Checkpoint Protein BubR1 Acts Synergistically with Mad2 to Inhibit Anaphase-promoting Complex

The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC.

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Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524551113 ◽

2016 ◽

Vol 113 (4) ◽

pp. 966-971 ◽

Cited By ~ 10

Author(s):

Sharon Kaisari ◽

Danielle Sitry-Shevah ◽

Shirly Miniowitz-Shemtov ◽

Avram Hershko

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Checkpoint Proteins ◽

Sister Chromatids ◽

Unknown Species ◽

Mitotic Checkpoint Complex

The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system.

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Driving chromosome segregation: lessons from the human and Drosophila centromere–kinetochore machinery

Biochemical Society Transactions ◽

10.1042/bst0381667 ◽

2010 ◽

Vol 38 (6) ◽

pp. 1667-1675 ◽

Cited By ~ 3

Author(s):

Bernardo Orr ◽

Olga Afonso ◽

Tália Feijão ◽

Claudio E. Sunkel

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Genomic Stability ◽

Mitotic Progression ◽

Chromosomal Locus ◽

Microtubule Attachment ◽

Sister Chromatids ◽

And Function ◽

Drosophila Cells

The kinetochore is a complex molecular machine that serves as the interface between sister chromatids and the mitotic spindle. The kinetochore assembles at a particular chromosomal locus, the centromere, which is essential to maintain genomic stability during cell division. The kinetochore is a macromolecular puzzle of subcomplexes assembled in a hierarchical manner and fulfils three main functions: microtubule attachment, chromosome and sister chromatid movement, and regulation of mitotic progression though the spindle assembly checkpoint. In the present paper we compare recent results on the assembly, organization and function of the kinetochore in human and Drosophila cells and conclude that, although essential functions are highly conserved, there are important differences that might help define what is a minimal chromosome segregation machinery.

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Merotelic kinetochores in mammalian tissue cells

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2004.1610 ◽

2005 ◽

Vol 360 (1455) ◽

pp. 553-568 ◽

Cited By ~ 77

Author(s):

E.D Salmon ◽

D Cimini ◽

L.A Cameron ◽

J.G DeLuca

Keyword(s):

Mitotic Spindle ◽

Binding Sites ◽

Metaphase Plate ◽

Mammalian Tissue ◽

Anaphase Onset ◽

Sister Chromatids ◽

Pulling Forces ◽

Tissue Cells ◽

Early Mitosis

Merotelic kinetochore attachment is a major source of aneuploidy in mammalian tissue cells in culture. Mammalian kinetochores typically have binding sites for about 20–25 kinetochore microtubules. In prometaphase, kinetochores become merotelic if they attach to microtubules from opposite poles rather than to just one pole as normally occurs. Merotelic attachments support chromosome bi-orientation and alignment near the metaphase plate and they are not detected by the mitotic spindle checkpoint. At anaphase onset, sister chromatids separate, but a chromatid with a merotelic kinetochore may not be segregated correctly, and may lag near the spindle equator because of pulling forces toward opposite poles, or move in the direction of the wrong pole. Correction mechanisms are important for preventing segregation errors. There are probably more than 100 times as many PtK1 tissue cells with merotelic kinetochores in early mitosis, and about 16 times as many entering anaphase as the 1% of cells with lagging chromosomes seen in late anaphase. The role of spindle mechanics and potential functions of the Ndc80/Nuf2 protein complex at the kinetochore/microtubule interface is discussed for two correction mechanisms: one that functions before anaphase to reduce the number of kinetochore microtubules to the wrong pole, and one that functions after anaphase onset to move merotelic kinetochores based on the ratio of kinetochore microtubules to the correct versus incorrect pole.

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Role of Polo-like kinase 1 in the regulation of the action of p31comet in the disassembly of mitotic checkpoint complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902970116 ◽

2019 ◽

pp. 201902970 ◽

Cited By ~ 4

Author(s):

Sharon Kaisari ◽

Pnina Shomer ◽

Tamar Ziv ◽

Danielle Sitry-Shevah ◽

Shirly Miniowitz-Shemtov ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Hela Cells ◽

Mitotic Spindle ◽

Joint Action ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Futile Cycle ◽

Mitotic Checkpoint Complex ◽

Bi 2536

The Mad2-binding protein p31comet has important roles in the inactivation of the mitotic checkpoint system, which delays anaphase until chromosomes attach correctly to the mitotic spindle. The activation of the checkpoint promotes the assembly of a Mitotic Checkpoint Complex (MCC), which inhibits the action of the ubiquitin ligase APC/C (Anaphase-Promoting Complex/Cyclosome) to degrade inhibitors of anaphase initiation. The inactivation of the mitotic checkpoint requires the disassembly of MCC. p31comet promotes the disassembly of mitotic checkpoint complexes by liberating their Mad2 component in a joint action with the ATPase TRIP13. Here, we investigated the regulation of p31comet action. The release of Mad2 from checkpoint complexes in extracts from nocodazole-arrested HeLa cells was inhibited by Polo-like kinase 1 (Plk1), as suggested by the effects of selective inhibitors of Plk1. Purified Plk1 bound to p31comet and phosphorylated it, resulting in the suppression of its activity (with TRIP13) to disassemble checkpoint complexes. Plk1 phosphorylated p31comet on S102, as suggested by the prevention of the phosphorylation of this residue in checkpoint extracts by the selective Plk1 inhibitor BI-2536 and by the phosphorylation of S102 with purified Plk1. An S102A mutant of p31comet had a greatly decreased sensitivity to inhibition by Plk1 of its action to disassemble mitotic checkpoint complexes. We propose that the phosphorylation of p31comet by Plk1 prevents a futile cycle of MCC assembly and disassembly during the active mitotic checkpoint.

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Uncoupling Anaphase-Promoting Complex/Cyclosome Activity from Spindle Assembly Checkpoint Control by Deregulating Polo-Like Kinase 1

Molecular and Cellular Biology ◽

10.1128/mcb.25.5.2031-2044.2005 ◽

2005 ◽

Vol 25 (5) ◽

pp. 2031-2044 ◽

Cited By ~ 41

Author(s):

Barbara C. M. van de Weerdt ◽

Marcel A. T. M. van Vugt ◽

Catherine Lindon ◽

Jos J. W. Kauw ◽

Marieke J. Rozendaal ◽

...

Keyword(s):

Double Mutant ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Catastrophe ◽

Anaphase Promoting Complex ◽

Checkpoint Control ◽

Mitotic Progression ◽

Specific Antibodies ◽

Mitotic Entry

ABSTRACT Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.

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

The Journal of Cell Biology ◽

10.1083/jcb.200801049 ◽

2008 ◽

Vol 183 (2) ◽

pp. 267-277 ◽

Cited By ~ 42

Author(s):

Evan C. Osmundson ◽

Dipankar Ray ◽

Finola E. Moore ◽

Qingshen Gao ◽

Gerald H. Thomsen ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Spindle Assembly Checkpoint ◽

Spindle Checkpoint ◽

E3 Ligase ◽

Spindle Assembly ◽

Cyclin B ◽

Carboxyl Terminus ◽

Anaphase Promoting Complex ◽

Mitotic Spindles ◽

Anaphase Onset

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

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Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase

The Journal of Cell Biology ◽

10.1083/jcb.200411056 ◽

2005 ◽

Vol 169 (1) ◽

pp. 61-71 ◽

Cited By ~ 14

Author(s):

Jessica B. Casaletto ◽

Leta K. Nutt ◽

Qiju Wu ◽

Jonathan D. Moore ◽

Laurence D. Etkin ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Spindle Assembly Checkpoint ◽

Cyclin B1 ◽

Specific Protein ◽

Anaphase Promoting Complex ◽

Protein Substrates ◽

Ubiquitin Ligase Activity ◽

Egg Extracts ◽

Core Components ◽

Cytostatic Factor

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

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The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

Molecular and Cellular Biology ◽

10.1128/mcb.01867-08 ◽

2009 ◽

Vol 29 (14) ◽

pp. 3975-3990 ◽

Cited By ~ 98

Author(s):

Laura O'Regan ◽

Andrew M. Fry

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Mitotic Spindles ◽

Mitotic Progression ◽

Serine Threonine Kinase ◽

Spindle Formation ◽

Spindle Poles ◽

And Function ◽

Interfering Rna ◽

Reduced Activity

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

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EFFECTORS OF THE SPINDLE ASSEMBLY CHECKPOINT BUT NOT THE MITOTIC EXIT NETWORK ARE CONFINED WITHIN THE NUCLEUS OF SACCHAROMYCES CEREVISIAE

10.1101/340091 ◽

2018 ◽

Author(s):

Lydia R Heasley ◽

Jennifer G DeLuca ◽

Steven M Markus

Keyword(s):

Nuclear Envelope ◽

Mammalian Cells ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Mitotic Exit ◽

Cell Cycle Checkpoints ◽

Anaphase Promoting Complex ◽

Mitotic Exit Network ◽

And Function

The Spindle Assembly Checkpoint (SAC) prevents erroneous chromosome segregation by delaying mitotic progression when chromosomes are incorrectly attached to the mitotic spindle. This delay is mediated by Mitotic Checkpoint Complexes (MCCs), which assemble at unattached kinetochores and repress the activity of the Anaphase Promoting Complex/Cyclosome (APC/C). The cellular localizations of MCCs are likely critical for proper SAC function, yet remain poorly defined. We recently demonstrated that in mammalian cells, in which the nuclear envelope disassembles during mitosis, MCCs diffuse throughout the spindle region and cytoplasm. Here, we employed binucleate yeast zygotes to examine the localization dynamics of SAC effectors required for MCC assembly and function in budding yeast, in which the nuclear envelope remains intact throughout mitosis. Our findings indicate that in yeast MCCs are confined to the nuclear compartment and excluded from the cytoplasm during mitosis. In contrast, we find that effectors of the Mitotic Exit Network (MEN) - a pathway that initiates disassembly of the anaphase spindle only when it is properly oriented - are in fact freely exchanged between multiple nuclei within a shared cytoplasm. Our study provides insight into how cell cycle checkpoints have evolved to function in diverse cellular contexts.

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The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells

Open Biology ◽

10.1098/rsob.150160 ◽

2015 ◽

Vol 5 (11) ◽

pp. 150160 ◽

Cited By ~ 42

Author(s):

Gina V. Caldas ◽

Tina R. Lynch ◽

Ryan Anderson ◽

Sana Afreen ◽

Dileep Varma ◽

...

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Protein S ◽

Human Cells ◽

Anaphase Promoting Complex ◽

Anaphase Onset ◽

N Terminus ◽

Independent Mechanism ◽

Direct Linkage ◽

Surveillance Mechanism

The spindle assembly checkpoint is a surveillance mechanism that blocks anaphase onset until all chromosomes are properly attached to microtubules of the mitotic spindle. Checkpoint activity requires kinetochore localization of Mad1/Mad2 to inhibit activation of the anaphase promoting complex/cyclosome in the presence of unattached kinetochores. In budding yeast and Caenorhabditis elegans , Bub1, recruited to kinetochores through KNL1, recruits Mad1/Mad2 by direct linkage with Mad1. However, in human cells it is not yet established which kinetochore protein(s) function as the Mad1/Mad2 receptor. Both Bub1 and the RZZ complex have been implicated in Mad1/Mad2 kinetochore recruitment; however, their specific roles remain unclear. Here, we investigate the contributions of Bub1, RZZ and KNL1 to Mad1/Mad2 kinetochore recruitment. We find that the RZZ complex localizes to the N-terminus of KNL1, downstream of Bub1, to mediate robust Mad1/Mad2 kinetochore localization. Our data also point to the existence of a KNL1-, Bub1-independent mechanism for RZZ and Mad1/Mad2 kinetochore recruitment. Based on our results, we propose that in humans, the primary mediator for Mad1/Mad2 kinetochore localization is the RZZ complex.

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