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

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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A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

eLife ◽

10.7554/elife.22513 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 78

Author(s):

Zhejian Ji ◽

Haishan Gao ◽

Luying Jia ◽

Bing Li ◽

Hongtao Yu

Keyword(s):

Spindle Checkpoint ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Checkpoint Activation ◽

Checkpoint Signaling ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Phosphorylation Cascade ◽

Mitotic Checkpoint Complex

The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.

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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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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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Bub3 promotes Cdc20-dependent activation of the APC/C in S. cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.201412036 ◽

2015 ◽

Vol 209 (4) ◽

pp. 519-527 ◽

Cited By ~ 13

Author(s):

Yang Yang ◽

Dai Tsuchiya ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Unknown Function ◽

Budding Yeast ◽

Spindle Checkpoint ◽

Anaphase Promoting Complex ◽

Mitotic Progression ◽

Checkpoint Activation ◽

Anaphase Onset ◽

Impaired Binding

The spindle checkpoint ensures accurate chromosome segregation by sending a signal from an unattached kinetochore to inhibit anaphase onset. Numerous studies have described the role of Bub3 in checkpoint activation, but less is known about its functions apart from the spindle checkpoint. In this paper, we demonstrate that Bub3 has an unexpected role promoting metaphase progression in budding yeast. Loss of Bub3 resulted in a metaphase delay that was not a consequence of aneuploidy or the activation of a checkpoint. Instead, bub3Δ cells had impaired binding of the anaphase-promoting complex/cyclosome (APC/C) with its activator Cdc20, and the delay could be rescued by Cdc20 overexpression. Kinetochore localization of Bub3 was required for normal mitotic progression, and Bub3 and Cdc20 colocalized at the kinetochore. Although Bub1 binds Bub3 at the kinetochore, bub1Δ cells did not have compromised APC/C and Cdc20 binding. The results demonstrate that Bub3 has a previously unknown function at the kinetochore in activating APC/C-Cdc20 for normal mitotic progression.

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Bub1 is not required for the checkpoint response to unattached kinetochores in diploid human cells

10.1101/278820 ◽

2018 ◽

Cited By ~ 4

Author(s):

Cerys E. Currie ◽

Mar Mora-Santos ◽

Chris Smith ◽

Andrew D. McAinsh ◽

Jonathan B.A. Millar

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Human Cells ◽

Anaphase Promoting Complex ◽

Pathway Model ◽

Checkpoint Response ◽

Conflicting Evidence ◽

Dual Pathway ◽

Mitotic Checkpoint Complex

AbstractError-free chromosome segregation during mitosis depends on a functional spindle assembly checkpoint (SAC). The SAC is a multi-component signaling system that is recruited to incorrectly attached kinetochores to catalyze the formation of a soluble inhibitor, known as the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex [1]. We have previously proposed that two separable pathways, composed of KNL1-Bub3-Bub1 (KBB) and Rod-Zwilch-Zw10 (RZZ), recruit Mad1-Mad2 complexes to human kinetochores to activate the SAC [2]. We refer to this as the dual pathway model. Although Bub1 is absolutely required for MCC formation in yeast (which lack RZZ), there is conflicting evidence as to whether this is also the case in human cells based on siRNA studies [2–5]. Here we report, using genome editing, that Bub1 is not strictly required for the SAC response to unattached kinetochores in human diploid hTERT-RPE1 cells, consistent with the dual pathway model.

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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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Role of CCT chaperonin in the disassembly of mitotic checkpoint complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1620451114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 956-961 ◽

Cited By ~ 23

Author(s):

Sharon Kaisari ◽

Danielle Sitry-Shevah ◽

Shirly Miniowitz-Shemtov ◽

Adar Teichner ◽

Avram Hershko

Keyword(s):

Mitotic Checkpoint ◽

Biologically Active ◽

Anaphase Promoting Complex ◽

Checkpoint Proteins ◽

Aaa Atpase ◽

Sister Chromatids ◽

Ring Complex ◽

Combined Action ◽

Mitotic Checkpoint Complex

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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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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The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

The Journal of Cell Biology ◽

10.1083/jcb.200801049 ◽

2008 ◽

Vol 183 (2) ◽

pp. 267-277 ◽

Cited By ~ 42

Author(s):

Evan C. Osmundson ◽

Dipankar Ray ◽

Finola E. Moore ◽

Qingshen Gao ◽

Gerald H. Thomsen ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Spindle Assembly Checkpoint ◽

Spindle Checkpoint ◽

E3 Ligase ◽

Spindle Assembly ◽

Cyclin B ◽

Carboxyl Terminus ◽

Anaphase Promoting Complex ◽

Mitotic Spindles ◽

Anaphase Onset

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

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