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降解关键细胞周期调节蛋白。后期促进复合物（APC/C）是一种大型多亚基泛素连接酶，控制几个细胞周期转换，包括中期-后期转换。在所有姐妹染色单体都实现了与有丝分裂纺锤体的双极附着后，APC/C与其激活剂Cdc 20一起泛素化securin，securin是分离酶的抑制剂。securin的降解激活分离酶，然后分离酶切割粘连蛋白的亚基并触发姐妹染色单体分离和分裂后期的开始。APC/C-Cdc 20的活性受到多种机制的严格控制，以防止过早的姐妹染色单体分离。纺锤体检查点是一种这样的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-Cdc 20的活性，并延迟有丝分裂细胞内单个未附着动粒的后期发作。本研究的重点是APC/C的重要检查点抑制剂--有丝分裂检查点复合物（MCC）的组装和分解机制。MCC含有BubR 1、Bub 3、Cdc 20和Mad 2。BubR 1和Bub 3在整个细胞周期中组成性地相互结合，而BubR 1-Cdc 20和Mad 2-Cdc 20相互作用在有丝分裂期间增强。该提议的具体目的是：（1）确定Mad 1辅助的Mad 2构象变化在MCC组装中的作用;（2）确定Bub 1对Cdc 20的磷酸化在MCC组装中的作用;以及（3）确定Cdc 20自泛素化和降解在MCC分解中的作用。纺锤体检查点的功能障碍与人类癌症有关。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.