scholarly journals APC15 mediates CDC20 autoubiquitylation by APC/CMCC and disassembly of the mitotic checkpoint complex

2012 ◽  
Vol 19 (11) ◽  
pp. 1116-1123 ◽  
Author(s):  
Kristina Uzunova ◽  
Billy T Dye ◽  
Hannelore Schutz ◽  
Rene Ladurner ◽  
Georg Petzold ◽  
...  
Keyword(s):  
Mitotic Checkpoint ◽  
Mitotic Checkpoint Complex

scholarly journals Aneuploid abortion correlates positively with MAD1 overexpression and miR-125b down-regulation

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Juan Zhao ◽  
Hui Li ◽  
Guangxin Chen ◽  
Lijun Du ◽  
Peiyan Xu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Checkpoint ◽  
Early Embryo ◽  
Vital Role ◽  
Control Group ◽  
Embryo Abortion ◽  
Protein Levels ◽  
Mitotic Checkpoint Complex

Abstract Background Aneuploidy is the most frequent cause of early-embryo abortion. Any defect in chromosome segregation would fail to satisfy the spindle assembly checkpoint (SAC) during mitosis, halting metaphase and causing aneuploidy. The mitotic checkpoint complex (MCC), comprising MAD1, MAD2, Cdc20, BUBR1 and BUB3, plays a vital role in SAC activation. Studies have confirmed that overexpression of MAD2 and BUBR1 can facilitate correct chromosome segregation and embryo stability. Research also proves that miR-125b negatively regulates MAD1 expression by binding to its 3′UTR. However, miR-125b, Mad1 and Bub3 gene expression in aneuploid embryos of spontaneous abortion has not been reported to date. Methods In this study, embryonic villi from miscarried pregnancies were collected and divided into two groups (aneuploidy and euploidy) based on High-throughput ligation-dependent probe amplification (HLPA) and Fluorescence in situ hybridization (FISH) analyses. RNA levels of miR-125b, MAD1 and BUB3 were detected by Quantitative real-time PCR (qRT-PCR); protein levels of MAD1 and BUB3 were analysed by Western blotting. Results statistical analysis (p < 0.05) showed that miR-125b and BUB3 were significantly down-regulated in the aneuploidy group compared to the control group and that MAD1 was significantly up-regulated. Additionally, the MAD1 protein level was significantly higher in aneuploidy abortion villus, but BUB3 protein was only mildly increased. Correlation analysis revealed that expression of MAD1 correlated negatively with miR-125b. Conclusion These results suggest that aneuploid abortion correlates positively with MAD1 overexpression, which might be caused by insufficient levels of miR-125b. Taken together, our findings first confirmed the negative regulatory mode between MAD1 and miR-125b, providing a basis for further mechanism researches in aneuploid abortion.

CDC20 assists its catalytic incorporation in the mitotic checkpoint complex

Science ◽  
2020 ◽  
Vol 371 (6524) ◽  
pp. 67-71 ◽  
Author(s):  
Valentina Piano ◽  
Amal Alex ◽  
Patricia Stege ◽  
Stefano Maffini ◽  
Gerardo A. Stoppiello ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mitotic Checkpoint ◽  
Catalytic Complex ◽  
Controlled Systems ◽  
Cellular Pathways ◽  
Mitotic Checkpoint Complex ◽  
Rate Limiting

Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.

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

2019 ◽  
pp. 201902970 ◽  
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.

scholarly journals Mechanism of APC/CCDC20 activation by mitotic phosphorylation

2016 ◽  
Vol 113 (19) ◽  
pp. E2570-E2578 ◽  
Author(s):  
Renping Qiao ◽  
Florian Weissmann ◽  
Masaya Yamaguchi ◽  
Nicholas G. Brown ◽  
Ryan VanderLinden ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Checkpoint ◽  
Anaphase Promoting Complex ◽  
Loop Region ◽  
Evolutionarily Conserved ◽  
Terminal Loop ◽  
Different Types ◽  
Mitotic Phosphorylation ◽  
Mitotic Checkpoint Complex

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

scholarly journals Basis of catalytic assembly of the mitotic checkpoint complex

Nature ◽  
2017 ◽  
Vol 542 (7642) ◽  
pp. 498-502 ◽  
Author(s):  
Alex C. Faesen ◽  
Maria Thanasoula ◽  
Stefano Maffini ◽  
Claudia Breit ◽  
Franziska Müller ◽  
...  
Keyword(s):  
Mitotic Checkpoint ◽  
Mitotic Checkpoint Complex

scholarly journals Roles of different pools of the mitotic checkpoint complex and the mechanisms of their disassembly

2013 ◽  
Vol 110 (26) ◽  
pp. 10568-10573 ◽  
Author(s):  
E. Eytan ◽  
D. Sitry-Shevah ◽  
A. Teichner ◽  
A. Hershko
Keyword(s):  
Mitotic Checkpoint ◽  
Mitotic Checkpoint Complex

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 Role of phosphorylation of Cdc20 in p31comet-stimulated disassembly of the mitotic checkpoint complex

2012 ◽  
Vol 109 (21) ◽  
pp. 8056-8060 ◽  
Author(s):  
S. Miniowitz-Shemtov ◽  
E. Eytan ◽  
D. Ganoth ◽  
D. Sitry-Shevah ◽  
E. Dumin ◽  
...  
Keyword(s):  
Mitotic Checkpoint ◽  
Mitotic Checkpoint Complex
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