USP9X Limits Mitotic Checkpoint Complex Turnover to Strengthen the Spindle Assembly Checkpoint and Guard against Chromosomal Instability

Abstract Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been previously linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with the leukemic transformation of myelodysplastic syndromes.

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Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex

Oncogene ◽

10.1038/s41388-021-02106-8 ◽

2021 ◽

Author(s):

Lan Yu ◽

Yue Lang ◽

Ching-Cheng Hsu ◽

Wei-Min Chen ◽

Jui-Chung Chiang ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Independent Manner ◽

Chromosome Separation ◽

Surveillance Mechanism ◽

Mitotic Phosphorylation ◽

Mitotic Checkpoint Complex ◽

Mitotic Regulation

AbstractChromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP’s interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

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BubR1 recruitment to the kinetochore via Bub1 enhances Spindle Assembly Checkpoint signaling

10.1101/2021.08.12.456107 ◽

2021 ◽

Author(s):

Anand Banerjee ◽

Chu Chen ◽

Lauren Humphrey ◽

John J. Tyson ◽

Ajit Joglekar

Keyword(s):

Mathematical Modeling ◽

Dose Response ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Local Concentration ◽

Checkpoint Signaling ◽

Response Characteristics ◽

Anaphase Onset ◽

Mitotic Checkpoint Complex

During mitosis, unattached kinetochores in a dividing cell generate the anaphase-inhibitory Mitotic Checkpoint Complex (MCC) to activate the Spindle Assembly Checkpoint (SAC) and delay anaphase onset. To generate MCC, these kinetochores recruit MCC constituent proteins including the protein BubR1. The increased local concentration of BubR1 resulting from this recruitment should enhance MCC generation, but prior studies found this not to be the case. We analyzed the contribution of two BubR1 recruitment pathways to MCC generation in human kinetochores. For these analyses, we isolated a subset of the MCC generation reactions to the cytosol using ectopic SAC activation systems. These analyses and mathematical modeling show that BubR1 binding to the SAC protein Bub1, but not to the 'KI' motifs in the kinetochore protein Knl1, significantly enhances the rate of Bub1-mediated MCC generation in the kinetochore. Our work also suggests that Bub1-BubR1 stoichiometry will strongly influence the dose-response characteristics of SAC signaling.

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Coupling of Cdc20 inhibition and activation by BubR1

10.1101/2020.12.18.423461 ◽

2020 ◽

Author(s):

Jamin Hein ◽

Dimitriya H Garvanska ◽

Isha Nasa ◽

Arminja Kettenbach ◽

Jakob Nilsson

Keyword(s):

Ubiquitin Ligase ◽

Cross Talk ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cyclin B1 ◽

Mitotic Checkpoint ◽

Anaphase Onset ◽

Mitotic Checkpoint Complex

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.

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Spindle assembly checkpoint: the third decade

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2011.0072 ◽

2011 ◽

Vol 366 (1584) ◽

pp. 3595-3604 ◽

Cited By ~ 126

Author(s):

Andrea Musacchio

Keyword(s):

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Checkpoint Control ◽

Checkpoint Response ◽

The Third ◽

Binding Interface ◽

Mitotic Checkpoint Complex

The spindle assembly checkpoint controls cell cycle progression during mitosis, synchronizing it with the attachment of chromosomes to spindle microtubules. After the discovery of the mitotic arrest deficient ( MAD ) and budding uninhibited by benzymidazole ( BUB ) genes as crucial checkpoint components in 1991, the second decade of checkpoint studies (2001–2010) witnessed crucial advances in the elucidation of the mechanism through which the checkpoint effector, the mitotic checkpoint complex, targets the anaphase-promoting complex (APC/C) to prevent progression into anaphase. Concomitantly, the discovery that the Ndc80 complex and other components of the microtubule-binding interface of kinetochores are essential for the checkpoint response finally asserted that kinetochores are crucial for the checkpoint response. Nevertheless, the relationship between kinetochores and checkpoint control remains poorly understood. Crucial advances in this area in the third decade of checkpoint studies (2011–2020) are likely to be brought about by the characterization of the mechanism of kinetochore recruitment, activation and inactivation of checkpoint proteins, which remains elusive for the majority of checkpoint components. Here, we take a molecular view on the main challenges hampering this task.

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Explaining the oligomerization properties of the spindle assembly checkpoint protein Mad2

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2004.1618 ◽

2005 ◽

Vol 360 (1455) ◽

pp. 637-648 ◽

Cited By ~ 26

Author(s):

Anna DeAntoni ◽

Valeria Sala ◽

Andrea Musacchio

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Checkpoint Activation ◽

Anaphase Onset ◽

Open Conformation ◽

Chromosome Attachment ◽

A Subunit ◽

Mitotic Checkpoint Complex ◽

Spindle Assembly Checkpoint Protein

Mad2 is an essential component of the spindle assembly checkpoint (SAC), a molecular device designed to coordinate anaphase onset with the completion of chromosome attachment to the spindle. Capture of chromosome by microtubules occur on protein scaffolds known as kinetochores. The SAC proteins are recruited to kinetochores in prometaphase where they generate a signal that halts anaphase until all sister chromatid pairs are bipolarly oriented. Mad2 is a subunit of the mitotic checkpoint complex, which is regarded as the effector of the spindle checkpoint. Its function is the sequestration of Cdc20, a protein required for progression into anaphase. The function of Mad2 in the checkpoint correlates with a dramatic conformational rearrangement of the Mad2 protein. Mad2 adopts a closed conformation (C-Mad2) when bound to Cdc20, and an open conformation (O-Mad2) when unbound to this ligand. Checkpoint activation promotes the conversion of O-Mad2 to Cdc20-bound C-Mad2. We show that this conversion requires a C-Mad2 template and we identify this in Mad1-bound Mad2. In our proposition, Mad1-bound C-Mad2 recruits O-Mad2 to kinetochores, stimulating Cdc20 capture, implying that O-Mad2 and C-Mad2 form dimers. We discuss Mad2 oligomerization and link our discoveries to previous observations related to Mad2 oligomerization.

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PIGN spatiotemporally regulates the spindle assembly checkpoint proteins in leukemia transformation and progression

Scientific Reports ◽

10.1038/s41598-021-98218-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Emmanuel K. Teye ◽

Shasha Lu ◽

Fangyuan Chen ◽

Wenrui Yang ◽

Thomas Abraham ◽

...

Keyword(s):

Chromosomal Instability ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Vital Role ◽

The Novel ◽

Checkpoint Activation ◽

Checkpoint Proteins ◽

Mitotic Kinase ◽

Related Proteins

AbstractPhosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with leukemic transformation and progression.

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Loss of BubR1 acetylation causes defects in spindle assembly checkpoint signaling and promotes tumor formation

The Journal of Cell Biology ◽

10.1083/jcb.201210099 ◽

2013 ◽

Vol 202 (2) ◽

pp. 295-309 ◽

Cited By ~ 36

Author(s):

Inai Park ◽

Hae-ock Lee ◽

Eunhee Choi ◽

Yoo-Kyung Lee ◽

Mi-Sun Kwon ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

P53 Mutation ◽

Tumor Formation ◽

Embryonic Fibroblasts ◽

Primary Tumors ◽

Checkpoint Signaling ◽

Treated Condition ◽

Mitotic Checkpoint Complex

BubR1 acetylation is essential in mitosis. Mice heterozygous for the acetylation-deficient BubR1 allele (K243R/+) spontaneously developed tumors with massive chromosome missegregations. K243R/+ mouse embryonic fibroblasts (MEFs) exhibited a weakened spindle assembly checkpoint (SAC) with shortened mitotic timing. The generation of the SAC signal was intact, as Mad2 localization to the unattached kinetochore (KT) was unaltered; however, because of the premature degradation of K243R-BubR1, the mitotic checkpoint complex disassociated prematurely in the nocodazole-treated condition, suggesting that maintenance of the SAC is compromised. BubR1 acetylation was also required to counteract excessive Aurora B activity at the KT for stable chromosome–spindle attachments. The association of acetylation-deficient BubR1 with PP2A-B56α phosphatase was reduced, and the phosphorylated Ndc80 at the KT was elevated in K243R/+ MEFs. In relation, there was a marked increase of micronuclei and p53 mutation was frequently detected in primary tumors of K243R/+ mice. Collectively, the combined effects of failure in chromosome–spindle attachment and weakened SAC cause genetic instability and cancer in K243R/+ mice.

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Coupling of Cdc20 inhibition and activation by BubR1

The Journal of Cell Biology ◽

10.1083/jcb.202012081 ◽

2021 ◽

Vol 220 (5) ◽

Author(s):

Jamin B. Hein ◽

Dimitriya H. Garvanska ◽

Isha Nasa ◽

Arminja N. Kettenbach ◽

Jakob Nilsson

Keyword(s):

Ubiquitin Ligase ◽

Cross Talk ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cyclin B1 ◽

Mitotic Checkpoint ◽

Anaphase Onset ◽

Mitotic Checkpoint Complex

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.

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

Molecular Cytogenetics ◽

10.1186/s13039-021-00538-1 ◽

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.

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