scholarly journals 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

2001 ◽  
Vol 153 (1) ◽  
pp. 137-148 ◽  
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.

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

2019 ◽  
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.

scholarly journals AML1/RUNX1 Phosphorylation by Cyclin-Dependent Kinases Regulates the Degradation of AML1/RUNX1 by the Anaphase-Promoting Complex

2006 ◽  
Vol 26 (20) ◽  
pp. 7420-7429 ◽  
Author(s):  
Joseph R. Biggs ◽  
Luke F. Peterson ◽  
Youhong Zhang ◽  
Andrew S. Kraft ◽  
Dong-Er Zhang
Keyword(s):  
Transcriptional Activation ◽  
Muscle Function ◽  
Cyclin A ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Wild Type ◽  
Cyclin Dependent Kinases

ABSTRACT AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Here, we report that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein which cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree.

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

2008 ◽  
Vol 183 (2) ◽  
pp. 267-277 ◽  
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.

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 Recruitment of Cdc20 to the Kinetochore Requires BubR1 but Not Mad2 in Drosophila melanogaster

2010 ◽  
Vol 30 (13) ◽  
pp. 3384-3395 ◽  
Author(s):  
Deyu Li ◽  
Gary Morley ◽  
Michael Whitaker ◽  
Jun-Yong Huang
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Stable Complex ◽  
S2 Cells ◽  
Anaphase Promoting Complex ◽  
Surveillance Mechanism ◽  
Initial Binding

ABSTRACT To prevent aneuploidy, cells require a mitotic surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC prevents metaphase/anaphase transition by blocking the ubiquitylation and destruction of cyclin B and securin via the Cdc20-activated anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway. This checkpoint involves the kinetochore proteins Mad2, BubR1, and Cdc20. Mad2 and BubR1 are inhibitors of the APC/C, but Cdc20 is an activator. Exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear; in vertebrates, most current models suggest that kinetochore-bound Mad2 is required for initial binding to Cdc20 to form a stable complex that includes BubR1. Here, we show that the Mad2 kinetochore dimerization recruitment mechanism is conserved and that the recruitment of Cdc20 to kinetochores in Drosophila requires BubR1 but not Mad2. BubR1 and Mad2 can bind to Cdc20 independently, and the interactions are enhanced after cells are arrested at mitosis by the depletion of Cdc27 using RNA interference (RNAi) in S2 cells or by MG132 treatment in syncytial embryos. These findings offer an explanation of why BubR1 is more important than Mad2 for SAC function in flies. These findings could lead to a better understanding of vertebrate SAC mechanisms.

scholarly journals How cyclin A destruction escapes the spindle assembly checkpoint

2010 ◽  
Vol 190 (4) ◽  
pp. 501-509 ◽  
Author(s):  
Barbara Di Fiore ◽  
Jonathon Pines
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Cyclin A ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
N Terminus ◽  
Specific Proteins ◽  
Necessary And Sufficient

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A–Cdc20 to the APC/C.

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 Kinetochore stretching inactivates the spindle assembly checkpoint

2009 ◽  
Vol 184 (3) ◽  
pp. 383-390 ◽  
Author(s):  
Kazuhiko S.K. Uchida ◽  
Kentaro Takagaki ◽  
Kazuki Kumada ◽  
Youko Hirayama ◽  
Tetsuo Noda ◽  
...  
Keyword(s):  
Hela Cell ◽  
Live Cell Imaging ◽  
Spindle Assembly Checkpoint ◽  
Cell Imaging ◽  
Protein A ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Hela Cell Line ◽  
Centromere Protein A

The spindle assembly checkpoint (SAC) monitors the attachment of microtubules to the kinetochore and inhibits anaphase when microtubule binding is incomplete. The SAC might also respond to tension; however, how cells can sense tension and whether its detection is important to satisfy the SAC remain controversial. We generated a HeLa cell line in which two components of the kinetochore, centromere protein A and Mis12, are labeled with green and red fluorophores, respectively. Live cell imaging of these cells reveals repetitive cycles of kinetochore extension and recoiling after biorientation. Under conditions in which kinetochore stretching is suppressed, cells fail to silence the SAC and enter anaphase after a delay, regardless of centromere stretching. Monitoring cyclin B levels as a readout for anaphase-promoting complex/cyclosome activity, we find that suppression of kinetochore stretching delays and decelerates cyclin B degradation. These observations suggest that the SAC monitors stretching of kinetochores rather than centromeres and that kinetochore stretching promotes silencing of the SAC signal.

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

Reproduction ◽  
2012 ◽  
Vol 144 (3) ◽  
pp. 331-338 ◽  
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.

scholarly journals The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes

2004 ◽  
Vol 167 (6) ◽  
pp. 1037-1050 ◽  
Author(s):  
Chizuko Tsurumi ◽  
Steffen Hoffmann ◽  
Stephan Geley ◽  
Ralph Graeser ◽  
Zbigniew Polanski
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Dominant Negative ◽  
Meiotic Spindle ◽  
Anaphase Promoting Complex ◽  
Mouse Oocytes ◽  
Cytostatic Factor ◽  
C Complex ◽  
Meiosis I

In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.

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