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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, Sue Biggins, 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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identification of a sgo2 dependent but mad2 independent pathway controlling anaphase onset in fission yeast
Cell Reports, 2017Co-Authors: John C Meadows, Kevin G. Hardwick, Maria Morasantos, Theresa C Lancaster, Alicja M Sochaj, Graham J Buttrick, Liam J Messin, Jonathan B A MillarAbstract:Summary The onset of anaphase is triggered by activation of the anaphase-promoting complex/cyclosome (APC/C) following silencing of the spindle assembly checkpoint (SAC). APC/C triggers ubiquitination of Securin and Cyclin B, which leads to loss of sister chromatid cohesion and inactivation of Cyclin B/Cdk1, respectively. This promotes relocalization of Aurora B kinase and other components of the chromosome passenger complex (CPC) from centromeres to the spindle midzone. In fission yeast, this is mediated by Clp1 phosphatase-dependent interaction of CPC with Klp9/MKLP2 (kinesin-6). When this interaction is disrupted, kinetochores bi-orient normally, but APC/C activation is delayed via a mechanism that requires Sgo2 and some (Bub1, Mph1/Mps1, and Mad3), but not all (Mad1 and Mad2), components of the SAC and the first, but not second, lysine, glutamic acid, asparagine (KEN) box in Mad3. These data indicate that interaction of CPC with Klp9 terminates a Sgo2-dependent, but Mad2-independent, APC/C-inhibitory pathway that is distinct from the canonical SAC.
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phosphodependent recruitment of bub1 and bub3 to spc7 knl1 by mph1 kinase maintains the spindle checkpoint
Current Biology, 2012Co-Authors: Lindsey A Shepperd, Kevin G. Hardwick, Theresa C Lancaster, John C Meadows, Alicja M Sochaj, Juri Rappsilber, Graham J Buttrick, Juan Zou, Jonathan B A MillarAbstract:Summary The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B [1]. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension [2–5]. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression [6–8]. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC [9–12]. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.
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kinase activity of fission yeast mph1 is required for mad2 and mad3 to stably bind the anaphase promoting complex
Current Biology, 2012Co-Authors: Judith Zich, Alicja M Sochaj, Heather M Syred, Laura Milne, Atlanta G Cook, Hiro Ohkura, Juri Rappsilber, Kevin G. HardwickAbstract:Summary Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6–8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.
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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.
Andrea Musacchio - One of the best experts on this subject based on the ideXlab platform.
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sustained mps1 activity is required in mitosis to recruit o mad2 to the mad1 c mad2 core complex
Journal of Cell Biology, 2010Co-Authors: Laura Hewi, Anthony Tighe, Stefano Santaguida, Anne White, Clifford David Jones, Andrea Musacchio, Stephe Gree, Stephe S TayloAbstract:Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression.
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accumulation of mad2 cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of mad2 in saccharomyces cerevisiae
Journal of Cell Biology, 2006Co-Authors: Luigi Nezi, Anna De Antoni, Simonetta Piatti, Giulia Rancati, Sebastiano Pasqualato, Andrea MusacchioAbstract:The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.
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Explaining the oligomerization properties of the spindle assembly checkpoint protein Mad2
Philosophical Transactions of the Royal Society B: Biological Sciences, 2005Co-Authors: Anna Deantoni, Valeria Sala, Andrea MusacchioAbstract: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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Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint
The EMBO Journal, 2002Co-Authors: Lucia Sironi, Anna De Antoni, Marina Mapelli, Stefan Knapp, Kuan-teh Jeang, Andrea MusacchioAbstract:The spindle checkpoint protein Mad1 recruits Mad2 to unattached kinetochores and is essential for Mad2–Cdc20 complex formation in vivo but not in vitro. The crystal structure of the Mad1–Mad2 complex reveals an asymmetric tetramer, with elongated Mad1 monomers parting from a coiled-coil to form two connected sub-complexes with Mad2. The Mad2 C-terminal tails are hinged mobile elements wrapping around the elongated ligands like molecular ‘safety belts’. We show that Mad1 is a competitive inhibitor of the Mad2–Cdc20 complex, and propose that the Mad1–Mad2 complex acts as a regulated gate to control Mad2 release for Cdc20 binding. Mad1–Mad2 is strongly stabilized in the tetramer, but a 1:1 Mad1–Mad2 complex slowly releases Mad2 for Cdc20 binding, driven by favourable binding energies. Thus, the rate of Mad2 binding to Cdc20 during checkpoint activation may be regulated by conformational changes that destabilize the tetrameric Mad1–Mad2 assembly to promote Mad2 release. We also show that unlocking the Mad2 C-terminal tail is required for ligand release from Mad2, and that the ‘safety belt’ mechanism may prolong the lifetime of Mad2–ligand complexes.
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bub3 interaction with mad2 mad3 and cdc20 is mediated by wd40 repeats and does not require intact kinetochores
The EMBO Journal, 2001Co-Authors: Roberta Fraschini, Lucia Sironi, Andrea Musacchio, Alessia Beretta, Giovanna Lucchini, Simonetta PiattiAbstract:The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3, Bub1, Bub3 and Mps1 proteins, prevents anaphase entry and mitotic exit by inhibiting the anaphase promoting complex activator Cdc20 in response to monopolar attachment of sister kinetochores to spindle fibres. We show here that Cdc20, which had previously been shown to interact physically with Mad2 and Mad3, associates also with Bub3 and association is up-regulated upon checkpoint activation. Moreover, co-fractionation experiments suggest that Mad2, Mad3 and Bub3 may be concomitantly present in protein complexes with Cdc20. Formation of the Bub3-Cdc20 complex requires all kinetochore checkpoint proteins but, surprisingly, not intact kinetochores. Conversely, point mutations altering the conserved WD40 motifs of Bub3, which might be involved in the formation of a beta-propeller fold devoted to protein-protein interactions, disrupt its association with Mad2, Mad3 and Cdc20, as well as proper checkpoint response. We suggest that Bub3 could serve as a platform for interactions between kinetochore checkpoint proteins, and its association with Mad2, Mad3 and Cdc20 might be instrumental for checkpoint activation.
Rey-huei Chen - One of the best experts on this subject based on the ideXlab platform.
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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.
Xuelian Luo - One of the best experts on this subject based on the ideXlab platform.
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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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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.
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p31comet blocks Mad2 activation through structural mimicry.
Cell, 2007Co-Authors: Maojun Yang, Josep Rizo, Diana R. Tomchick, Mischa Machius, Xuelian LuoAbstract:Summary The status of spindle checkpoint signaling depends on the balance of two opposing dynamic processes that regulate the highly unusual two-state behavior of Mad2. In mitosis, a Mad1-Mad2 core complex recruits cytosolic Mad2 to kinetochores through Mad2 dimerization and converts Mad2 to a conformer amenable to Cdc20 binding, thereby facilitating checkpoint activation. p31 comet inactivates the checkpoint through binding to Mad1- or Cdc20-bound Mad2, thereby preventing Mad2 activation and promoting the dissociation of the Mad2-Cdc20 complex. Here, we report the crystal structure of the Mad2-p31 comet complex. The C-terminal region of Mad2 that undergoes rearrangement in different Mad2 conformers is a major structural determinant for p31 comet binding, explaining the specificity of p31 comet toward Mad1- or Cdc20-bound Mad2. p31 comet adopts a fold strikingly similar to that of Mad2 and binds at the dimerization interface of Mad2. Thus, p31 comet exploits the two-state behavior of Mad2 to block its activation by acting as an "anti-Mad2."
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The Mad2 Spindle Checkpoint Protein Undergoes Similar Major Conformational Changes Upon Binding to Either Mad1 or Cdc20
Molecular Cell, 2002Co-Authors: Xuelian Luo, Zhanyun Tang, Josep RizoAbstract:Abstract Mad2 participates in spindle checkpoint inhibition of APC Cdc20 . We show that RNAi-mediated suppression of Mad1 function in mammalian cells causes loss of Mad2 kinetochore localization and impairment of the spindle checkpoint. Mad1 and Cdc20 contain Mad2 binding motifs that share a common consensus. We have identified a class of Mad2 binding peptides with a similar consensus. Binding of one of these ligands, MBP1, triggers an extensive rearrangement of the tertiary structure of Mad2. Mad2 also undergoes a similar striking structural change upon binding to a Mad1 or Cdc20 binding motif peptide. Our data suggest that, upon checkpoint activation, Mad1 recruits Mad2 to unattached kinetochores and may promote binding of Mad2 to Cdc20.
Jonathan B A Millar - One of the best experts on this subject based on the ideXlab platform.
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identification of a sgo2 dependent but mad2 independent pathway controlling anaphase onset in fission yeast
Cell Reports, 2017Co-Authors: John C Meadows, Kevin G. Hardwick, Maria Morasantos, Theresa C Lancaster, Alicja M Sochaj, Graham J Buttrick, Liam J Messin, Jonathan B A MillarAbstract:Summary The onset of anaphase is triggered by activation of the anaphase-promoting complex/cyclosome (APC/C) following silencing of the spindle assembly checkpoint (SAC). APC/C triggers ubiquitination of Securin and Cyclin B, which leads to loss of sister chromatid cohesion and inactivation of Cyclin B/Cdk1, respectively. This promotes relocalization of Aurora B kinase and other components of the chromosome passenger complex (CPC) from centromeres to the spindle midzone. In fission yeast, this is mediated by Clp1 phosphatase-dependent interaction of CPC with Klp9/MKLP2 (kinesin-6). When this interaction is disrupted, kinetochores bi-orient normally, but APC/C activation is delayed via a mechanism that requires Sgo2 and some (Bub1, Mph1/Mps1, and Mad3), but not all (Mad1 and Mad2), components of the SAC and the first, but not second, lysine, glutamic acid, asparagine (KEN) box in Mad3. These data indicate that interaction of CPC with Klp9 terminates a Sgo2-dependent, but Mad2-independent, APC/C-inhibitory pathway that is distinct from the canonical SAC.
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bub3 bub1 binding to spc7 knl1 toggles the spindle checkpoint switch by licensing the interaction of bub1 with mad1 mad2
Current Biology, 2016Co-Authors: Maria Morasantos, Katharina Sewart, America Hervasaguilar, Theresa C Lancaster, John C Meadows, Jonathan B A MillarAbstract:The spindle assembly checkpoint (SAC) ensures that sister chromatids do not separate until all chromosomes are attached to spindle microtubules and bi-oriented. Spindle checkpoint proteins, including Mad1, Mad2, Mad3 (BubR1), Bub1, Bub3, and Mph1 (Mps1), are recruited to unattached and/or tensionless kinetochores. SAC activation catalyzes the conversion of soluble Mad2 (O-Mad2) into a form (C-Mad2) that binds Cdc20, BubR1, and Bub3 to form the mitotic checkpoint complex (MCC), a potent inhibitor of the anaphase-promoting complex (APC/C). SAC silencing de-represses Cdc20-APC/C activity allowing poly-ubiquitination of Securin and Cyclin B, leading to the dissolution of sister chromatids and anaphase onset [1]. Understanding how microtubule interaction at kinetochores influences the timing of anaphase requires an understanding of how spindle checkpoint protein interaction with the kinetochore influences spindle checkpoint signaling. We, and others, recently showed that Mph1 (Mps1) phosphorylates multiple conserved MELT motifs in the Spc7 (Spc105/KNL1) protein to recruit Bub1, Bub3, and Mad3 (BubR1) to kinetochores [2-4]. In budding yeast, Mps1 phosphorylation of a central non-catalytic region of Bub1 promotes its association with the Mad1-Mad2 complex, although this association has not yet been detected in other organisms [5]. Here we report that multisite binding of Bub3 to the Spc7 MELT array toggles the spindle checkpoint switch by permitting Mph1 (Mps1)-dependent interaction of Bub1 with Mad1-Mad2.
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phosphodependent recruitment of bub1 and bub3 to spc7 knl1 by mph1 kinase maintains the spindle checkpoint
Current Biology, 2012Co-Authors: Lindsey A Shepperd, Kevin G. Hardwick, Theresa C Lancaster, John C Meadows, Alicja M Sochaj, Juri Rappsilber, Graham J Buttrick, Juan Zou, Jonathan B A MillarAbstract:Summary The spindle assembly checkpoint (SAC) is the major surveillance system that ensures that sister chromatids do not separate until all chromosomes are correctly bioriented during mitosis. Components of the checkpoint include Mad1, Mad2, Mad3 (BubR1), Bub3, and the kinases Bub1, Mph1 (Mps1), and Aurora B [1]. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension [2–5]. Kinetochore association of Mad2 causes it to undergo a conformational change, which promotes its association to Mad3 and Cdc20 to form the mitotic checkpoint complex (MCC). The MCC inhibits the anaphase-promoting complex/cyclosome (APC/C) until the checkpoint is satisfied. SAC silencing derepresses Cdc20-APC/C activity. This triggers the polyubiquitination of securin and cyclin, which promotes the dissolution of sister chromatid cohesion and mitotic progression [6–8]. We, and others, recently showed that association of PP1 to the Spc7/Spc105/KNL1 family of kinetochore proteins is necessary to stabilize microtubule-kinetochore attachments and silence the SAC [9–12]. We now report that phosphorylation of the conserved MELT motifs in Spc7 by Mph1 (Mps1) recruits Bub1 and Bub3 to the kinetochore and that this is required to maintain the SAC signal.
