Faculty Bibliography

The Mad2-binding protein p31^comet has important roles in the inactivation of the mitotic checkpoint system, which delays anaphase until chromosomes attach correctly to the mitotic spindle. The activation of the checkpoint promotes the assembly of a Mitotic Checkpoint Complex (MCC), which inhibits the action of the ubiquitin ligase APC/C (Anaphase-Promoting Complex/Cyclosome) to degrade inhibitors of anaphase initiation. The inactivation of the mitotic checkpoint requires the disassembly of MCC. p31^comet promotes the disassembly of mitotic checkpoint complexes by liberating their Mad2 component in a joint action with the ATPase TRIP13. Here, we investigated the regulation of p31^comet action. The release of Mad2 from checkpoint complexes in extracts from nocodazole-arrested HeLa cells was inhibited by Polo-like kinase 1 (Plk1), as suggested by the effects of selective inhibitors of Plk1. Purified Plk1 bound to p31^comet and phosphorylated it, resulting in the suppression of its activity (with TRIP13) to disassemble checkpoint complexes. Plk1 phosphorylated p31^comet on S102, as suggested by the prevention of the phosphorylation of this residue in checkpoint extracts by the selective Plk1 inhibitor BI-2536 and by the phosphorylation of S102 with purified Plk1. An S102A mutant of p31^comet had a greatly decreased sensitivity to inhibition by Plk1 of its action to disassemble mitotic checkpoint complexes. We propose that the phosphorylation of p31^comet by Plk1 prevents a futile cycle of MCC assembly and disassembly during the active mitotic checkpoint.

The mitotic checkpoint system ensures the fidelity of chromosome segregation in mitosis by preventing premature initiation of anaphase until correct bipolar attachment of chromosomes to the mitotic spindle is reached. It promotes the assembly of a mitotic checkpoint complex (MCC), composed of BubR1, Bub3, Cdc20, and Mad2, which inhibits the activity of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. When the checkpoint is satisfied, anaphase is initiated by the disassembly of MCC. Previous studies indicated that the dissociation of APC/C-bound MCC requires ubiquitylation and suggested that the target of ubiquitylation is the Cdc20 component of MCC. However, it remained unknown how ubiquitylation causes the release of MCC from APC/C and its disassembly and whether ubiquitylation of additional proteins is involved in this process. We find that ubiquitylation causes the dissociation of BubR1 from Cdc20 in MCC and suggest that this may lead to the release of MCC components from APC/C. BubR1 in MCC is ubiquitylated by APC/C, although to a lesser degree than Cdc20. The extent of BubR1 ubiquitylation was markedly increased in recombinant MCC that contained a lysine-less mutant of Cdc20. Mutation of lysine residues to arginines in the N-terminal region of BubR1 partially inhibited its ubiquitylation and slowed down the release of MCC from APC/C, provided that Cdc20 ubiquitylation was also blocked. It is suggested that ubiquitylation of both Cdc20 and BubR1 may be involved in their dissociation from each other and in the release of MCC components from APC/C.

The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. When this checkpoint is active, a mitotic checkpoint complex (MCC), composed of the checkpoint proteins Mad2, BubR1, Bub3, and Cdc20, is assembled. MCC inhibits the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), whose action is necessary for anaphase initiation. When the checkpoint signal is turned off, MCC is disassembled, a process required for exit from checkpoint-arrested state. Different moieties of MCC are disassembled by different ATP-requiring processes. Previous work showed that Mad2 is released from MCC by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31^comet Now we have isolated from extracts of HeLa cells an ATP-dependent factor that releases Cdc20 from MCC and identified it as chaperonin containing TCP1 or TCP1-Ring complex (CCT/TRiC chaperonin), a complex known to function in protein folding. Bacterially expressed CCT5 chaperonin subunits, which form biologically active homooligomers [Sergeeva, et al. (2013) J Biol Chem 288(24):17734-17744], also promote the disassembly of MCC. CCT chaperonin further binds and disassembles subcomplexes of MCC that lack Mad2. Thus, the combined action of CCT chaperonin with that of TRIP13 ATPase promotes the complete disassembly of MCC, necessary for the inactivation of the mitotic checkpoint.

The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system.

The AAA-ATPase thyroid hormone receptor interacting protein 13 (TRIP13), jointly with the Mad2-binding protein p31comet, promotes the inactivation of the mitotic (spindle assembly) checkpoint by disassembling the mitotic checkpoint complex (MCC). This checkpoint system ensures the accuracy of chromosome segregation by delaying anaphase until correct bipolar attachment of chromatids to the mitotic spindle is achieved. MCC inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3, in association with the APC/C activator Cdc20. The assembly of MCC in active checkpoint is initiated by the conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformation, which then binds tightly to Cdc20. Conversely, the disassembly of MCC that takes place when the checkpoint is turned off involves the conversion of C-Mad2 back to O-Mad2. Previously, we found that the latter process is mediated by TRIP13 together with p31comet, but the mode of their interaction remained unknown. Here, we report that the oligomeric form of TRIP13 binds both p31comet and MCC. Furthermore, p31comet and checkpoint complexes mutually promote the binding of each other to oligomeric TRIP13. We propose that p31comet bound to C-Mad2-containing checkpoint complex is the substrate for the ATPase and that the substrate-binding site of TRIP13 is composed of subsites specific for p31comet and C-Mad2-containing complex. The simultaneous occupancy of both subsites is required for high-affinity binding to TRIP13.

The mitotic (or spindle assembly) checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is active, a Mitotic Checkpoint Complex (MCC) assembles and inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C). MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3 associated with the APC/C activator Cdc20. When the checkpoint signal is turned off, MCC is disassembled and the checkpoint is inactivated. The mechanisms of the disassembly of MCC are not sufficiently understood. We have previously observed that ATP hydrolysis is required for the action of the Mad2-binding protein p31comet to disassemble MCC. We now show that HeLa cell extracts contain a factor that promotes ATP- and p31comet-dependent disassembly of a Cdc20-Mad2 subcomplex and identify it as Thyroid Receptor Interacting Protein 13 (TRIP13), an AAA-ATPase known to interact with p31comet. The joint action of TRIP13 and p31comet also promotes the release of Mad2 from MCC, participates in the complete disassembly of MCC and abrogates checkpoint inhibition of APC/C. We propose that TRIP13 plays centrally important roles in the sequence of events leading to MCC disassembly and checkpoint inactivation.

The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis. When the checkpoint is turned on, the mitotic checkpoint complex (MCC) inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 associated with the APC/C activator Cdc20. The mechanisms of the assembly of MCC when the checkpoint is turned on, and of its disassembly when the checkpoint is inactivated, are not sufficiently understood. Previous reports indicated that APC/C-mediated polyubiquitylation of Cdc20 in MCC is required for the dissociation of APC/C-associated MCC, but not of free MCC. The pool of free MCC is disassembled by an ATP-dependent process stimulated by the Mad2-binding protein p31(comet). It remained unknown whether free MCC is the precursor or the dissociation product of APC/C-bound MCC. By characterizing the mechanisms of the disassembly of APC/C-bound MCC in a purified system, we find that it cannot be the source of free MCC, because it is bound at high affinity and is released only in ubiquitylated or partially disassembled forms. By the use of a cell-free system from Xenopus eggs that reproduces the mitotic checkpoint, we show that MCC can be assembled in the absence of APC/C in a checkpoint-dependent manner. We propose that when the checkpoint is turned on, free MCC is the precursor of APC/C-bound MCC. When the mitotic checkpoint is extinguished, both APC/C-bound and free MCC pools have to be disassembled to release APC/C from inhibition.