Role of phosphorylation of Cdc20 in p31 ^comet -stimulated disassembly of the mitotic checkpoint complex

Abstract

The mitotic checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is turned on, it promotes the formation of the mitotic checkpoint complex (MCC), which inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 bound to the APC/C activator Cdc20. When the checkpoint is satisfied, MCC is disassembled and APC/C becomes active. Previous studies have shown that the Mad2-binding protein p31 comet promotes the dissociation of Cdc20 from BubR1 in MCC in a process that requires ATP. We now show that a part of MCC dissociation is blocked by inhibitors of cyclin-dependent kinases (Cdks) and that purified Cdk1–cyclin B stimulates this process. The mutation of all eight potential Cdk phosphorylation sites of Cdc20 partially prevented its release from BubR1. Furthermore, p31 comet stimulated Cdk-catalyzed phosphorylation of Cdc20 in MCC. It is suggested that the binding of p31 comet to Mad2 in MCC may trigger a conformational change in Cdc20 that facilitates its phosphorylation by Cdk, and that the latter process may promote its dissociation from BubR1.

Disassembly of mitotic checkpoint complexes by the joint action of the AAA-ATPase TRIP13 and p31 comet

“…p31 comet inactivates the mitotic checkpoint in vivo and in vitro in a manner that requires its interaction with Mad2 (12,14). We found that p31 comet stimulates the phosphorylation of Cdc20 in MCC by cyclin-dependent kinase (Cdk), a process that promotes MCC disassembly (15). However, this explained only a part of ATP requirement for p31 comet action: We noted the existence of an additional pathway of MCC disassembly that required p31 comet and ATP, but not phosphorylation (15).…”

“…To generate Cdc20-Mad2 substrate for the assay, we used the subcomplex released from immunopurified MCC by incubation with p31 comet , ATP, and Cdk, as described (10,15). The disassembly of Cdc20-Mad2 was determined by immunoprecipitation with anti-Cdc20, followed by immunoblotting for Mad2.…”

“…We have previously observed that a Cdc20-Mad2 subcomplex is the intermediary product of MCC disassembly in a pathway that requires the action of p31 comet and Cdc20 phosphorylation (10,15). The initial aim of the present investigation was to explore the mechanisms by which this subcomplex is further dissociated into its individual components.…”

“…The role of p31 comet here may be the destabilization of MCC structure as suggested (7), by disrupting the binding of BubR1 to C-Mad2 through competition of p31 comet with BubR1 on a similar binding interface of C-Mad2. This dissociation is aided by Cdk-catalyzed phosphorylation of Cdc20 (15). The second type of dissociation process involves joint action of TRIP13 and p31 comet and causes the release of free Mad2 from Cdc20-Mad2 (Fig.…”

“…We found that p31 comet stimulates the phosphorylation of Cdc20 in MCC by cyclin-dependent kinase (Cdk), a process that promotes MCC disassembly (15). However, this explained only a part of ATP requirement for p31 comet action: We noted the existence of an additional pathway of MCC disassembly that required p31 comet and ATP, but not phosphorylation (15). By the use of a purified in vitro system, we found that the phosphorylation-dependent MCC disassembly pathway promoted the dissociation of Cdc20 from BubR1, resulting in the release of a subcomplex in which Cdc20 was still bound to Mad2 (10).…”