Regulation of the Anaphase-Promoting Complex by the Spindle Checkpoint

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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.