Role of CCT chaperonin in the disassembly of mitotic checkpoint complexes

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 p31comet. 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 Chem288(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.

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Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524551113 ◽

2016 ◽

Vol 113 (4) ◽

pp. 966-971 ◽

Cited By ~ 10

Author(s):

Sharon Kaisari ◽

Danielle Sitry-Shevah ◽

Shirly Miniowitz-Shemtov ◽

Avram Hershko

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Checkpoint Proteins ◽

Sister Chromatids ◽

Unknown Species ◽

Mitotic Checkpoint Complex

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.

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Role of Polo-like kinase 1 in the regulation of the action of p31comet in the disassembly of mitotic checkpoint complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902970116 ◽

2019 ◽

pp. 201902970 ◽

Cited By ~ 4

Author(s):

Sharon Kaisari ◽

Pnina Shomer ◽

Tamar Ziv ◽

Danielle Sitry-Shevah ◽

Shirly Miniowitz-Shemtov ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Hela Cells ◽

Mitotic Spindle ◽

Joint Action ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Futile Cycle ◽

Mitotic Checkpoint Complex ◽

Bi 2536

The Mad2-binding protein p31comet 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. p31comet 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 p31comet 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 p31comet and phosphorylated it, resulting in the suppression of its activity (with TRIP13) to disassemble checkpoint complexes. Plk1 phosphorylated p31comet 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 p31comet had a greatly decreased sensitivity to inhibition by Plk1 of its action to disassemble mitotic checkpoint complexes. We propose that the phosphorylation of p31comet by Plk1 prevents a futile cycle of MCC assembly and disassembly during the active mitotic checkpoint.

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Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31comet

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0389 ◽

2011 ◽

Vol 22 (22) ◽

pp. 4227-4235 ◽

Cited By ~ 39

Author(s):

Luying Jia ◽

Bing Li ◽

Ross T. Warrington ◽

Xing Hao ◽

Shixuan Wang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Spindle Checkpoint ◽

Functional Redundancy ◽

Mitotic Checkpoint ◽

Human Cells ◽

Unresolved Issue ◽

Anaphase Promoting Complex ◽

Anaphase Onset ◽

Mitotic Checkpoint Complex

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase–anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31comet delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31comet and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.

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Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2

The Journal of Cell Biology ◽

10.1083/jcb.200102093 ◽

2001 ◽

Vol 154 (5) ◽

pp. 925-936 ◽

Cited By ~ 558

Author(s):

Valery Sudakin ◽

Gordon K.T. Chan ◽

Tim J. Yen

Keyword(s):

Hela Cells ◽

Inhibitory Activity ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Checkpoint Proteins ◽

Ubiquitin Ligase Activity ◽

Interphase Cells ◽

Mitotic Checkpoint Complex ◽

Sustained Inhibition

The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

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Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint

The Journal of Cell Biology ◽

10.1083/jcb.201311113 ◽

2014 ◽

Vol 204 (6) ◽

pp. 901-908 ◽

Cited By ~ 41

Author(s):

Edward R. Ballister ◽

Michelle Riegman ◽

Michael A. Lampson

Keyword(s):

Protein Localization ◽

Mitotic Checkpoint ◽

Active Conformation ◽

Anaphase Promoting Complex ◽

Concomitant Increase ◽

Checkpoint Proteins ◽

Microtubule Attachment ◽

Chemically Induced ◽

Chemically Induced Dimerization ◽

Mitotic Checkpoint Complex

The mitotic checkpoint monitors kinetochore–microtubule attachment and prevents anaphase until all kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to kinetochores. Unattached, checkpoint-active kinetochores accumulate multiple checkpoint proteins, which are depleted from kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached kinetochores, it is not known what changes at kinetochores are essential for anaphase promoting complex/cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, we show that recruiting the checkpoint protein Mad1 to metaphase kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 or BubR1. Furthermore, Mad2 binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of our checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at kinetochores.

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Role of ubiquitylation of components of mitotic checkpoint complex in their dissociation from anaphase-promoting complex/cyclosome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1720312115 ◽

2018 ◽

Vol 115 (8) ◽

pp. 1777-1782 ◽

Cited By ~ 4

Author(s):

Danielle Sitry-Shevah ◽

Sharon Kaisari ◽

Adar Teichner ◽

Shirly Miniowitz-Shemtov ◽

Avram Hershko

Keyword(s):

Chromosome Segregation ◽

Ubiquitin Ligase ◽

Mitotic Spindle ◽

Mitotic Checkpoint ◽

Terminal Region ◽

Anaphase Promoting Complex ◽

Lysine Residues ◽

Mitotic Checkpoint Complex

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.

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Mode of interaction of TRIP13 AAA-ATPase with the Mad2-binding protein p31comet and with mitotic checkpoint complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515358112 ◽

2015 ◽

Vol 112 (37) ◽

pp. 11536-11540 ◽

Cited By ~ 34

Author(s):

Shirly Miniowitz-Shemtov ◽

Ety Eytan ◽

Sharon Kaisari ◽

Danielle Sitry-Shevah ◽

Avram Hershko

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Thyroid Hormone Receptor ◽

Binding Protein ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Interacting Protein ◽

Aaa Atpase ◽

Oligomeric Form ◽

Mitotic Checkpoint Complex

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.

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TRIP13 promotes the cell proliferation, migration and invasion of glioblastoma through the FBXW7/c-MYC axis

British Journal of Cancer ◽

10.1038/s41416-019-0633-0 ◽

2019 ◽

Vol 121 (12) ◽

pp. 1069-1078 ◽

Cited By ~ 5

Author(s):

Guanghui Zhang ◽

Qingzong Zhu ◽

Gang Fu ◽

Jianbing Hou ◽

Xiaosong Hu ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Therapeutic Target ◽

Biological Effect ◽

Thyroid Hormone Receptor ◽

E3 Ubiquitin Ligase ◽

Mitotic Checkpoint ◽

Migration And Invasion ◽

Cell Models ◽

Aaa Atpase

Abstract Background Thyroid hormone receptor interactor 13 (TRIP13) is an AAA + ATPase that plays an important role in the mitotic checkpoint. TRIP13 is highly expressed in various human tumours and promotes tumorigenesis. However, the biological effect of TRIP13 in GBM cells remains unclear. Methods We generated GBM cell models with overexpressed or silenced TRIP13 via lentivirus-mediated overexpression and RNAi methods. The biological role of TRIP13 in the proliferation, migration and invasion of GBM cells has been further explored. Results Our research indicated that TRIP13 was highly expressed in GBM tissues and cells. We found that the proliferation, migration and invasion abilities were inhibited in TRIP13-knockdown GBM cells. These results indicated that TRIP13 plays an important role in the tumorigenesis of GBM. Moreover, we found that TRIP13 first stabilised c-MYC by inhibiting the transcription of FBXW7, which is an E3 ubiquitin ligase of c-MYC, by directly binding to the promoter region of FBXW7. Therefore, our study indicated that the TRIP13/FBXW7/c-MYC pathway might provide a prospective therapeutic target in the treatment of GBM. Conclusions These results indicated that TRIP13 plays an oncogenic role in GBM. The TRIP13/FBXW7/c-MYC pathway might act as a prospective therapeutic target for GBM patients.

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Mechanism of APC/CCDC20 activation by mitotic phosphorylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604929113 ◽

2016 ◽

Vol 113 (19) ◽

pp. E2570-E2578 ◽

Cited By ~ 69

Author(s):

Renping Qiao ◽

Florian Weissmann ◽

Masaya Yamaguchi ◽

Nicholas G. Brown ◽

Ryan VanderLinden ◽

...

Keyword(s):

Chromosome Segregation ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Loop Region ◽

Evolutionarily Conserved ◽

Terminal Loop ◽

Different Types ◽

Mitotic Phosphorylation ◽

Mitotic Checkpoint Complex

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

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