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Kevin G. Hardwick - One of the best experts on this subject based on the ideXlab platform.
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The Bub1-TPR Domain Interacts Directly with Mad3 to Generate Robust Spindle Checkpoint Arrest.
Current biology : CB, 2019Co-Authors: Ioanna Leontiou, Karen M May, Nitobe London, Lucile Grzesiak, Bethan Medina-pritchard, Priya Amin, A. Arockia Jeyaprakash, Kevin G. HardwickAbstract:Summary The Spindle Checkpoint monitors kinetochore-microtubule interactions and generates a “wait anaphase” delay when any defects are apparent [ 1 , 2 , 3 ]. This provides time for cells to correct chromosome attachment errors and ensure high-fidelity chromosome segregation. Checkpoint signals are generated at unattached chromosomes during mitosis. To activate the Checkpoint, Mps1 Mph1 kinase phosphorylates the kinetochore component KNL1 Spc105/Spc7 on conserved MELT motifs to recruit Bub3-Bub1 complexes [ 4 , 5 , 6 ] via a direct Bub3 interaction with phospho-MELT motifs [ 7 , 8 ]. Mps1 Mph1 then phosphorylates Bub1, which strengthens its interaction with Mad1-Mad2 complexes to produce a signaling platform [ 9 , 10 ]. The Bub1-Mad1 platform is thought to recruit Mad3, Cdc20, and Mad2 to produce the mitotic Checkpoint complex (MCC), which is the diffusible wait anaphase signal [ 9 , 11 , 12 ]. The MCC binds and inhibits the mitotic E3 ubiquitin ligase, known as Cdc20-anaphase promoting complex/cyclosome (APC/C), and stabilizes securin and cyclin to delay anaphase onset [ 13 , 14 , 15 , 16 , 17 ]. Here we demonstrate, in both budding and fission yeast, that kinetochores and KNL1 Spc105/Spc7 can be bypassed; simply inducing heterodimers of Mps1 Mph1 kinase and Bub1 is sufficient to trigger metaphase arrest that is dependent on Mad1, Mad2, and Mad3. We use this to dissect the domains of Bub1 necessary for arrest, highlighting the need for Bub1-CD1, which binds Mad1 [ 9 ], and Bub1’s highly conserved N-terminal tetratricopeptide repeat (TPR) domain [ 18 , 19 ]. We demonstrate that the Bub1 TPR domain is both necessary and sufficient to bind and recruit Mad3. We propose that this brings Mad3 into close proximity to Mad1-Mad2 and Mps1 Mph1 kinase, enabling efficient generation of MCC complexes.
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a novel protein phosphatase 1 dependent Spindle Checkpoint silencing mechanism
Current Biology, 2009Co-Authors: Vincent Vanoosthuyse, Kevin G. HardwickAbstract:Summary The Spindle Checkpoint is a surveillance system acting in mitosis to delay anaphase onset until all chromosomes are properly attached to the mitotic Spindle [1, 2]. When the Checkpoint is activated, the Mad2 and Mad3 proteins directly bind and inhibit Cdc20, which is an essential activator of an E3 ubiquitin ligase known as the anaphase-promoting complex (APC) [3]. When the Checkpoint is satisfied, Cdc20-APC is activated and polyubiquitinates securin and cyclin, leading to the dissolution of sister chromatid cohesion and mitotic progression. Several protein kinases play critical roles in Spindle Checkpoint signaling, but the mechanism (or mechanisms) by which they inhibit mitotic progression remains unclear [4]. Furthermore, it is not known whether their activity needs to be reversed by protein phosphatases before anaphase onset can occur. Here we employ fission yeast to show that Aurora (Ark1) kinase activity is directly required to maintain Spindle Checkpoint arrest, even in the presence of many unattached kinetochores. Upon Ark1 inhibition, Checkpoint complexes are disassembled and cyclin B is rapidly degraded. Importantly, Checkpoint silencing and cyclin B degradation require the kinetochore-localized isoform of protein phosphatase 1 (PP1 Dis2 ). We propose that PP1 Dis2 -mediated dephosphorylation of Checkpoint components forms a novel Spindle Checkpoint silencing mechanism.
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the Spindle Checkpoint functions of mad3 and mad2 depend on a mad3 ken box mediated interaction with cdc20 anaphase promoting complex apc c
Journal of Biological Chemistry, 2008Co-Authors: Matylda Sczaniecka, Julie Blyth, Jun Song Chen, Anna Feoktistova, Kathleen L Gould, Karen M May, Kevin G. HardwickAbstract:Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The Spindle assembly Checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain Checkpoint proteins interact directly with Cdc20, but it remains unclear how the Checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 Spindle Checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the Spindle Checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic Checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.
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mad3 ken boxes mediate both cdc20 and mad3 turnover and are critical for the Spindle Checkpoint
PLOS ONE, 2007Co-Authors: Emma M J King, Sjaak Van Der Sar, Kevin G. HardwickAbstract:Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/CCdc20 is inhibited by the Spindle Checkpoint, thereby delaying anaphase onset and providing more time for Spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C Cdc20 inhibition remain unclear. The mitotic Checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble ‘degron’ signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for Spindle Checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced Checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for Spindle Checkpoint signaling and high fidelity chromosome segregation.
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the Spindle Checkpoint structural insights into dynamic signalling
Nature Reviews Molecular Cell Biology, 2002Co-Authors: Andrea Musacchio, Kevin G. HardwickAbstract:Chromosome segregation is a complex and astonishingly accurate process whose inner working is beginning to be understood at the molecular level. The Spindle Checkpoint plays a key role in ensuring the fidelity of this process. It monitors the interactions between chromosomes and microtubules, and delays mitotic progression to allow extra time to correct defects. Here, we review and integrate findings on the dynamics of Checkpoint proteins at kinetochores with structural information about signalling complexes.
Rey-huei Chen - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of cdc20 is required for its inhibition by the Spindle Checkpoint
Nature Cell Biology, 2003Co-Authors: Eunah Chung, Rey-huei ChenAbstract:The Spindle Checkpoint delays anaphase until all chromosomes are properly attached to Spindle microtubules. When the Spindle Checkpoint is activated at unattached kinetochores, the Checkpoint proteins BubR1, Bub3 and Mad2 bind and inhibit Cdc20, an activator of the anaphase-promoting complex (APC). Here, we show that Xenopus laevis Cdc20 is phosphorylated at Ser 50, Thr 64, Thr 68 and Thr 79 during mitosis and that mitogen-activated protein kinase (MAPK) contributes to the phosphorylation at Thr 64 or Thr 68. Cdc20 mutants that are phosphorylation-deficient are able to activate the APC in X. laevis egg extracts. However, Cdc20 mutants in which any of the four phosphorylation sites were altered to Ala or Val failed to respond to the Spindle Checkpoint signal, owing to their reduced affinity for the Spindle Checkpoint proteins. This study demonstrates that the Spindle Checkpoint stops anaphase by inhibiting fully-phosphorylated Cdc20. Our results also have implications for the Spindle Checkpoint silencing mechanism.
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bubr1 is essential for kinetochore localization of other Spindle Checkpoint proteins and its phosphorylation requires mad1
Journal of Cell Biology, 2002Co-Authors: Rey-huei ChenAbstract:The Spindle Checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic Spindle. Checkpoint signal is generated at kinetochores that are not bound with Spindle microtubules or not under tension. Unattached kinetochores associate with several Checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the Spindle Checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the Spindle Checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other Spindle Checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.
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Spindle Checkpoint requires Mad1-bound and Mad1-free Mad2.
Molecular Biology of the Cell, 2002Co-Authors: Eunah Chung, Rey-huei ChenAbstract:The Spindle Checkpoint prevents anaphase from occurring until all chromosomes have attached properly to the mitotic Spindle. The Checkpoint components Mad1 and Mad2 associate with unattached kinetochores and are probably involved in triggering the Checkpoint. We now demonstrate that in Xenopus egg extracts Mad1 and Mad2 form a stable complex, whereas a fraction of Mad2 molecules is not bound to Mad1. The Checkpoint establishment and maintenance are lost upon titrating out free Mad2 with an excess of Mad1 or a truncated Mad1 (amino acids 326–718, Mad1C) that contains the Mad2-binding region. Mad1N (amino acids 1–445) that binds kinetochores, but not Mad2, reduces Mad1 and Mad2 at kinetochores and abolishes Checkpoint maintenance. Furthermore, the association between Mad2 and Cdc20, the activator for the anaphase-promoting complex, is enhanced under Checkpoint-active condition compared with that at metaphase. Immunodepletion analysis shows that the Mad1-free Mad2 protein is unable to bind Cdc20, consistent with the model that kinetochore localization of Mad2 facilitates the formation of Mad2–Cdc20 complex. This study demonstrates that the ratio between Mad1 and Mad2 is critical for maintaining a pool of Mad1-free Mad2 that is necessary for the Spindle Checkpoint. We propose that Mad2 may become activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1-free Mad2.
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Spindle Checkpoint protein bub1 is required for kinetochore localization of mad1 mad2 bub3 and cenp e independently of its kinase activity
Journal of Cell Biology, 2001Co-Authors: Hilary E Sharpbaker, Rey-huei ChenAbstract:The Spindle Checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic Spindle. We have isolated a Xenopus homologue of the Spindle Checkpoint component Bub1, and investigated its role in the Spindle Checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the Checkpoint and is localized to kinetochores, similar to the Spindle Checkpoint complex Mad1–Mad2. However, Bub1 differs from Mad1–Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the Spindle Checkpoint and the kinetochore binding of the Checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the Checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the Spindle Checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the Spindle Checkpoint in Xenopus egg extracts.
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the Spindle Checkpoint of budding yeast depends on a tight complex between the mad1 and mad2 proteins
Molecular Biology of the Cell, 1999Co-Authors: Kevin G. Hardwick, Andrew W Murray, Rey-huei Chen, Dana L Smith, Michelle D BradyAbstract:The Spindle Checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic Spindle or in the attachment of chromosomes to the Spindle. When Spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability. We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle. Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another Spindle Checkpoint component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known Checkpoint proteins. In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE. Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for Checkpoint function and for hyperphosphorylation of Mad1p.
Andrew W Murray - One of the best experts on this subject based on the ideXlab platform.
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the Spindle Checkpoint rescues the meiotic segregation of chromosomes whose crossovers are far from the centromere
Nature Genetics, 2007Co-Authors: Soni Lacefield, Andrew W MurrayAbstract:The Spindle Checkpoint rescues the meiotic segregation of chromosomes whose crossovers are far from the centromere
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a small molecule inhibitor of mps1 blocks the Spindle Checkpoint response to a lack of tension on mitotic chromosomes
Current Biology, 2005Co-Authors: Russell K Dorer, Sheng Zhong, John A Tallarico, Wing Hung Wong, Timothy J Mitchison, Andrew W MurrayAbstract:Summary The Spindle Checkpoint prevents chromosome loss by preventing chromosome segregation in cells with improperly attached chromosomes [1–3]. The Checkpoint senses defects in the attachment of chromosomes to the mitotic Spindle [4] and the tension exerted on chromosomes by Spindle forces in mitosis [5–7]. Because many cancers have defects in chromosome segregation, this Checkpoint may be required for survival of tumor cells and may be a target for chemotherapy. We performed a phenotype-based chemical-genetic screen in budding yeast and identified an inhibitor of the Spindle Checkpoint, called cincreasin. We used a genome-wide collection of yeast gene-deletion strains and traditional genetic and biochemical analysis to show that the target of cincreasin is Mps1, a protein kinase required for Checkpoint function [8]. Despite the requirement for Mps1 for sensing both the lack of microtubule attachment and tension at kinetochores, we find concentrations of cincreasin that selectively inhibit the tension-sensitive branch of the Spindle Checkpoint. At these concentrations, cincreasin causes lethal chromosome missegregation in mutants that display chromosomal instability. Our results demonstrate that Mps1 can be exploited as a target and that inhibiting the tension-sensitive branch of the Spindle Checkpoint may be a way of selectively killing cancer cells that display chromosomal instability.
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Spindle Checkpoint component mad2 contributes to biorientation of homologous chromosomes
Current Biology, 2003Co-Authors: Marion A Shonn, Amara L Murray, Andrew W MurrayAbstract:Abstract Cell cycle Checkpoints sense defects in chromosome metabolism, halt the cell cycle, and activate pathways that repair the defects. The Spindle Checkpoint arrests the cell cycle in response to defects in the interaction between microtubules and kinetochores (the proteinaceous complex assembled on centromeric DNA), but no repair function has been demonstrated for this Checkpoint. We show that the roles of two Spindle Checkpoint components, Mad2 and Mad3, differ in meiosis I. In the absence of Mad2, meiosis I nondisjunction occurs at a high frequency and can be corrected by delaying the onset of anaphase [1]. The absence of Mad3 does not induce nondisjunction, even though mad3 Δ cells cannot arrest the cell cycle in response to kinetochores that lack either microtubules or tension on the linkage between chromosomes and microtubules. The two proteins have different roles in chromosome alignment. Compared to wild type and mad3 Δ cells, mad2 Δ mutants are slower to attach homologous chromosomes to opposite poles of the Spindle. This observation suggests that Mad2 plays a role in reorienting chromosomes that are incorrectly attached to the Spindle as well as delaying the cell cycle, whereas Mad3 is needed for the cell cycle delay, but not for chromosome reorientation.
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the budding yeast protein kinase ipl1 aurora allows the absence of tension to activate the Spindle Checkpoint
Genes & Development, 2001Co-Authors: Sue Biggins, Andrew W MurrayAbstract:The Spindle Checkpoint prevents cell cycle progression in cells that have mitotic Spindle defects. Although several Spindle defects activate the Spindle Checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of Spindle Checkpoint arrests. Ipl1p is required to maintain the Spindle Checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the Spindle Checkpoint is not a consequence of kinetochore and/or Spindle defects. The requirement for Ipl1p distinguishes two different activators of the Spindle Checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by Spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore.
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lack of tension at kinetochores activates the Spindle Checkpoint in budding yeast
Current Biology, 2001Co-Authors: Bodo M Stern, Andrew W MurrayAbstract:The Spindle Checkpoint delays the onset of anaphase until all pairs of sister chromatids are attached to the mitotic Spindle. The Checkpoint could monitor the attachment of microtubules to kinetochores, the tension that results from the two sister chromatids attaching to opposite Spindle poles, or both. We tested the role of tension by allowing cells to enter mitosis without a prior round of DNA replication. The unreplicated chromatids are attached to Spindle microtubules but are not under tension since they lack a sister chromatid that could attach to the opposite pole. Because the Spindle Checkpoint is activated in these cells, we conclude that the absence of tension at the yeast kinetochore is sufficient to activate the Spindle Checkpoint in mitosis.
Xuelian Luo - One of the best experts on this subject based on the ideXlab platform.
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Mechanistic insight into TRIP13-catalyzed Mad2 structural transition and Spindle Checkpoint silencing
Nature Communications, 2017Co-Authors: Melissa L. Brulotte, Byung Cheon Jeong, Chad A. Brautigam, Xuelian LuoAbstract:The Spindle Checkpoint maintains genomic stability and prevents aneuploidy. Unattached kinetochores convert the latent open conformer of the Checkpoint protein Mad2 (O-Mad2) to the active closed conformer (C-Mad2), bound to Cdc20. C-Mad2–Cdc20 is incorporated into the mitotic Checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C). The C-Mad2-binding protein p31comet and the ATPase TRIP13 promote MCC disassembly and Checkpoint silencing. Here, using nuclear magnetic resonance (NMR) spectroscopy, we show that TRIP13 and p31comet catalyze the conversion of C-Mad2 to O-Mad2, without disrupting its stably folded core. We determine the crystal structure of human TRIP13, and identify functional TRIP13 residues that mediate p31comet–Mad2 binding and couple ATP hydrolysis to local unfolding of Mad2. TRIP13 and p31comet prevent APC/C inhibition by MCC components, but cannot reactivate APC/C already bound to MCC. Therefore, TRIP13–p31comet intercepts and disassembles free MCC not bound to APC/C through mediating the local unfolding of the Mad2 C-terminal region. The Spindle Checkpoint ensures the fidelity of chromosome segregation during mitosis and meiosis. Here the authors use a combination of biochemical and structural biology approaches to show how the TRIP13 ATPase and its adaptor, p31comet, catalyze the conversion of the Checkpoint protein Mad2 between latent and active forms
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Structure of an intermediate conformer of the Spindle Checkpoint protein Mad2
Proceedings of the National Academy of Sciences, 2015Co-Authors: Mayuko Hara, Hongbin Sun, Engin Özkan, Xuelian LuoAbstract:The Spindle Checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The Checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.
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the cdc20 binding phe box of the Spindle Checkpoint protein bubr1 maintains the mitotic Checkpoint complex during mitosis
Journal of Biological Chemistry, 2015Co-Authors: Laura A Diazmartinez, Luying Jia, Chad A. Brautigam, Ross Warrington, Wei Tian, Xuelian LuoAbstract:The Spindle Checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the Checkpoint and enhance the production of the mitotic Checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical Checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting Spindle-Checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the Spindle Checkpoint might be due to functional duality or redundant, competing mechanisms.
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Phosphorylation of the Spindle Checkpoint protein Mad2 regulates its conformational transition
Proceedings of the National Academy of Sciences, 2010Co-Authors: Soonjoung Kim, Katja Wassmann, Hongbin Sun, Haydn L. Ball, Xuelian LuoAbstract:Regulated conformational changes of proteins are critical for cellular signal transduction. The Spindle Checkpoint protein Mad2 is an unusual protein with two native folds: the latent open conformer (O-Mad2) and the activated closed conformer (C-Mad2). During mitosis, cytosolic O-Mad2 binds to the Mad1-Mad2 core complex at unattached kinetochores and undergoes conformational activation to become C-Mad2. C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2(S195D) mutant and the phospho-S195 Mad2 protein obtained using intein-mediated semisynthesis do not form C-Mad2 on their own. Mad2(S195D) fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-Mad2. Overexpression of Mad2(S195D) in human cells causes Checkpoint defects. Our results indicate that Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of Mad2 is regulated by posttranslational mechanisms.
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Insights Into MAD2 Regulation in the Spindle Checkpoint Revealed by the Crystal Structure of the Symmetric MAD2 Dimer.
PLoS Biology, 2008Co-Authors: Maojun Yang, Josep Rizo, Diana R. Tomchick, Mischa Machius, Chyong Jy Liu, Xuelian LuoAbstract:In response to misaligned sister chromatids during mitosis, the Spindle Checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/CCdc20 inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2–C-Mad2 (C–C) or asymmetric O-Mad2–C-Mad2 (O–C) dimers. We also report the crystal structure of the symmetric C–C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C–C dimer is functional in vitro and in living cells. Finally, the Mad1–Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the Spindle Checkpoint.
Gary J Gorbsky - One of the best experts on this subject based on the ideXlab platform.
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ska3 is required for Spindle Checkpoint silencing and the maintenance of chromosome cohesion in mitosis
Current Biology, 2009Co-Authors: John R Daum, Gary J Gorbsky, Jonathan D Wren, Jeremy J Daniel, Sushama Sivakumar, Jennifer N Mcavoy, Tamara A PotapovaAbstract:The mitotic Spindle Checkpoint monitors proper bipolar attachment of chromosomes to the mitotic Spindle. Previously, depletion of the novel kinetochore complex Ska1/Ska2 was found to induce a metaphase delay. By using bioinformatics, we identified C13orf3, predicted to associate with kinetochores. Recently, three laboratories independently indentified C13orf3 as an additional Ska complex component, and therefore we adopted the name Ska3. We found that cells depleted of Ska3 by RNAi achieve metaphase alignment but fail to silence the Spindle Checkpoint or enter anaphase. After hours of metaphase arrest, chromatids separate but retain robust kinetochore-microtubule attachments. Ska3-depleted cells accumulate high levels of the Checkpoint protein Bub1 at kinetochores. Ska3 protein accumulation at kinetochores in prometaphase is dependent on Sgo1 protein. Sgo1, which accumulates at the centromeres earlier, in prophase, is not dependent on Ska3. Sgo1-depleted cells show a stronger premature chromatid separation phenotype than those depleted of Ska3. We hypothesize that Ska3 functions to coordinate Checkpoint signaling from the microtubule binding sites within a kinetochore by laterally linking the individual binding sites. We suggest that this network plays a major role in silencing the Spindle Checkpoint when chromosomes are aligned at metaphase to allow timely anaphase onset and mitotic exit.
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dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic Spindle Checkpoint
Carcinogenesis, 2009Co-Authors: Annaleena Salmela, Jeroen Pouwels, Asta Varis, Anu M Kukkonen, Pauliina Toivonen, Pasi Halonen, Merja Perala, Olli Kallioniemi, Gary J GorbskyAbstract:Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2-160 microg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs Spindle Checkpoint signaling, which may contribute to the antiproliferative effects of the compound.
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a high throughput whole cell screen for small molecule inhibitors of the mitotic Spindle Checkpoint identifies om137 a novel aurora kinase inhibitor
Cancer Research, 2009Co-Authors: Joanna H Demoe, Stefano Santaguida, Andrea Musacchio, John R Daum, Gary J GorbskyAbstract:In mitosis, the kinetochores of chromosomes that lack full microtubule attachments and/or mechanical tension activate a signaling pathway called the mitotic Spindle Checkpoint that blocks progression into anaphase and prevents premature segregation of the chromatids until chromosomes become aligned at the metaphase plate. The Spindle Checkpoint is responsible for arresting cells in mitosis in response to chemotherapeutic Spindle poisons such as paclitaxel or vinblastine. Some cancer cells show a weakened Checkpoint signaling system that may contribute to chromosome instability in tumors. Because complete absence of the Spindle Checkpoint leads to catastrophic cell division, we reasoned that drugs targeting the Checkpoint might provide a therapeutic window in which the Checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the Spindle Checkpoint. Most cells respond to microtubule drugs by activating the Spindle Checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of Checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study, we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects of subnanomolar concentrations of paclitaxel. [Cancer Res 2009;69(4):1509–16]
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polo like kinase 1 creates the tension sensing 3f3 2 phosphoepitope and modulates the association of Spindle Checkpoint proteins at kinetochores
Current Biology, 2005Co-Authors: Leena J Ahonen, Todd P Stukenberg, Marko J. Kallio, John R Daum, Margaret A Bolton, Isaac A Manke, Michael B Yaffe, Gary J GorbskyAbstract:Summary Background : In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the Spindle Checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression. Results : We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a Spindle-Checkpoint-mediated mitotic arrest. Conclusions : Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in Checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.
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inhibition of aurora b kinase blocks chromosome segregation overrides the Spindle Checkpoint and perturbs microtubule dynamics in mitosis
Current Biology, 2002Co-Authors: Marko J. Kallio, Todd P Stukenberg, Mark L Mccleland, Gary J GorbskyAbstract:Abstract How kinetochores correct improper microtubule attachments and regulate the Spindle Checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila , inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the Spindle Checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the Spindle Checkpoint, and cytokinesis.
