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1.
Eukaryotic cells rely on a surveillance mechanism, the "Spindle Assembly Checkpoint"SACM in order to ensure accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. In different organisms, a mitotic checkpoint complex (MCC) composed of Mad2, Bub3, BubR1/Mad3, and Cdc20 inhibits the anaphase promoting complex (APC/C) to initiate promotion into anaphase. The mechanism of MCC formation and its regulation by the kinetochore are unclear. Here, we constructed dynamical models of MCC formation involving different kinetochore control mechanisms including amplification as well as inhibition effects, and analysed their quantitative properties. In particular, in this system, fast and stable metaphase to anaphase transition can only be triggered when the kinetochore controls the Bub3:BubR1-related reactions; signal amplification and inhibition play a subordinate role. Furthermore, when introducing experimentally determined parameter values into the models analysed here, we found that effective MCC formation is not combined with complete Cdc20 sequestering. Instead, the MCC might bind and completely block the APC/C. The SACM might function by an MCC:APC/C complex rearrangement.  相似文献   

2.
Tang Z  Shu H  Oncel D  Chen S  Yu H 《Molecular cell》2004,16(3):387-397
To ensure the fidelity of chromosome segregation, the spindle checkpoint blocks the ubiquitin ligase activity of APC/C(Cdc20) in response to a single chromatid not properly attached to the mitotic spindle. Here we show that HeLa cells depleted for Bub1 by RNA interference are defective in checkpoint signaling. Bub1 directly phosphorylates Cdc20 in vitro and inhibits the ubiquitin ligase activity of APC/C(Cdc20) catalytically. A Cdc20 mutant with all six Bub1 phosphorylation sites removed is refractory to Bub1-mediated phosphorylation and inhibition in vitro. Upon checkpoint activation, Bub1 itself is hyperphosphorylated and its kinase activity toward Cdc20 is stimulated. Ectopic expression of the nonphosphorylatable Cdc20 mutant allows HeLa cells to escape from mitosis in the presence of spindle damage. Therefore, Bub1-mediated phosphorylation of Cdc20 is required for proper checkpoint signaling. We speculate that inhibition of APC/C(Cdc20) by Bub1 in a catalytic fashion may partly account for the exquisite sensitivity of the spindle checkpoint.  相似文献   

3.
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.  相似文献   

4.
Regulation of BubR1 is central to the control of APC/C activity. We have found that BubR1 forms a complex with PCAF and is acetylated at lysine 250. Using mass spectrometry and acetylated BubR1-specific antibodies, we have confirmed that BubR1 acetylation occurs at prometaphase. Importantly, BubR1 acetylation was required for checkpoint function, through the inhibition of ubiquitin-dependent BubR1 degradation. BubR1 degradation began before the onset of anaphase. It was noted that the pre-anaphase degradation was regulated by BubR1 acetylation. Degradation of an acetylation-mimetic form, BubR1–K250Q, was inhibited and chromosome segregation in cells expressing BubR1–K250Q was markedly delayed. By contrast, the acetylation-deficient mutant, BubR1–K250R, was unstable, and mitosis was accelerated in BubR1–K250R-expressing cells. Furthermore, we found that APC/C–Cdc20 was responsible for BubR1 degradation during mitosis. On the basis of our collective results, we propose that the acetylation status of BubR1 is a molecular switch that converts BubR1 from an inhibitor to a substrate of the APC/C complex, thus providing an efficient way to modulate APC/C activity and mitotic timing.  相似文献   

5.
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.  相似文献   

6.
Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053–32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C.  相似文献   

7.
The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.  相似文献   

8.
Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble 'degron' signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
The spindle checkpoint is a surveillance mechanism that regulates the metaphase-anaphase transition during somatic cell division through inhibition of the APC/C ensuring proper chromosome segregation. We show that the conserved spindle checkpoint protein BubR1 is required during early embryonic development. BubR1 is maternally provided and localises to kinetochores from prophase to metaphase during syncytial divisions similarly to somatic cells. To determine BubR1 function during embryogenesis, we generated a new hypomorphic semi-viable female sterile allele. Mutant females lay eggs containing undetectable levels of BubR1 show early developmental arrest, abnormal syncytial nuclear divisions, defects in chromosome congression, premature sister chromatids separation, irregular chromosome distribution and asynchronous divisions. Nuclei in BubR1 mutant embryos do not arrest in response to spindle damage suggesting that BubR1 performs a checkpoint function during syncytial divisions. Furthermore, we find that in wild-type embryos BubR1 localises to the kinetochores of condensed polar body chromosomes. This localisation is functional because in mutant embryos, polar body chromatin undergoes cycles of condensation-decondensation with additional rounds of DNA replication. Our results suggest that BubR1 is required for normal synchrony and progression of syncytial nuclei through mitosis and to maintain the mitotic arrest of the polar body chromosomes after completion of meiosis.  相似文献   

12.
The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.  相似文献   

13.
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.  相似文献   

14.
The mitotic checkpoint complex (MCC) ensures the fidelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins.  相似文献   

15.
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.  相似文献   

16.
Regulation of APC-Cdc20 by the spindle checkpoint   总被引:26,自引:0,他引:26  
The spindle checkpoint ensures the fidelity of chromosome segregation in mitosis and meiosis. In response to defects in the mitotic apparatus, it blocks the activity of the anaphase-promoting complex, a large ubiquitin ligase required for chromosome segregation. Recent studies indicate that the spindle checkpoint monitors both the attachment of chromosomes to the mitotic spindle and the tension across the sister chromatid generated by microtubules. Upon checkpoint activation, checkpoint protein complexes containing BubR1(Mad3), Bub3, Mad2 and Cdc20 directly bind to the anaphase-promoting complex and inhibit its ligase activity. Therefore, the checkpoint proteins form a complex intracellular signalling network to inhibit the anaphase-promoting complex.  相似文献   

17.
The mitotic checkpoint blocks the activation of the anaphase-promoting complex (APC) until all sister chromatids have achieved bipolar attachment to the spindle. A checkpoint complex containing BubR1 and Bub3 has been purified from mitotic human cells. Upon checkpoint activation, the BubR1-Bub3 complex interacts with Cdc20. In the absence of Mad2, BubR1 inhibits the activity of APC by blocking the binding of Cdc20 to APC. Surprisingly, the kinase activity of BubR1 is not required for the inhibition of APCCdc20. BubR1 also prevents the activation of APCCdc20 in Xenopus egg extracts, and restores the mitotic arrest in Cdc20-overexpressing cells treated with nocodazole. Because BubR1 also interacts with the mitotic motor CENP-E, the ability of BubR1 to inhibit APC may be regulated by kinetochore tension or occupancy.  相似文献   

18.
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.  相似文献   

19.
Equal partitioning of the duplicated chromosomes into two daughter cells during cell division is a coordinated process and is initiated only after completion of DNA synthesis. However, this strict order of execution breaks down in CDC6-deficient cells. Cdc6, an evolutionarily conserved protein, is required for the assembly of pre-replicative complexes (pre-RCs) and is essential for the initiation of DNA replication. Yeast cells lacking Cdc6 function, though unable to initiate DNA replication, proceed to undergo “reductional anaphase” by partitioning the unreplicated chromosomes and lose viability rapidly. This extreme form of genomic instability in cdc6 cells is thought to be due to inactivation of a pre-RC based, Cdc6-dependent checkpoint mechanism that, during normal cell cycle, inhibits premature onset of mitosis until pre-RC is assembled. Here, we show that chromosome segregation in cdc6 mutant is caused not by precocious initiation of mitosis in the absence of a checkpoint, but by the deregulation of spindle dynamics induced via a regulatory network involving the ubiquitin-conjugating enzyme Cdc34, microtubule-associated proteins (MAPs) and the anaphase-promoting complex (APC) activator Cdh1. This regulatory circuit governs spindle behavior in the early part of the division cycle and precipitates catastrophic chromosome segregation in the absence of DNA replication.  相似文献   

20.
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.  相似文献   

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