scholarly journals Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation

2012 ◽  
Vol 199 (1) ◽  
pp. 27-37 ◽  
Author(s):  
Daisuke Izawa ◽  
Jonathon Pines
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Genomic Stability ◽  
Cyclin B1 ◽  
Mitotic Exit ◽  
Anaphase Promoting Complex ◽  
Chromosome Attachment ◽  
A Site

The spindle assembly checkpoint (SAC) is essential to ensure proper chromosome segregation and thereby maintain genomic stability. The SAC monitors chromosome attachment, and any unattached chromosomes generate a “wait anaphase” signal that blocks chromosome segregation. The target of the SAC is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C) that triggers anaphase and mitotic exit by ubiquitylating securin and cyclin B1. The inhibitory complex formed by the SAC has recently been shown to inhibit Cdc20 by acting as a pseudosubstrate inhibitor, but in this paper, we show that Mad2 also inhibits Cdc20 by binding directly to a site required to bind the APC/C. Mad2 and the APC/C competed for Cdc20 in vitro, and a Cdc20 mutant that does not bind stably to Mad2 abrogated the SAC in vivo. Thus, we provide insights into how Cdc20 binds the APC/C and uncover a second mechanism by which the SAC inhibits the APC/C.

scholarly journals Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

2002 ◽  
Vol 13 (7) ◽  
pp. 2289-2300 ◽  
Author(s):  
Brett K. Kaiser ◽  
Zachary A. Zimmerman ◽  
Harry Charbonneau ◽  
Peter K. Jackson
Keyword(s):  
Cell Cycle ◽  
Phosphatase Activity ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Genomic Stability ◽  
Mitotic Exit ◽  
Protein Abundance ◽  
Cyclin Dependent Kinase ◽  
Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

scholarly journals PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing

2014 ◽  
Vol 206 (7) ◽  
pp. 833-842 ◽  
Author(s):  
Antonio Espert ◽  
Pelin Uluocak ◽  
Ricardo Nunes Bastos ◽  
Davinderpreet Mangat ◽  
Philipp Graab ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Feedback Loop ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Eukaryotic Cells ◽  
Aurora B ◽  
Safeguard Mechanism

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.

scholarly journals PP2A-Cdc55 phosphatase coordinates actomyosin ring contraction and septum formation during cytokinesis

2020 ◽  
Author(s):  
Yolanda Moyano-Rodríguez ◽  
Odena Vilalta-Castany ◽  
Magdalena Foltman ◽  
Alberto Sanchez-Diaz ◽  
Ethel Queralt
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Exit ◽  
In Vitro Assays ◽  
Ring Contraction ◽  
Mitotic Kinases ◽  
Septum Formation ◽  
Primary Septum ◽  
Complete Cell

SummaryEukaryotic cells divide and separate all their components after chromosome segregation by a process called cytokinesis to complete cell division. Cytokinesis is regulated by exclusive elements of the process, and by some mitotic exit regulators. The mitotic kinases Cdc28-Clb2, Cdc5, and Dbf2-Mob1 phosphorylate cytokinetic proteins in budding yeast, but very little is known about the phosphatases regulating cytokinesis. The PP2A-Cdc55 phosphatase regulates mitosis counteracting Cdk1- and Cdc5-dependent phosphorylations. This prompted us to propose that PP2A-Cdc55 could also regulate cytokinesis by counteracting the mitotic kinases. Here, we demonstrate by in vivo and in vitro assays that PP2A-Cdc55 dephosphorylates the F-BAR protein Hof1 and the chitin synthase Chs2, two components of the Ingression Progression Complexes (IPC) involved in cytokinesis regulation. Primary septum formation and actomyosin ring contraction are impaired in absence of PP2A-Cdc55. Interestingly, the non-phosphorylable version of Chs2 rescue the asymmetric AMR contraction observed in absence of Cdc55, indicating that timely dephosphorylation of the IPC proteins by PP2A-Cdc55 is crucial for proper actomyosin ring contraction and septum formation. These findings reveal a new mechanism of cytokinesis regulation by the PP2A-Cdc55 phosphatase and extend our knowledge in the involvement of multiple phosphatases during cytokinesis.

scholarly journals Chromosome biorientation and APC activity remain uncoupled in oocytes with reduced volume

2017 ◽  
Vol 216 (12) ◽  
pp. 3949-3957 ◽  
Author(s):  
Simon I.R. Lane ◽  
Keith T. Jones
Keyword(s):  
Cell Volume ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Anaphase Promoting Complex ◽  
Chromosome Missegregation ◽  
Anaphase Onset ◽  
Reduced Volume ◽  
Chromosome Attachment ◽  
Meiosis I

The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity.

scholarly journals Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis

2009 ◽  
Vol 284 (24) ◽  
pp. 16501-16512 ◽  
Author(s):  
Kyung Uk Hong ◽  
Hyun-Jun Kim ◽  
Hyo-Sil Kim ◽  
Yeon-Sun Seong ◽  
Kyeong-Man Hong ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Cyclin B1 ◽  
Mutant Form ◽  
Metaphase Chromosomes ◽  
C Terminus ◽  
Mitotic Spindle Assembly ◽  
Bipolar Spindles

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.

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 Anaphase-Promoting Complex/Cyclosome Controls the Stability of TPX2 during Mitotic Exit

2005 ◽  
Vol 25 (23) ◽  
pp. 10516-10527 ◽  
Author(s):  
Scott Stewart ◽  
Guowei Fang
Keyword(s):  
Cell Cycle ◽  
Spindle Assembly ◽  
Mitotic Exit ◽  
Anaphase Promoting Complex ◽  
Discrete Elements ◽  
Protein Levels ◽  
Recognition Motifs ◽  
The Stability

ABSTRACT TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/CCdh1: a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/CCdh1 in vitro and for its degradation in vivo. We conclude that APC/CCdh1 controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.

scholarly journals FANCA Fine-Tunes Chromosome Segregation By Controlling BUBR1K250 Acetylation at the Kinetochores

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1040-1040
Author(s):  
Zahi Abdul Sater ◽  
Richa Sharma ◽  
Elizabeth Sierra Potchanant ◽  
Ying He ◽  
Grzegorz Nalepa
Keyword(s):  
Genomic Instability ◽  
Chromosome Segregation ◽  
Molecular Mechanisms ◽  
Spindle Assembly Checkpoint ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Cyclin B1 ◽  
Dependent Manner ◽  
Anaphase Onset

Abstract Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with genomic instability, high risk of acute myeloid leukemia (AML) and other malignancies. Somatic mutations within the FA/BRCA signaling network occur in AML in the general population, reflecting the importance of FA genes in tumor suppression. While the role of FA signaling in DNA damage repair and replication is well-established, we and others found that the FA network is essential for error-free chromosome segregation during cell division. Both interphase and mitotic errors contribute to the evolution of genomic instability during FA-/- human and murine hematopoiesis in vivo. However, the molecular mechanisms of FA pathway-dependent genome housekeeping during mitosis are incompletely understood. Through a synthetic lethal kinome-wide shRNA screen in FANCA patient cells, we discovered interphase and mitotic phosphosignaling networks that FANCA-/- cells depend on for survival, including the BUB1-BUBR1 axis of the spindle assembly checkpoint (SAC). BUB1 and BUBR1 are essential SAC kinases that prevent premature anaphase onset and chromosome mis-segregation by inhibiting the APC (anaphase-promoting complex) ubiquitin ligase at the centromeres until all kinetochores achieve correct attachment to the spindle microtubules. Our super-resolution microscopy and biochemistry experiments revealed that FANCA shuttles to kinetochores upon mitotic entry and physically interacts with BUB1 and BUBR1 at the kinetochore-microtubule attachment sites in attachment- and tension-dependent manner. Consistent with impaired SAC, we found that that anaphase onset as well as APC-mediated degradation of cyclin B1, BUBR1 and CDC20 all occur prematurely in FANCA-/- cells. We found that FANCA is essential for BUBR1 lysine 250 (K-250) acetylation at prometaphase kinetochores, and we confirmed that endogenous BUBR1K250 acetylation is disrupted in FANCA-/- primary patient cells using a validated acetyl-specific antibody. BUBR1K250 acetylation event works as a molecular switch in which BUBR1 is converted from a degradation target to a potent inhibitor of the APC ligase. Further, we observed that loss of FANCA disrupts kinetochore recruitment of the BUBR1K250 acetyltransferase PCAF and its upstream regulator, FANCD1/BRCA2. Our findings establish the first mechanistic connection between FANCA, the canonical SAC tumor suppressor cascade and the FA effector FANCD1/BRCA2. These findings further our understanding of the mechanisms of genomic instability and carcinogenesis resulting from loss of FA signaling. Since impaired BUBR1K250 acetylation causes chromosomal instability and cancer in vivo, our results have a direct translational relevance. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 489-503 ◽  
Author(s):  
Karen E Ross ◽  
Orna Cohen-Fix
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Chromosome Loss ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Growth Defects ◽  
Genes Encoding ◽  
Specificity Factor

Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

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