Orchestration of the spindle assembly checkpoint by CDK1‐cyclin B1

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.

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Slip slidin’ away of mitosis with CRL2Zyg11

The Journal of Cell Biology ◽

10.1083/jcb.201609086 ◽

2016 ◽

Vol 215 (2) ◽

pp. 143-145 ◽

Cited By ~ 1

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.

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Cyclin B1 scaffolds MAD1 at the corona to activate the spindle assembly checkpoint

10.1101/726224 ◽

2019 ◽

Cited By ~ 2

Author(s):

Lindsey A Allan ◽

Magda Reis ◽

Yahui Liu ◽

Pim Huis in ’t Veld ◽

Geert JPL Kops ◽

...

Keyword(s):

Genome Stability ◽

Spindle Assembly Checkpoint ◽

Point Mutations ◽

Spindle Assembly ◽

Cyclin B1 ◽

Cyclin B ◽

Mitotic Progression ◽

Mitotic Kinase ◽

Binding Interface

ABSTRACTThe Cyclin B:CDK1 kinase complex is the master regulator of mitosis that phosphorylates hundreds of proteins to coordinate mitotic progression. We show here that, in addition to these kinase functions, Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminus of MAD1, and point mutations in this region remove corona MAD1 and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness, in this context, arises because Cyclin B1-MAD1 localisation becomes MPS1-independent after the corona has been established. We demonstrate that this allows corona-MAD1 to persist at kinetochores when MPS1 activity falls, ensuring that it can still be phosphorylated on a key C-terminal catalytic site by MPS1. Therefore, this study explains how corona MAD1 generates a robust SAC signal and why stripping of this pool by dynein is essential for SAC silencing. It also reveals that the key mitotic kinase, Cyclin B1-Cdk1, scaffolds the pathway that inhibits its own degradation.

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Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

PLoS ONE ◽

10.1371/journal.pone.0013037 ◽

2010 ◽

Vol 5 (9) ◽

pp. e13037 ◽

Cited By ~ 24

Author(s):

Ana Lúcia Mena ◽

Eric W.-F. Lam ◽

Sukalyan Chatterjee

Keyword(s):

Spindle Assembly Checkpoint ◽

De Novo ◽

Spindle Assembly ◽

Cyclin B1 ◽

Checkpoint Response

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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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CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes

10.1101/587238 ◽

2019 ◽

Author(s):

Zi-Yun Yi ◽

Tie-Gang Meng ◽

Xue-Shan Ma ◽

Jian Li ◽

Chun-Hui Zhang ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Time Lapse ◽

Cyclin B1 ◽

Meiotic Spindle ◽

G2 Arrest ◽

Mouse Oocytes ◽

Summary Statement ◽

Initiation Of Dna Replication ◽

Oocyte Meiotic Maturation

AbstractCell division cycle protein CDC6 is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from Pro-MI to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (GV breakdown, GVBD) through regulation of Cdh1 and cyclin B1 expression and CDK1 phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation and spindle assembly checkpoint (SAC) activation, leading to significant Pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.Summary statementWe show that CDC6 is indispensable for maintaining G2 arrest of mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.

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Discovery of Novel Agents on Spindle Assembly Checkpoint to Sensitize Vinorelbine-Induced Mitotic Cell Death against Human Non-Small Cell Lung Cancers

International Journal of Molecular Sciences ◽

10.3390/ijms21165608 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5608

Author(s):

Ya-Ching Chang ◽

Yu-Ling Tseng ◽

Wohn-Jenn Leu ◽

Chi-Min Du ◽

Yi-Huei Jiang ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

B Cell Lymphoma ◽

Cyclin B1 ◽

Mitotic Arrest ◽

Small Cell ◽

Nsclc Cell Line ◽

Synergistic Activity ◽

Small Cell Lung ◽

Lung Cancers

Non-small cell lung cancer (NSCLC) accounts about 80% of all lung cancers. More than two-thirds of NSCLC patients have inoperable, locally advanced or metastatic tumors. Non-toxic agents that synergistically potentiate cancer-killing activities of chemotherapeutic drugs are in high demand. YL-9 was a novel and non-cytotoxic compound with the structure related to sildenafil but showing much less activity against phosphodiesterase type 5 (PDE5). NCI-H460, an NSCLC cell line with low PDE5 expression, was used as the cell model. YL-9 synergistically potentiated vinorelbine-induced anti-proliferative and apoptotic effects in NCI-H460 cells. Vinorelbine induced tubulin acetylation and Bub1-related kinase (BUBR1) phosphorylation, a necessary component in spindle assembly checkpoint. These effects, as well as BUBR1 cleavage, were substantially enhanced in co-treatment with YL-9. Several mitotic arrest signals were enhanced under combinatory treatment of vinorelbine and YL-9, including an increase of mitotic spindle abnormalities, increased cyclin B1 expression, B-cell lymphoma 2 (Bcl-2) phosphorylation and increased phosphoproteins. Moreover, YL-9 also displayed synergistic activity in combining with vinorelbine to induce apoptosis in A549 cells which express PDE5. In conclusion. the data suggest that YL-9 is a novel agent that synergistically amplifies vinorelbine-induced NSCLC apoptosis through activation of spindle assembly checkpoint and increased mitotic arrest of the cell cycle. YL-9 shows the potential for further development in combinatory treatment against NSCLC.

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Anaphase-Promoting Complex/Cyclosome–Dependent Proteolysis of Human Cyclin a Starts at the Beginning of Mitosis and Is Not Subject to the Spindle Assembly Checkpoint

The Journal of Cell Biology ◽

10.1083/jcb.153.1.137 ◽

2001 ◽

Vol 153 (1) ◽

pp. 137-148 ◽

Cited By ~ 266

Author(s):

Stephan Geley ◽

Edgar Kramer ◽

Christian Gieffers ◽

Julian Gannon ◽

Jan-Michael Peters ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cyclin A ◽

Cyclin B1 ◽

Cyclin B ◽

Anaphase Promoting Complex ◽

Wild Type ◽

Destruction Box

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The “destruction box” (D-box) of cyclin A is 10–20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.

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Spindle assembly checkpoint-related meiotic defect in oocytes from LT/Sv mice has cytoplasmic origin and diminishes in older females

Reproduction ◽

10.1530/rep-11-0362 ◽

2012 ◽

Vol 144 (3) ◽

pp. 331-338 ◽

Cited By ~ 4

Author(s):

Steffen Hoffmann ◽

Marzena Król ◽

Zbigniew Polanski

Keyword(s):

Spindle Assembly Checkpoint ◽

Germinal Vesicle ◽

Spindle Assembly ◽

Cyclin B1 ◽

Meiotic Metaphase ◽

Wild Type ◽

Anaphase Onset ◽

Spindle Microtubules ◽

Old Mice ◽

Segregation Of Chromosomes

The spindle assembly checkpoint (SAC) ensures proper segregation of chromosomes by delaying anaphase onset until all kinetochores are properly attached to the spindle microtubules. Oocytes from the mouse strain LT/Sv arrest at the first meiotic metaphase (MI) due to, as reported recently, enormously prolonged activity of the SAC. We compared the dynamics of cyclin B1–GFP degradation, the process which is a measure of the SAC activity, in chromosomal and achromosomal halves of LT/Sv oocytes. In chromosome-containing oocyte halves arrested at MI, cyclin B1–GFP was not degraded indicating active SAC. However, in the halves lacking chromosomes, which is a condition precluding the SAC function, degradation always occurred confirming that MI arrest in LT/Sv oocytes is SAC dependent. Transferring the germinal vesicle (GV) from LT/Sv oocytes into the enucleated oocytes from wild-type mice resulted in the progression through meiosis one, indicating that a SAC-activating defect in LT/Sv oocytes is cytoplasmic, yet can be rescued by foreign cytoplasm. These results may help to define the etiology of the human infertility related to the oocyte MI arrest, indicating the involvement of the SAC as likely candidate, and point to GV transfer as the possible therapy. Finally, we found that majority of oocytes isolated from old LT/Sv mice complete the first meiosis. Reciprocal transfers of the GV between the oocytes from young and old LT/Sv females suggest that the factor(s) responsible for the reversal of the phenotype in oocytes from old mice is located both in the GV and in the cytoplasm.

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Cyclin B1-Cdk1 binding to MAD1 links nuclear pore disassembly to chromosomal stability

10.1101/701474 ◽

2019 ◽

Cited By ~ 3

Author(s):

Mark Jackman ◽

Chiara Marcozzi ◽

Mercedes Pardo ◽

Lu Yu ◽

Adam L. Tyson ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cyclin B1 ◽

Nuclear Pore ◽

Major Question ◽

Nuclear Envelope Breakdown ◽

Kinetochore Assembly ◽

Regulatory Machinery ◽

Multiple Protein ◽

Spindle Assembly Checkpoint Protein

AbstractHow the cell completely reorganises its architecture when it divides is a problem that has fascinated researchers for almost 150 years. We now know that the core regulatory machinery is highly conserved in eukaryotes but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated to remodel the cell in a matter of minutes remains a major question. Cyclin B-CDK is the primary kinase that drives mitotic remodelling and here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the spindle assembly checkpoint protein, MAD1. This localised Cyclin B1-CDK1 activity coordinates NPC disassembly with kinetochore assembly: it is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore, which enables MAD1 to be recruited to kinetochores before nuclear envelope breakdown, thereby strengthening the spindle assembly checkpoint to maintain genomic stability.

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