Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint

The spindle checkpoint delays anaphase until all chromosomes are properly attached to spindle microtubules. When the spindle checkpoint is activated at unattached kinetochores, the checkpoint proteins BubR1, Bub3 and Mad2 bind and inhibit Cdc20, an activator of the anaphase-promoting complex (APC). Here, we show that Xenopus laevis Cdc20 is phosphorylated at Ser 50, Thr 64, Thr 68 and Thr 79 during mitosis and that mitogen-activated protein kinase (MAPK) contributes to the phosphorylation at Thr 64 or Thr 68. Cdc20 mutants that are phosphorylation-deficient are able to activate the APC in X. laevis egg extracts. However, Cdc20 mutants in which any of the four phosphorylation sites were altered to Ala or Val failed to respond to the spindle checkpoint signal, owing to their reduced affinity for the spindle checkpoint proteins. This study demonstrates that the spindle checkpoint stops anaphase by inhibiting fully-phosphorylated Cdc20. Our results also have implications for the spindle checkpoint silencing mechanism.     

The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction

The spindle assembly checkpoint (SAC) is required to block sister chromatid separation until all chromosomes are properly attached to the mitotic apparatus. The SAC prevents cells from entering anaphase by inhibiting the ubiquitylation of cyclin B1 and securin by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The target of the SAC is the essential APC/C activator Cdc20. It is unclear how the SAC inactivates Cdc20 but most current models suggest that Cdc20 forms a stable complex with the Mad2 checkpoint protein. Here we show that most Cdc20 is not in a complex with Mad2; instead Mad2 is required for Cdc20 to form a complex with another checkpoint protein, BubR1. We further show that during the SAC, the APC/C ubiquitylates Cdc20 to target it for degradation. Thus, ubiquitylation of human Cdc20 is not required to release it from the checkpoint complex, but to degrade it to maintain mitotic arrest.     

Dual inhibition of Cdc20 by the spindle checkpoint

The metaphase-to-anaphase transition is triggered by the Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase that targets proteins for degradation, leading to sister chromatid separation and mitotic exit. The function of APC is controlled by the spindle checkpoint that delays anaphase onset in the presence of any chromosome that has not established bipolar attachment to the mitotic spindle. In this way, the checkpoint ensures accurate chromosome segregation. The spindle checkpoint is mostly activated from kinetochores that are not attached to microtubules or not under tension that is normally generated from bipolar attachment. These kinetochores recruit several spindle checkpoint proteins to assemble an inhibitory complex composed of checkpoint proteins Mad2, Bub3, and Mad3/BubR1. This complex binds and inhibits Cdc20, an activator and substrate adaptor for APC. In addition, the checkpoint complex promotes Cdc20 degradation, thus lowering Cdc20 protein level upon checkpoint activation. This dual inhibition on Cdc20 likely ensures that the spindle checkpoint is sustained even when the cell contains only a single unattached kinetochore.     

Molecular mechanisms of APC/C release from spindle assembly checkpoint inhibition by APC/C SUMOylation

1. Download : Download high-res image (146KB)
2. Download : Download full-size image

     

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.     

Phosphorylation of Cdc20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint

The cyclosome/anaphase promoting complex (APC) is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. It is activated at the end of mitosis by phosphorylation and association with the WD-40 protein Cdc20/Fizzy and is then kept active in the G1 phase by association with Cdh1/Hct1. The mitotic checkpoint system that keeps cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory action on the cyclosome. The activity of Cdh1 is negatively regulated by phosphorylation, while the abundance of Cdc20 is cell cycle regulated, with a peak in M-phase. Cdc20 is also phosphorylated in G2/M and in mitotically arrested cells, but the role of phosphorylation remained unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B abrogates its ability to activate cyclosome/APC from mitotic HeLa cells. A nonphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than does wild-type Cdc20. It is suggested that inhibitory phosphorylation of Cdc20/Fizzy may have a role in keeping the cyclosome inactive in early mitosis and under conditions of mitotic checkpoint arrest.     

APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation

The spindle assembly checkpoint (SAC) is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores. Cdc20, an activator of the Anaphase Promoting Complex/Cyclosome (APC/C), is known as a major downstream target for inhibition by the SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1. Here, we report that the SAC also negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation. Once the checkpoint is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2. We propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/C^Cdc20 in response to the SAC.     

Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.     

Phosphorylation and activation of Bub1 on unattached chromosomes facilitate the spindle checkpoint

The spindle checkpoint inhibits anaphase until all kinetochores have attached properly to spindle microtubules. The protein kinase Bub1 is an essential checkpoint component that resides at kinetochores during mitosis. It is shown herein that Xenopus Bub1 becomes hyperphosphorylated and the kinase is activated on unattached chromosomes. MAP kinase (MAPK) contributes to this phosphorylation, as inhibiting MAPK or altering MAPK consensus sites in Bub1 to alanine or valine (Bub1(5AV)) abolishes the phosphorylation and activation on chromosomes. Both Bub1 and Bub1(5AV) support the checkpoint under an optimal condition for spindle checkpoint activation. However, Bub1, but not Bub1(5AV), supports the checkpoint at a relatively low concentration of nuclei or the microtubule inhibitor nocodazole. Similar to Bub1(5AV), Bub1 without the kinase domain (Bub1(deltaKD)) is also partially compromised in its checkpoint function and in its ability to recruit other checkpoint proteins to kinetochores. This study suggests that activation of Bub1 at kinetochores enhances the efficiency of the spindle checkpoint and is probably important in maintaining the checkpoint toward late prometaphase when the cell contains only a few or a single unattached kinetochore.     

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

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.     

Bub1-mediated adaptation of the spindle checkpoint

During cell division, the spindle checkpoint ensures accurate chromosome segregation by monitoring the kinetochore-microtubule interaction and delaying the onset of anaphase until each pair of sister chromosomes is properly attached to microtubules. The spindle checkpoint is deactivated as chromosomes start moving toward the spindles in anaphase, but the mechanisms by which this deactivation and adaptation to prolonged mitotic arrest occur remain obscure. Our results strongly suggest that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for the degradation of Bub1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest.     

Mutual regulation between the spindle checkpoint and APC/C

Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The spindle checkpoint is a cellular surveillance system that ensures the fidelity of chromosome segregation. In response to sister chromatids not properly captured by spindle microtubules, the spindle checkpoint interferes with the functions of Cdc20, the mitotic activator of the anaphase-promoting complex or cyclosome (APC/C), thereby blocking APC/C-mediated degradation of securin and cyclin B to delay anaphase onset. This review summarizes the recent progress on the mechanisms by which checkpoint proteins inhibit APC/C, the conformational and enzymatic activation of checkpoint proteins, and the emerging roles of APC/C-dependent ubiquitination in checkpoint inactivation.     

Cdc20 proteolysis requires p38 MAPK signaling and Cdh1‐independent APC/C ubiquitination during spindle assembly checkpoint activation by cadmium

Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, initiates the destruction of key mitotic regulators to facilitate mitosis, while it is negatively regulated by the spindle assembly checkpoint (SAC) to prevent premature anaphase entry. Activation of the p38 mitogen‐activated protein kinase could contribute to mitotic arrest, but the underlying mechanism is unknown. Here we report a novel pathway in which the p38 signaling triggers Cdc20 destruction under SAC elicited by cadmium, a human carcinogen. We found that the cadmium‐induced prometaphase arrest was linked to decreased Cdc20 and accumulated cyclin A protein levels in human cells, whereas the activity of cyclin B1–Cdk1 was unaffected. The Cdc20 half‐life was markedly shortened along with its ubiquitination and degradation via 26S proteasome in cadmium‐treated asynchronous or G₂‐enriched cells. Depletion of APC3 markedly suppressed the cadmium‐induced Cdc20 ubiquitination and proteolysis, while depletion of Cdh1, another activator of APC/C, did not. Intriguingly, blockage of p38 activity restored the Cdc20 levels for continuing mitosis under cadmium, while inhibition of JNK activity had no effect. The cadmium‐induced Cdc20 proteolysis was also suppressed during transient depletion of p38α or stable expression a dominant negative form of p38. Inhibition of p38 abolished the induction of Mad2–Cdc20–APC3 complex by cadmium. Moreover, forced expression of MKK6–p38 signaling could promote Cdc20 degradation in a Cdh1‐independent APC/C pathway. In summary, accelerated ubiquitination and proteolysis of Cdc20 is essential for prometaphase arrest that is mediated via the p38 signaling during SAC activation. J. Cell. Physiol. 223: 327–334, 2010. © 2010 Wiley‐Liss, Inc.     

Bub1 maintains centromeric cohesion by activation of the spindle checkpoint

Bub1 is a component of the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures genome stability by delaying anaphase until all the chromosomes are stably attached to spindle microtubules via their kinetochores. To define Bub1's role in chromosome segregation, embryogenesis, and tissue homeostasis, we generated a mouse strain in which BUB1 can be inactivated by administration of tamoxifen, thereby bypassing the preimplantation lethality associated with the Bub1 null phenotype. We show that Bub1 is essential for postimplantation embryogenesis and proliferation of primary embryonic fibroblasts. Bub1 inactivation in adult males inhibits proliferation in seminiferous tubules, reducing sperm production and causing infertility. In culture, Bub1-deficient fibroblasts fail to align their chromosomes or sustain SAC function, yielding a highly aberrant mitosis that prevents further cell divisions. Centromeres in Bub1-deficient cells also separate prematurely; however, we show that this is a consequence of SAC dysfunction rather than a direct role for Bub1 in protecting centromeric cohesion.     

Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint

Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast. We have found that the spindle checkpoint kinase Bub1p becomes phosphorylated by Cdc2p during spindle damage in mitotic cells. Cdc2p directly phosphorylates Bub1p in vitro at the CDK consensus sites. A Bub1p mutant that cannot be phosphorylated by Cdc2p is checkpoint defective, indicating that Cdc2p-dependent Bub1p phosphorylation is required to activate the checkpoint after spindle damage. The kinase activity of Bub1p is required, but is not sufficient, for complete spindle checkpoint function. The role of Bub1p in maintaining centromeric localization of Rec8p during meiosis I is entirely dependent upon its kinase activity, suggesting that Bub1p kinase activity is essential for establishing proper kinetochore function. Finally, we show that there is a Bub1p-dependent meiotic checkpoint, which is activated in recombination mutants.     

KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome

The spindle checkpoint is a cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a protein serine-threonine kinase that plays multiple roles in chromosome segregation and the spindle checkpoint. In response to misaligned chromosomes, Bub1 directly inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator Cdc20. The protein level and the kinase activity of Bub1 are regulated during the cell cycle; they peak in mitosis and are low in G1/S phase. Here we show that Bub1 is degraded during mitotic exit and that degradation of Bub1 is mediated by APC/C in complex with its activator Cdh1 (APC/C(Cdh1)). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1, whereas depletion of Cdh1 by RNA interference increases the level of the endogenous Bub1 protein. Bub1 is ubiquitinated by immunopurified APC/C(Cdh1) in vitro. We further identify two KEN-box motifs on Bub1 that are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells but fails to elicit a cell cycle phenotype, indicating that degradation of Bub1 by APC/C(Cdh1) is not required for mitotic exit. Nevertheless, our study clearly demonstrates that Bub1, an APC/C inhibitor, is also an APC/C substrate. The antagonistic relationship between Bub1 and APC/C may help to prevent the premature accumulation of Bub1 during G1.     

Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint

The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.     

Bub1, a gatekeeper for Cdc20-dependent mitotic exit

The mitotic spindle assembly checkpoint arrests cells at metaphase by suppressing Cdc20, a protein required to trigger ubiquitination and consequent degradation of cyclin B. New evidence from Tang et al. appearing in the November 5th issue of Molecular Cell finds that one of the checkpoint proteins, Bub1, specifically phosphorylates Cdc20 to suppress APC/C activation.