The Cdc20-binding Phe Box of the Spindle Checkpoint Protein BubR1 Maintains the Mitotic Checkpoint Complex During Mitosis

The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.     

BUBR1 and closed MAD2 (C-MAD2) interact directly to assemble a functional mitotic checkpoint complex

The mitotic checkpoint maintains genomic stability by ensuring that chromosomes are accurately segregated during mitosis. When the checkpoint is activated, the mitotic checkpoint complex (MCC), assembled from BUBR1, BUB3, CDC20, and MAD2, directly binds and inhibits the anaphase-promoting complex/cyclosome (APC/C) until all chromosomes are properly attached and aligned. The mechanisms underlying MCC assembly and MCC-APC/C interaction are not well characterized. Here, we show that a novel interaction between BUBR1 and closed MAD2 (C-MAD2) is essential for MCC-mediated inhibition of APC/C. Intriguingly, Arg(133) and Gln(134) in C-MAD2 are required for BUBR1 interaction. The same residues are also critical for MAD2 dimerization and MAD2 binding to p31(comet), a mitotic checkpoint silencing protein. Along with previously characterized BUBR1-CDC20 and C-MAD2-CDC20 interactions, our results underscore the integrity of the MCC for its activity and suggest the fundamental importance of the MAD2 αC helix in modulating mitotic checkpoint activation and silencing.     

The AAA+ ATPase TRIP13 remodels HORMA domains through N‐terminal engagement and unfolding

Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed “closure motifs”. The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain–closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X‐ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31^comet. We show that p31^comet binding to the TRIP13 N‐terminal domain positions the disordered MAD2 N‐terminus for engagement by the TRIP13 “pore loops”, which then unfold MAD2 in the presence of ATP. N‐terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13‐mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain–closure motif complexes by TRIP13.     

Dynein intermediate chain 2c (DNCI2c) complex is essential for exiting Mad2-dependent spindle assembly checkpoint

The Mad2 protein plays a key role in the spindle assembly checkpoint (SAC) function. The SAC pathway delays mitotic progression into anaphase until all kinetochores attach to the spindle during mitosis. The formation of the Mad2–p31^comet complex correlates with the completion of spindle attachment and the entry into anaphase during mitosis.Herein, we showed that dynein intermediate chain 2c (DNCI2c)—a subunit of dynein motor protein—forms an immunocomplex with p31^comet during mitosis. DNCI2c-knockdown resulted in prolonged mitotic arrest in a Mad2-dependent manner. Furthermore, DNCI2c-knockdown-induced mitotic arrest was not rescued by p31^comet overexpression. However, the combination of p31^comet overexpression with the mitotic drug treatment reversed the mitotic arrest in DNCI2c-knockdown. Together, these results indicate that the DNCI2c–p31^comet complex plays an important role in exiting Mad2-dependent SAC.     

Monopolar Spindle 1 (MPS1) Kinase Promotes Production of Closed MAD2 (C-MAD2) Conformer and Assembly of the Mitotic Checkpoint Complex

MPS1 kinase is an essential component of the spindle assembly checkpoint (SAC), but its functioning mechanisms are not fully understood. We have shown recently that direct interaction between BUBR1 and MAD2 is critical for assembly and function of the human mitotic checkpoint complex (MCC), the SAC effector. Here we report that inhibition of MPS1 kinase activity by reversine disrupts BUBR1-MAD2 as well as CDC20-MAD2 interactions, causing premature activation of the anaphase-promoting complex/cyclosome. The effect of MPS1 inhibition is likely due to reduction of closed MAD2 (C-MAD2), as expressing a MAD2 mutant (MAD2^L13A) that is locked in the C conformation rescued the checkpoint defects. In the presence of reversine, exogenous C-MAD2 does not localize to unattached kinetochores but is still incorporated into the MCC. Contrary to a previous report, we found that sustained MPS1 activity is required for maintaining both the MAD1·C-MAD2 complex and open MAD2 (O-MAD2) at unattached kinetochores to facilitate C-MAD2 production. Additionally, mitotic phosphorylation of BUBR1 is also affected by MPS1 inhibition but seems dispensable for MCC assembly. Our results support the notion that MPS1 kinase promotes C-MAD2 production and subsequent MCC assembly to activate the SAC.     

Phosphorylation of Xenopus p31comet potentiates mitotic checkpoint exit

p31^comet plays an important role in spindle assembly checkpoint (SAC) silencing. However, how p31^comet''s activity is regulated remains unclear. Here we show that the timing of M-phase exit in Xenopus egg extracts (XEEs) depends upon SAC activity, even under conditions that are permissive for spindle assembly. p31^comet antagonizes the SAC, promoting XEE progression into anaphase after spindles are fully formed. We further show that mitotic p31^comet phosphorylation by Inhibitor of nuclear factor κ-B kinase-β (IKK-β) enhances this role in SAC silencing. Together, our findings implicate IKK-β in the control of anaphase timing in XEE through p31^comet activation and SAC downregulation.     

p31comet-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31^comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31^comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31^comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31^comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31^comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31^comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31^comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31^comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31^comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31^comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.     

Depletion of p31comet protein promotes sensitivity to antimitotic drugs

Antimitotic spindle poisons are among the most important chemotherapeutic agents available. However, precocious mitotic exit by mitotic slippage limits the cytotoxicity of spindle poisons. The MAD2-binding protein p31(comet) is implicated in silencing the spindle assembly checkpoint after all kinetochores are attached to spindles. In this study, we report that the levels of p31(comet) and MAD2 in different cell lines are closely linked with susceptibility to mitotic slippage. Down-regulation of p31(comet) increased the sensitivity of multiple cancer cell lines to spindle poisons, including nocodazole, vincristine, and Taxol. In the absence of p31(comet), lower concentrations of spindle poisons were required to induce mitotic block. The delay in checkpoint silencing was induced by an accumulation of mitotic checkpoint complexes. The increase in the duration of mitotic block after p31(comet) depletion resulted in a dramatic increase in mitotic cell death upon challenge with spindle poisons. Significantly, cells that are normally prone to mitotic slippage and resistant to spindle disruption-mediated mitotic death were also sensitized after p31(comet) depletion. These results highlight the importance of p31(comet) in checkpoint silencing and its potential as a target for antimitotic therapies.     

Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint

The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase in response to misaligned sister chromatids during mitosis. Upon checkpoint activation, Mad2 binds directly to Cdc20 and inhibits the anaphase-promoting complex or cyclosome (APC/C). Cdc20 binding triggers a dramatic conformational change of Mad2. Consistent with an earlier report, we show herein that depletion of p31(comet) (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31(comet) protein antagonizes the ability of Mad2 to inhibit APC/C(Cdc20) in vitro and in Xenopus egg extracts. Interestingly, p31(comet) binds selectively to the Cdc20-bound conformation of Mad2. Binding of p31(comet) to Mad2 does not prevent the interaction between Mad2 and Cdc20 in vitro. During checkpoint inactivation in HeLa cells, p31(comet) forms a transient complex with APC/C(Cdc20)-bound Mad2. Purified p31(comet) enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2-Cdc20 interaction. Therefore, our results suggest that p31(comet) counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint.     

Orderly inactivation of the key checkpoint protein mitotic arrest deficient 2 (MAD2) during mitotic progression

Anaphase is promoted by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) only when all the chromosomes have achieved bipolar attachment to the mitotic spindles. Unattached kinetochores or the absence of tension between the paired kinetochores activates a surveillance mechanism termed the spindle-assembly checkpoint. A fundamental principle of the checkpoint is the activation of mitotic arrest deficient 2 (MAD2). MAD2 then forms a diffusible complex called mitotic checkpoint complex (designated as MAD2(MCC)) before it is recruited to APC/C (designated as MAD2(APC/C)). Large gaps in our knowledge remain on how MAD2 is inactivated after the checkpoint is satisfied. In this study, we have investigated the regulation of MAD2-containing complexes during mitotic progression. Using selective immunoprecipitation of checkpoint components and gel filtration chromatography, we found that MAD2(MCC) and MAD2(APC/C) were regulated very differently during mitotic exit. Temporally, MAD2(MCC) was broken down ahead of MAD2(APC/C). The inactivation of the two complexes also displayed different requirements of proteolysis; although APC/C and proteasome activities were dispensable for MAD2(MCC) inactivation, they are required for MAD2(APC/C) inactivation. In fact, the degradation of CDC20 is inextricably linked to the breakdown of MAD2(APC/C). These data extended our understanding of the checkpoint complexes during checkpoint silencing.     

Phosphorylation Regulates the p31Comet-Mitotic Arrest-deficient 2 (Mad2) Interaction to Promote Spindle Assembly Checkpoint (SAC) Activity

The spindle assembly checkpoint (SAC) ensures the faithful segregation of the genome during mitosis by ensuring that sister chromosomes form bipolar attachments with microtubules of the mitotic spindle. p31^Comet is an antagonist of the SAC effector Mad2 and promotes silencing of the SAC and mitotic progression. However, p31^Comet interacts with Mad2 throughout the cell cycle. We show that p31^Comet binds Mad2 solely in an inhibitory manner. We demonstrate that attenuating the affinity of p31^Comet for Mad2 by phosphorylation promotes SAC activity in mitosis. Specifically, phosphorylation of Ser-102 weakens p31^Comet-Mad2 binding and enhances p31^Comet-mediated bypass of the SAC. Our results provide the first evidence for regulation of p31^Comet and demonstrate a previously unknown event controlling SAC activity.     

A Wee1 checkpoint inhibits anaphase onset

Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APC^Cdc20). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25⁺) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2A^Cdc55 triggers anaphase onset.     

Anaphase-promoting complex/cyclosome-Cdc-20 promotes Zwint-1 degradation

ZW10 interactor (Zwint-1) is an important component of the centromere and can recruit the dynamic protein kinase and dynein to promote chromosome movement and regulate the spindle assembly checkpoint (SAC). Zwint-1 activity is tightly regulated during the cell cycle. However, how the stability of Zwint-1 is regulated has not been clarified. Here, we show that the relative levels of Zwint-1 expression gradually decreased with the progression of cell cycling and decline sharply during mitotic exit. Treatment with cycloheximide reduced the levels of Zwint-1 while treatment with MG132 to inhibit endogenous ubiquitin-proteasome elevated the levels of Zwint-1 in HEK293T cells or Hela cells. Such data suggest that Zwint-1 may be degraded by endogenous ubiquitin-proteasome. Furthermore, induction of cell-division cycle protein 20 (Cdc20) overexpression decreased the levels of Zwint-1, which was abrogated by MG132 treatment. In contrast, Cdc20 silencing promoted the accumulation of Zwint-1. in vivo ubiquitination assay revealed that Cdc20 promoted the formation of Zwint-1 and ubiquitin-proteasome conjugates. Cotransfection with Cdc20 and wild-type Zwint-1, but not Zwint-1^ΔD-box, reduced the levels of Zwint-1. Immunoprecipitation and western blot analyses showed that Cdc20 interacted with wild-type Zwint-1, but not Zwint-1^ΔD-box although both Zwint-1 and Zwint-1^ΔD-box overexpression did not induce mitotic arrest. Collectively, our data indicated that Zwint-1 was ubiquitinated by anaphase-promoting complex/cyclosome (APC/C)-Cdc20 in a D-box-dependent manner. Therefore, the APC/C-Cdc20 controls the stability of Zwint-1, ensuring accurate regulation of the spindle assembly during the cell cycling in HEK293T cells.     

Homeostatic control of mitotic arrest

The spindle assembly checkpoint (SAC) restricts mitotic exit to cells that have completed chromosome-microtubule attachment. Cdc20 is a bifunctional protein. In complex with SAC proteins Mad2, BubR1, and Bub3, Cdc20 forms the mitotic checkpoint complex (MCC), which binds the anaphase-promoting complex (APC/C) and inhibits its mitotic exit-promoting activity. When devoid of SAC proteins, Cdc20 serves as an APC/C coactivator and promotes mitotic exit. During mitotic arrest, Cdc20 is continuously degraded via ubiquitin-dependent proteolysis and resynthesized. It is believed that this cycle keeps the levels of Cdc20 below a threshold above which Cdc20 would promote mitotic exit. We report that p31(comet), a checkpoint antagonist, is necessary for mitotic destabilization of Cdc20. p31(comet) depletion stabilizes the MCC, super-inhibits the APC/C, and delays mitotic exit, indicating that Cdc20 proteolysis in prometaphase opposes the checkpoint. Our studies reveal a homeostatic network in which checkpoint-sustaining and -repressing forces oppose each other during mitotic arrest and suggest ways for enhancing the sensitivity of cancer cells to antitubulin chemotherapeutics.     

Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events

We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.     

Cdk1 is essential for mammalian cyclosome/APC regulation

The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro. Studying cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint. We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1. Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint. Cdk1-cyclin B is thus an inhibitor, activator, and substrate of the cyclosome.     

Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31(comet)

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 p31(comet) delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31(comet) and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.     

APOLLON Protein Promotes Early Mitotic CYCLIN A Degradation Independent of the Spindle Assembly Checkpoint

In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC.