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展需要泛素-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）是一种大型多亚基泛素连接酶，控制多种细胞周期转变，包括中期-后期转变。当所有姐妹染色单体都与有丝分裂纺锤体实现双极性附着后，APC/C与其激活剂Cdc20一起泛素化securin，一种分离酶抑制剂。securin的降解激活分离酶，然后分裂一个内聚蛋白亚基并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格控制，以防止姐妹染色单体过早分离。纺锤体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，并延迟有丝分裂细胞内单个未附着着丝点的后期开始。本文主要研究APC/C的重要检查点抑制剂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在整个细胞周期中相互结合，而BubR1- cdc20和Mad2-Cdc20的相互作用在有丝分裂期间增强。本提案的具体目的是：(1)确定Mad1辅助Mad2构象变化在MCC组装中的作用；(2)确定Bub1磷酸化Cdc20在MCC组装中的作用；(3)确定Cdc20自泛素化和降解在MCC分解中的作用。纺锤体检查点的故障与人类癌症有关。抗有丝分裂类抗癌药物，如紫杉醇，通过利用这一途径中的缺陷杀死癌细胞。这项提议中的实验将揭示纺锤体检查点的分子基础，这可能最终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.