaphase-promoting complex or cyclosome (APC/C) is a large multi-subunit ubiquitin ligase that controls several cell cycle transitions, including the metaphase-anaphase transition. After all sister chromatids have achieved bipolar attachment to the mitotic spindle, APC/C in conjunction with its activator Cdc20 ubiquitinates securin, an inhibitor of separase. Degradation of securin activates separase, which then cleaves a subunit of cohesin and triggers sister- chromatid separation and the onset of anaphase. The activity of APC/C-Cdc20 is tightly controlled by multiple mechanisms to prevent premature sister-chromatid separation. The spindle checkpoint is one such APC/C-regulatory mechanism. Our long-term goal is to understand how the spindle checkpoint inhibits the activity of APC/C-Cdc20 and delays the onset of anaphase in response to a single unattached kinetochore within a mitotic cell. This proposal focuses on the mechanism of assembly and disassembly of the mitotic checkpoint complex (MCC), an important checkpoint inhibitor of APC/C. MCC contains BubR1, Bub3, Cdc20, and Mad2. BubR1 and Bub3 bind to each other constitutively throughout the cell cycle whereas the BubR1-Cdc20 and Mad2-Cdc20 interactions are enhanced during mitosis. The specific aims of this proposal are: (1) to determine the role of Mad1 -assisted conformational change of Mad2 in MCC assembly; (2) to determine the role of phosphorylation of Cdc20 by Bub1 in MCC assembly; and (3) to determine the role of Cdc20 autoubiquitination and degradation in MCC disassembly. Malfunction of the spindle checkpoint has been implicated in human cancers. The antimitotic class of anti-cancer drugs, such as Taxol, kills cancer cells by exploiting defects in this pathway. Experiments in this proposal will shed light on the molecular basis of the spindle checkpoint, which might eventually lead to new strategies to treat human cancers.