scholarly journals Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer

2021 ◽  
Author(s):  
Owen J. Chen ◽  
Ester Castellsagué ◽  
Mohamed Moustafa-Kamal ◽  
Javad Nadaf ◽  
Barbara Rivera ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Familial Cancer ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Mitotic Slippage ◽  
Oncogenic Mutations ◽  
Whole Exome

SUMMARYCDC20 is a co-activator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with cancer in two families. Characterization of these mutants showed they retain APC/C activation activity but show reduced binding to BUBR1, a component of the SAC. Expression of L151R and N331K promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. CRISPR/Cas9 was used to generate mice carrying N331K. Homozygous mice carrying N331K were non-viable, however, heterozygotes displayed accelerated oncogenicity in Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor promoting genes in humans.

scholarly journals The ubiquitin ligase CRL2ZYG11 targets cyclin B1 for degradation in a conserved pathway that facilitates mitotic slippage

2016 ◽  
Vol 215 (2) ◽  
pp. 151-166 ◽  
Author(s):  
Riju S. Balachandran ◽  
Cassandra S. Heighington ◽  
Natalia G. Starostina ◽  
James W. Anderson ◽  
David L. Owen ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Cyclin B1 ◽  
Human Cells ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Animal Cells ◽  
C Elegans ◽  
Chemotherapy Drugs ◽  
Mitotic Slippage

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is known to target the degradation of cyclin B1, which is crucial for mitotic progression in animal cells. In this study, we show that the ubiquitin ligase CRL2ZYG-11 redundantly targets the degradation of cyclin B1 in Caenorhabditis elegans and human cells. In C. elegans, both CRL2ZYG-11 and APC/C are required for proper progression through meiotic divisions. In human cells, inactivation of CRL2ZYG11A/B has minimal effects on mitotic progression when APC/C is active. However, when APC/C is inactivated or cyclin B1 is overexpressed, CRL2ZYG11A/B-mediated degradation of cyclin B1 is required for normal progression through metaphase. Mitotic cells arrested by the spindle assembly checkpoint, which inactivates APC/C, often exit mitosis in a process termed “mitotic slippage,” which generates tetraploid cells and limits the effectiveness of antimitotic chemotherapy drugs. We show that ZYG11A/B subunit knockdown, or broad cullin–RING ubiquitin ligase inactivation with the small molecule MLN4924, inhibits mitotic slippage in human cells, suggesting the potential for antimitotic combination therapy.

The mitosis-regulating and protein-protein interaction activities of Astrin are controlled by Aurora-A-induced phosphorylation

2014 ◽  
Vol 307 (5) ◽  
pp. C466-C478 ◽  
Author(s):  
Shao-Chih Chiu ◽  
Jo-Mei Maureen Chen ◽  
Tong-You Wade Wei ◽  
Tai-Shan Cheng ◽  
Ya-Hui Candice Wang ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Spindle Assembly Checkpoint ◽  
Morphological Changes ◽  
Spindle Assembly ◽  
Aurora A ◽  
Protein Protein Interaction ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Complicated Process ◽  
Spindle Stability

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser115. The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

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

2005 ◽  
Vol 25 (5) ◽  
pp. 2031-2044 ◽  
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.

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

2016 ◽  
Vol 113 (4) ◽  
pp. 966-971 ◽  
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.

scholarly journals Slip slidin’ away of mitosis with CRL2Zyg11

2016 ◽  
Vol 215 (2) ◽  
pp. 143-145 ◽  
Author(s):  
Michael Brandeis
Keyword(s):  
Spindle Assembly Checkpoint ◽  
E3 Ligase ◽  
Spindle Assembly ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
Cell Biol ◽  
Mitotic Slippage ◽  
Mitotic Cells

The spindle assembly checkpoint arrests mitotic cells by preventing degradation of cyclin B1 by the anaphase-promoting complex/cyclosome, but some cells evade this checkpoint and slip out of mitosis. Balachandran et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601083) show that the E3 ligase CRL2ZYG11 degrades cyclin B1, allowing mitotic slippage.

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

2002 ◽  
Vol 13 (3) ◽  
pp. 755-766 ◽  
Author(s):  
Guowei Fang
Keyword(s):  
Ubiquitin Ligase ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Metaphase Plate ◽  
Anaphase Promoting Complex ◽  
Checkpoint Control ◽  
Sister Chromatids ◽  
Direct Binding ◽  
And Function

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.

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

2010 ◽  
Vol 38 (6) ◽  
pp. 1667-1675 ◽  
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.

scholarly journals Characterization of Vertebrate Cohesin Complexes and Their Regulation in Prophase

2000 ◽  
Vol 151 (4) ◽  
pp. 749-762 ◽  
Author(s):  
Izabela Sumara ◽  
Elisabeth Vorlaufer ◽  
Christian Gieffers ◽  
Beate H. Peters ◽  
Jan-Michael Peters
Keyword(s):  
Budding Yeast ◽  
Human Cells ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
A Subunit ◽  
Mitotic Regulation

In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

scholarly journals p31comet acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment

2011 ◽  
Vol 22 (22) ◽  
pp. 4236-4246 ◽  
Author(s):  
Robert S. Hagan ◽  
Michael S. Manak ◽  
Håkon Kirkeby Buch ◽  
Michelle G. Meier ◽  
Patrick Meraldi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Mitotic Exit ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Microtubule Attachment ◽  
And Function ◽  
High Cyclin

The spindle assembly checkpoint links the onset of anaphase to completion of chromosome-microtubule attachment and is mediated by the binding of Mad and Bub proteins to kinetochores of unattached or maloriented chromosomes. Mad2 and BubR1 traffic between kinetochores and the cytosol, thereby transmitting a “wait anaphase” signal to the anaphase-promoting complex. It is generally assumed that this signal dissipates automatically upon kinetochore-microtubule binding, but it has been shown that under conditions of nocodazole-induced arrest p31comet, a Mad2-binding protein, is required for mitotic progression. In this article we investigate the localization and function of p31comet during normal, unperturbed mitosis in human and marsupial cells. We find that, like Mad2, p31comet traffics on and off kinetochores and is also present in the cytosol. Cells depleted of p31comet arrest in metaphase with mature bipolar kinetochore-microtubule attachments, a satisfied checkpoint, and high cyclin B levels. Thus p31comet is required for timely mitotic exit. We propose that p31comet is an essential component of the machinery that silences the checkpoint during each cell cycle.

scholarly journals Recent Progress on the Localization of the Spindle Assembly Checkpoint Machinery to Kinetochores

Cells ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 278 ◽  
Author(s):  
Zhen Dou ◽  
Diogjena Prifti ◽  
Ping Gui ◽  
Xing Liu ◽  
Sabine Elowe ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Mitotic Progression ◽  
Cycle Progression ◽  
Daughter Cells ◽  
Microtubule Attachment ◽  
Surveillance Mechanism

Faithful chromosome segregation during mitosis is crucial for maintaining genome stability. The spindle assembly checkpoint (SAC) is a surveillance mechanism that ensures accurate mitotic progression. Defective SAC signaling leads to premature sister chromatid separation and aneuploid daughter cells. Mechanistically, the SAC couples the kinetochore microtubule attachment status to the cell cycle progression machinery. In the presence of abnormal kinetochore microtubule attachments, the SAC prevents the metaphase-to-anaphase transition through a complex kinase-phosphatase signaling cascade which results in the correct balance of SAC components recruited to the kinetochore. The correct kinetochore localization of SAC proteins is a prerequisite for robust SAC signaling and, hence, accurate chromosome segregation. Here, we review recent progresses on the kinetochore recruitment of core SAC factors.

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