Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1

The mitotic spindle assembly checkpoint delays anaphase until all chromosomes achieve bipolar attachment to the spindle microtubules. The spindle assembly checkpoint protein BubR1 is thought to act by forming an inhibitory complex with Cdc20. We here identify two Cdc20 binding sites on BubR1. A strong Cdc20 binding site is located between residues 490 and 560, but mutations that disrupt Cdc20 binding to this region have no effect upon checkpoint function. A second Cdc20 binding site present between residues 1 and 477 is highly specific for Cdc20 already bound to Mad2. Mutation of a conserved lysine in this region weakened Cdc20 binding and correspondingly reduced checkpoint function. Our results indicate that there may be more than one checkpoint complex containing BubR1, Mad2, and Cdc20. They also lead us to propose that in vivo checkpoint inhibition of Cdc20 is a two-step process in which prior binding of Mad2 to Cdc20 is required to make Cdc20 sensitive to inhibition by BubR1. Thus, Mad2 and BubR1 must cooperate to inhibit Cdc20 activity.     

Bub1 and the multilayered inhibition of Cdc20-APC/C in mitosis

To ensure the accuracy of chromosome segregation in mitosis, the spindle checkpoint blocks the activity of the anaphase-promoting complex APC/C until all chromosomes are properly bi-orientated on the metaphase spindle. How the checkpoint machinery actually inhibits the APC/C is still unclear. A new paper by Tang and coworkers helps further our understanding of this complex and fundamental process.     

Yeast Dam1p Is Required to Maintain Spindle Integrity during Mitosis and Interacts with the Mps1p Kinase

We have identified a mutant allele of the DAM1 gene in a screen for mutations that are lethal in combination with the mps1-1 mutation. MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint. Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel. The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p. DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex. We report here that cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature. Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function. We have observed that a Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis.     

Kinase activity of fission yeast Mph1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6-8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.     

Identification of an Overlapping Binding Domain on Cdc20 for Mad2 and Anaphase-Promoting Complex: Model for Spindle Checkpoint Regulation

Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis in mammalian cells. Cdc20, which specifically recognizes APC substrates involved in the metaphase-to-anaphase transition, plays a pivotal role in APC activation through direct interaction with the APC. The activation of the APC by Cdc20 is prevented by the interaction of Cdc20 with Mad2 when the spindle checkpoint is activated. Using deletion mutagenesis and peptide mapping, we have identified the sequences in Cdc20 that target it to Mad2 and the APC, respectively. These sequences are distinct but overlapping, providing a possible structural explanation for the internal modulation of the APC-Cdc20 complex by Mad2. In the course of these studies, a truncation mutant of Cdc20 (1-153) that constitutively binds Mad2 but fails to bind the APC was identified. Overexpression of this mutant induces the formation of multinucleated cells and increases their susceptibility to undergoing apoptosis when treated with microtubule-inhibiting drugs. Our experiments demonstrate that disruption of the Mad2-Cdc20 interaction perturbs the mitotic checkpoint, leading to premature activation of the APC, sensitizing the cells to the cytotoxic effects of microtubule-inhibiting drugs.     

The Bub1-TPR Domain Interacts Directly with Mad3 to Generate Robust Spindle Checkpoint Arrest

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Degradation of the Human Mitotic Checkpoint Kinase Mps1 Is Cell Cycle-regulated by APC-cCdc20 and APC-cCdh1 Ubiquitin Ligases

Mps1 is a dual specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into the G₁ phase. Although evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the anaphase-promoting complex (APC)-c^Cdc20 complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G₁ phase. First, we showed that treatment of HEK 293T cells with the proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell cycle-dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G₁ phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G₁ phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC-c^Cdc20 and APC-c^Cdh1 ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.     

A Yeast GSK-3 Kinase Mck1 Promotes Cdc6 Degradation to Inhibit DNA Re-Replication

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCF^CDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.     

Mad3/BubR1 Phosphorylation during Spindle Checkpoint Activation Depends on both Polo and Aurora Kinases in Budding Yeast

During mitosis the spindle assembly checkpoint (SAC) delays the onset of anaphase and mitotic exit until all chromosomes are bipolarly attached to spindle fibers. Both lack of attachment due to spindle/kinetochore defects and lack of tension across kinetochores generate the “wait anaphase” signal transmitted by the SAC, which involves the evolutionarily conserved Mad1, Mad2, Mad3/BubR1, Bub1, Bub3 and Mps1 proteins, and inhibits the activity of the ubiquitin ligase Cdc20/APC, that promotes both sister chromatid dissociation in anaphase and mitotic exit. In particular, Mad3/BubR1 is directly implicated, together with Mad2, in Cdc20 inactivation in both human and yeast cells, suggesting that its activity is likely finely regulated. We show that budding yeast Mad3, like its human orthologue BubR1, is a phosphoprotein that is hyperphosphorylated during mitosis and when SAC activation is triggered by microtubule depolymerizing agents, kinetochore defects or lack of kinetochore tension. In vivo Mad3 phosphorylation depends on the Polo kinase Cdc5 and, to a minor extent, the Aurora B kinase Ipl1. Accordingly, replacing with alanines five serine residues belonging to Polo kinase-dependent putative phosphorylation sites dramatically reduces Mad3 phosphorylation, suggesting that Mad3 is likely an in vivo target of Cdc5.     

The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C)

Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.     

Kinetochore localized Mad2 and Cdc20 is itself insufficient for triggering the mitotic checkpoint when Mps1 is low in Drosophila melanogaster neuroblasts

The relationships between the kinetochore and checkpoint control remain unresolved. Here, we report the characterization of the in vivo behavior of Cdc20 and Mad2 and the relevant spindle assembly checkpoint (SAC) functions in the neuroblasts of a Drosophila Mps1 weak allele (ald^B4–2). ald^B4–2 third instar larvae brain samples contain only around 16% endogenous Mps1 protein, and the SAC function is abolished. However, this does not lead to rapid anaphase onset and mitotic exit, in contrast to the loss of Mad2 alone in a mad2^EY mutant. The level of GFP-Cdc20 recruitment to the kinetochore is unaffected in ald^B4–2 neuroblasts, while the level of GFP-Mad2 is reduced to just about 20%. Cdc20 and Mad2 display only monophasic exponential kinetics at the kinetochores. The ald^B4–2 heterozygotes expressed approximately 65% of normal Mps1 protein levels, and this is enough to restore the SAC function. The kinetochore recruitment of GFP-Mad2 in response to SAC activation increases by around 80% in heterozygotes, compared with just about 20% in ald^B4–2 mutant. This suggests a correlation between Mps1 levels and Mad2 kinetochore localization and perhaps the existence of a threshold level at which Mps1 is fully functional. The failure to arrest the mitotic progression in ald^B4–2 neuroblasts in response to colchicine treatment suggests that when Mps1 levels are low, approximately 20% of normal GFP-Mad2, alongside normal levels of GFP-Cdc20 kinetochore recruitments, is insufficient for triggering SAC signal propagation.     

Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint.

Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis. The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos. Here we show that Mad2 forms incompatible complexes with Mad1 and Cdc20, neither of which requires Mad2 oligomerization. A monomeric point mutant of Mad2 can sustain a cell cycle arrest of comparable strength to that of the wild-type protein. We show that the interaction of Mad2 with Mad1 is crucial for the localization of Mad2 to kinetochores, where Mad2 interacts with Cdc20. We propose a model that features the kinetochore as a 'folding factory' for the formation of a Mad2-Cdc20 complex endowed with inhibitory activity on the anaphase promoting complex.     

A Cul3-Based E3 Ligase Regulates Mitosis and is Required to Maintain the Spindle Assembly Checkpoint in Human Cells

The spindle assembly checkpoint (SAC) is a mechanism that prevents premature chromosome segregation in anaphase before all chromosomes are correctly attached to the mitotic spindle. Errors in chromosome segregation lead to aneuploidy, which may be causally involved in tumorgenesis. Kinetochore complexes are the structural components of the SAC, which are tightly regulated by various mechanisms including phosphorylation and ubiquitin-dependent proteolysis. Recent studies shed new light on the regulatory pathways of the ubiquitin proteasome system involved in SAC signaling. Here we present evidence that a Cul3-based E3 ubiquitin-ligase is required to maintain SAC signaling in human cells. Inactivation of the Cul3/KLHL9/KLHL13 ligase leads to premature degradation of Cyclin B and exit from the mitotic state in the presence of microtubule poisons. We discuss possible mechanisms how Cul3 may be required to maintain SAC activity by ubiquitination of the chromosomal passenger protein Aurora B.     

An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast

For ordered mitotic progression, various proteins have to be regulated by an ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) with appropriate timing. Recent studies have implied that the activity of APC/C also contributes to release of mitotic checkpoint complexes (MCCs) from its target Cdc20 in the process of silencing the spindle assembly checkpoint (SAC). Here we describe a temperature-sensitive mutant (ubc11-P93L) in which cell cycle progression is arrested at mitosis. The mutant grows normally at the restrictive temperature when SAC is inactivated, suggesting that the arrest is not due to abnormal spindle assembly, but rather due to prolonged activation of SAC. Supporting this notion, MCCs remain bound to APC/C even when SAC is satisfied. The ubc11⁺ gene encodes one of the two E2 enzymes required for progression through mitosis in fission yeast. Remarkably, Slp1 (a fission yeast homolog of Cdc20), which is degraded in an APC/C-dependent manner, stays stable throughout the cell cycle in the ubc11-P93L mutant lacking the functional SAC. Other APC/C substrates, in contrast, were degraded on schedule. We have also found that a loss of Ubc4, the other E2 required for progression through mitosis, does not affect the stability of Slp1. We propose that each of the two E2 enzymes is responsible for collaborating with APC/C for a specific set of substrates, and that Ubc11 is responsible for regulating Slp1 with APC/C for silencing the SAC.     

Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, and Mph1p-dependent manner

The spindle checkpoint delays the metaphase-to-anaphase transition in response to spindle and kinetochore defects. Genetic screens in budding yeast identified the Mad and Bub proteins as key components of this conserved regulatory pathway. Here we present the fission yeast homologue of Mad3p. Cells devoid of mad3(+) are unable to arrest their cell cycle in the presence of microtubule defects. Mad3p coimmunoprecipitates Bub3p, Mad2p, and the spindle checkpoint effector Slp1/Cdc20p. We demonstrate that Mad3p function is required for the overexpression of Mad2p to result in a metaphase arrest. Mad1p, Bub1p, and Bub3p are not required for this arrest. Thus, Mad3p appears to have a crucial role in transducing the inhibitory "wait anaphase" signal to the anaphase-promoting complex (APC). Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. For the first time, we have systematically studied the dependency of Mad3/BubR1 protein recruitment to kinetochores. We find Mad3-GFP kinetochore localization to be dependent upon Bub1p, Bub3p, and the Mph1p kinase, but not upon Mad1p or Mad2p. We discuss the implications of these findings in the context of our current understanding of spindle checkpoint function.     

Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6- and E7-Expressing Keratinocytes

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G₂/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G₂ damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.Failure to activate cell cycle checkpoints in the presence of any DNA damage leads to genomic instability, polyploidy, and subsequently, aneuploidy, which is a hallmark of many cancers (26). Human papillomaviruses (HPVs) which cause various epithelial cancers, produce two proteins, E6 and E7, whose expression allows bypass or overriding of normal DNA damage and spindle checkpoint signals, primarily through inactivation of p53 and retinoblastoma family members, respectively (11, 16, 17). Our laboratory and others have previously shown that bypass of these arrest signals due to the presence of the viral genes gives rise to a significant population of cells that are polyploid (13, 16, 24, 32). Polyploid and aneuploid cells predominantly arise due to defects in the spindle assembly checkpoint (SAC) during mitosis. While we have some understanding of the mechanisms that lead to bypass of DNA damage arrest signals at the G₂/M stage of the cell cycle, it is not clear how the E6/E7-expressing cells with DNA damage and abnormal chromosomes are allowed to (i) to enter into mitosis and (ii) exit out of mitosis to initiate the next round of replication. Progression through mitosis is regulated by the ubiquitin-dependent degradation machinery, consisting of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit ubiquitin ligase. The activity of APC/C is dependent on the substrate-specifying proteins Cdc20 in metaphase and Cdh1 in telophase (25, 37). In normal cells, spindle checkpoint proteins Mad2 and BubR1 serve to inhibit APC/C until all the chromosomes are aligned correctly on the mitotic spindle by binding Cdc20 and preventing it from activating APC/C (5, 21, 31). In the event of DNA damage and/or unattached kinetochores, the SAC will arrest cells before exit from mitosis by inhibiting activation of APC/C. As a consequence of APC/C inhibition, cyclin B is not degraded, thus preventing cells from mitotic exit (6). Work by Chen''s group (11) has shown that E6- and E7-expressing cells (also referred to here as E6/E7 cells) adapt to an active SAC and are capable of mitotic slippage. So, what is the mechanism that underlies mitotic slippage in E6/E7 cells and allows them to enter the next round of cell cycle? Recent work by van Ree et al. (34) has shown that overexpression of E2 ubiquitin-conjugating enzyme Ubch10 leads to uncontrolled APC/C activity and degradation of cyclin B even in the presence of an active mitotic checkpoint, leading to mitotic slippage. In this report, we show that primary human foreskin keratinocytes (HFKs) expressing E6/E7 have high levels of cyclin B, which allows entry into mitosis in the presence of DNA damage. We show that these cells successfully exit mitosis by, in part, indirect activation of APC/C through upregulation of the E2-conjugating protein, Ubch10, and the substrate-specific component of APC/C, Cdc20, leading to the required degradation of cyclin B. In addition, Cdc20 is detected in different complexes; one includes the protein BubR1, indicating an active checkpoint, while other complexes are free of BubR1 and are thus free to activate APC/C. Upregulation of cyclin B and Ubch10 as well as Cdc20 is primarily through E6 and its ability to target p53 degradation, although inhibition of the pRb family members by E7 may also play a part.     

Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling

The spindle assembly checkpoint (SAC) in mammals uses cytosolic and kinetochore-based signaling pathways to inhibit anaphase. In this study, we use chemical genetics to show that the protein kinase Mps1 regulates both aspects of the SAC. Human MPS1-null cells were generated via gene targeting and reconstituted with either the wild-type kinase (Mps1^wt) or a mutant version (Mps1^as) sensitized to bulky purine analogues. Mps1 inhibition sharply accelerated anaphase onset, such that cells completed mitosis in 12 min, and prevented Cdc20’s association with either Mad2 or BubR1 during interphase, i.e., before the appearance of functional kinetochores. Furthermore, intramitotic Mps1 inhibition evicted Bub1 and all other known SAC transducers from the outer kinetochore, but contrary to a recent study, did not perturb aurora B–dependent phosphorylation. We conclude that Mps1 has two complementary roles in SAC regulation: (1) initial cytoplasmic activation of Cdc20 inhibitors and (2) recruitment of factors that promote sustained anaphase inhibition and chromosome biorientation to unattached kinetochores.