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Jonathan B A Millar - One of the best experts on this subject based on the ideXlab platform.
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BUB1 is not essential for the checkpoint response to unattached kinetochores in diploid human cells
Current biology : CB, 2018Co-Authors: Cerys E. Currie, Mar Mora-santos, Christopher A. Smith, Andrew D. Mcainsh, Jonathan B A MillarAbstract:Summary Error-free chromosome segregation during mitosis depends on a functional spindle assembly checkpoint (SAC). The SAC is a multi-component signalling system that is recruited to unattached or incorrectly attached kinetochores to catalyse the formation of a soluble inhibitor, known as the Mitotic Checkpoint Complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C) [1] . We have previously proposed that two separable pathways, composed of KNL1–Bub3–BUB1 (KBB) and Rod–Zwilch–Zw10 (RZZ), recruit Mad1–Mad2 complexes to human kinetochores to activate the SAC [2] . Although BUB1 is absolutely required for checkpoint signalling in yeast (which lack RZZ), there is conflicting evidence as to whether this is the case in human cells based on siRNA studies 2 , 3 , 4 , 5 . Here we show that, while BUB1 is required for recruitment of BubR1, it is not strictly required for the checkpoint response to unattached kinetochores in diploid human cells.
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BUB1 is not required for the checkpoint response to unattached kinetochores in diploid human cells
bioRxiv, 2018Co-Authors: Cerys E. Currie, Christopher A. Smith, Andrew D. Mcainsh, Maria Dm Morasantos, Jonathan B A MillarAbstract:Error-free chromosome segregation during mitosis depends on a functional spindle assembly checkpoint (SAC). The SAC is a multi-component signaling system that is recruited to incorrectly attached kinetochores to catalyze the formation of a soluble inhibitor, known as the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex [1]. We have previously proposed that two separable pathways, composed of KNL1-Bub3-BUB1 (KBB) and Rod-Zwilch-Zw10 (RZZ), recruit Mad1-Mad2 complexes to human kinetochores to activate the SAC [2]. We refer to this as the dual pathway model. Although BUB1 is absolutely required for MCC formation in yeast (which lack RZZ), there is conflicting evidence as to whether this is also the case in human cells based on siRNA studies [2-5]. Here we report, using genome editing, that BUB1 is not strictly required for the SAC response to unattached kinetochores in human diploid hTERT-RPE1 cells, consistent with the dual pathway model.
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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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knl1 bubs and rzz provide two separable pathways for checkpoint activation at human kinetochores
Developmental Cell, 2015Co-Authors: Virginia Silio, Andrew D. Mcainsh, Jonathan B A MillarAbstract:The spindle assembly checkpoint (SAC) ensures the accurate segregation of sister chromatids during mitosis. Activation of the SAC occurs through a series of ordered molecular events that result in recruitment of Mad1:Mad2 complexes to improperly attached kinetochores. The current model involves sequential phospho-dependent recruitment of Bub3:BUB1 to KNL1 followed by binding of Mad1:Mad2 to BUB1. Here, we show in non-transformed diploid human cells that the KNL1-Bub3-BUB1 (KBB) pathway is required during normal mitotic progression when kinetochores are misaligned but is nonessential for SAC activation and Mad2 loading when kinetochores are unattached from microtubules. We provide evidence that the Rod-ZW10-Zwilch (RZZ) complex is necessary to recruit Mad1:Mad2 to, and delay anaphase onset in response to, unattached kinetochores independently of the KBB pathway. These data suggest that the KBB and RZZ complexes provide two distinct kinetochore receptors for Mad1:Mad2 and reveal mechanistic differences between SAC activation by unattached and improperly attached kinetochores.
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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.
Stephen S. Taylor - One of the best experts on this subject based on the ideXlab platform.
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Inhibitors of the BUB1 spindle assembly checkpoint kinase: Synthesis of BAY-320 and comparison with 2OH-BNPP1
2020Co-Authors: Ilma Amalina, Anthony Tighe, Helen J. Whalley, David Perera, Joanne C. Mcgrail, David J. Procter, Stephen S. TaylorAbstract:Summary BUB1 is a serine/threonine kinase proposed to function centrally in both mitotic chromosome alignment and the spindle assembly checkpoint (SAC), however its role remains controversial. Although it is well documented that BUB1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors can be invaluable tools for investigation of kinase function, we decided to evaluate the relative potential of two agents (2OH-BNPPI and BAY-320) as BUB1 inhibitors. After confirming that both agents inhibit BUB1 in vitro, we developed a cell based-assay to specifically measure BUB1 inhibition in vivo. For this assay we overexpressed a fusion of Histone 2B and the BUB1 kinase region (BUB1C) tethering it in close proximity to H2A, which generated a strong ectopic H2ApT120 signal along chromosome arms. The ectopic signal generated from BUB1C activity was effectively inhibited by BAY-320, but not 2OH-BNPP1. In addition, only BAY-320 was able to inhibit endogenous BUB1-mediated Sgo1 localisation. Preliminary experiments using BAY-320 suggested a minor role for BUB1 kinase activity in chromosome alignment and the SAC, however results suggest that BAY-320 may exhibit off-target effects at the concentration required to demonstrate these outcomes. In conclusion, 2OH-BNPP1 may not be an effective BUB1 inhibitor in vivo, and while BAY-320 is able to inhibit BUB1 in vivo, the high concentrations required and potential for off-target effects highlight the ongoing need for improved BUB1 inhibitors.
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BUB1 Maintains Centromeric Cohesion by Activation of the Spindle Checkpoint
Developmental cell, 2007Co-Authors: David Perera, Valerie Tilston, Jane A. Hopwood, Marco Barchi, Ray Boot-handford, Stephen S. TaylorAbstract:BUB1 is a component of the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures genome stability by delaying anaphase until all the chromosomes are stably attached to spindle microtubules via their kinetochores. To define BUB1's role in chromosome segregation, embryogenesis, and tissue homeostasis, we generated a mouse strain in which BUB1 can be inactivated by administration of tamoxifen, thereby bypassing the preimplantation lethality associated with the BUB1 null phenotype. We show that BUB1 is essential for postimplantation embryogenesis and proliferation of primary embryonic fibroblasts. BUB1 inactivation in adult males inhibits proliferation in seminiferous tubules, reducing sperm production and causing infertility. In culture, BUB1-deficient fibroblasts fail to align their chromosomes or sustain SAC function, yielding a highly aberrant mitosis that prevents further cell divisions. Centromeres in BUB1-deficient cells also separate prematurely; however, we show that this is a consequence of SAC dysfunction rather than a direct role for BUB1 in protecting centromeric cohesion.
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BUB1 and aurora b cooperate to maintain bubr1 mediated inhibition of apc ccdc20
Journal of Cell Science, 2005Co-Authors: Christopher J. Morrow, Anthony Tighe, Victoria L. Johnson, Maria I. F. Scott, Claire Ditchfield, Stephen S. TaylorAbstract:The spindle checkpoint maintains genome stability by inhibiting Cdc20-mediated activation of the anaphase promoting complex/cyclosome (APC/C) until all the chromosomes correctly align on the microtubule spindle apparatus via their kinetochores. BubR1, an essential component of this checkpoint, localises to kinetochores and its kinase activity is regulated by the kinesin-related motor protein Cenp-E. BubR1 also inhibits APC/C(Cdc20) in vitro, thus providing a molecular link between kinetochore-microtubule interactions and the proteolytic machinery that regulates mitotic progression. Several other protein kinases, including BUB1 and members of the Ipl1/aurora family, also regulate anaphase onset. However, in human somatic cells BUB1 and aurora B kinase activity do not appear to be essential for spindle checkpoint function. Specifically, when BUB1 is inhibited by RNA interference, or aurora kinase activity is inhibited with the small molecule ZM447439, cells arrest transiently in mitosis following exposure to spindle toxins that prevent microtubule polymerisation. Here, we show that mitotic arrest of BUB1-deficient cells is dependent on aurora kinase activity, and vice versa. We suggest therefore that the checkpoint is composed of two arms, one dependent on BUB1, the other on aurora B. Analysis of BubR1 complexes suggests that both of these arms converge on the mitotic checkpoint complex (MCC), which includes BubR1, Bub3, Mad2 and Cdc20. Although it is known that MCC components can bind and inhibit the APC/C, we show here for the first time that the binding of the MCC to the APC/C is dependent on an active checkpoint signal. Furthermore, we show that both BUB1 and aurora kinase activity are required to promote binding of the MCC to the APC/C. These observations provide a simple explanation of why BubR1 and Mad2 are essential for checkpoint function following spindle destruction, yet BUB1 and aurora B kinase activity are not. Taken together with other observations, we suggest that these two arms respond to different spindle cues: whereas the BUB1 arm monitors kinetochore-microtubule attachment, the aurora B arm monitors biorientation. This bifurcation in the signalling mechanism may help explain why many tumour cells mount a robust checkpoint response following spindle damage, despite exhibiting chromosome instability.
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BUB1 is required for kinetochore localization of bubr1 cenp e cenp f and mad2 and chromosome congression
Journal of Cell Science, 2004Co-Authors: Victoria L. Johnson, Maria I. F. Scott, Deema Hussein, Sarah Holt, Stephen S. TaylorAbstract:During mitosis, the recruitment of spindle-checkpoint-associated proteins to the kinetochore occurs in a defined order. The protein kinase BUB1 localizes to the kinetochore very early during mitosis, followed by Cenp-F, BubR1, Cenp-E and finally Mad2. Using RNA interference, we have investigated whether this order of binding reflects a level of dependency in human somatic cells. Specifically, we show that BUB1 plays a key role in the assembly of checkpoint proteins at the kinetochore, being required for the subsequent localization of Cenp-F, BubR1, Cenp-E and Mad2. In contrast to studies in Xenopus, we also show that BubR1 is not required for kinetochore localization of BUB1. Repression of BUB1 increases the number of cells with lagging chromosomes at metaphase, suggesting that BUB1 plays a role in chromosome congression. However, repression of BUB1 does not appear to compromise spindle checkpoint function either during normal mitosis or in response to spindle damage. This raises the possibility that, in the absence of BUB1, other mechanisms contribute to spindle checkpoint function.
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kinetochore localisation and phosphorylation of the mitotic checkpoint components BUB1 and bubr1 are differentially regulated by spindle events in human cells
Journal of Cell Science, 2001Co-Authors: Stephen S. Taylor, Deema Hussein, Yunmei Wang, Sarah Elderkin, Christopher J. MorrowAbstract:BUB1 is a budding yeast gene required to ensure that progression through mitosis is coupled to correct spindle assembly. Two related human protein kinases, BUB1 and BubR1, both localise to kinetochores during mitosis, suggesting that they play a role in delaying anaphase until all chromosomes achieve correct, bipolar attachment to the spindle. However, how the activities of BUB1 and BubR1 are regulated by spindle events and how their activities regulate downstream cell cycle events is not known. To investigate how spindle events regulate BUB1 and BubR1, we characterised their relative localisations during mitosis in the presence and absence of microtubule toxins. In prometaphase cells, both kinases colocalise to the same domain of the kinetochore. However, whereas the localisation of BubR1 at sister kinetochores is symmetrical, localisation of BUB1 is often asymmetrical. This asymmetry is dependent on microtubule attachment, and the kinetochore exhibiting weaker BUB1 staining is typically closer to the nearest spindle pole. In addition, a 30 minute nocodazole treatment dramatically increases the amount of BUB1 localising to kinetochores but has little effect on BubR1. Furthermore, BUB1 levels increase at metaphase kinetochores following loss of tension caused by taxol treatment. Thus, these observations suggest that BUB1 localisation is sensitive to changes in both tension and microtubule attachment. Consistent with this, we also show that BUB1 is rapidly phosphorylated following brief treatments with nocodazole or taxol. In contrast, BubR1 is phosphorylated in the absence of microtubule toxins, and spindle damage has little additional effect. Although these observations indicate that BUB1 and BubR1 respond differently to spindle dynamics, they are part of a common complex during mitosis. We suggest therefore that BUB1 and BubR1 may integrate different ‘spindle assembly signals’ into a single signal which can then be interpreted by downstream cell cycle regulators.
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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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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BUB1 kinase targets sgo1 to ensure efficient chromosome biorientation in budding yeast mitosis
PLOS Genetics, 2005Co-Authors: Josefin Fernius, Kevin G. HardwickAbstract:During cell division all chromosomes must be segregated accurately to each daughter cell. Errors in this process give rise to aneuploidy, which leads to birth defects and is implicated in cancer progression. The spindle checkpoint is a surveillance mechanism that ensures high fidelity of chromosome segregation by inhibiting anaphase until all kinetochores have established bipolar attachments to spindle microtubules. BUB1 kinase is a core component of the spindle checkpoint, and cells lacking BUB1 fail to arrest in response to microtubule drugs and precociously segregate their DNA. The mitotic role(s) of BUB1 kinase activity remain elusive, and it is controversial whether this C-terminal domain of BUB1p is required for spindle checkpoint arrest. Here we make a detailed analysis of budding yeast cells lacking the kinase domain (BUB1ΔK). We show that despite being able to arrest in response to microtubule depolymerisation and kinetochore-microtubule attachment defects, BUB1ΔK cells are sensitive to microtubule drugs. This is because BUB1ΔK cells display significant chromosome mis-segregation upon release from nocodazole arrest. BUB1ΔK cells mislocalise Sgo1p, and we demonstrate that both the BUB1 kinase domain and Sgo1p are required for accurate chromosome biorientation after nocodazole treatment. We propose that BUB1 kinase and Sgo1p act together to ensure efficient biorientation of sister chromatids during mitosis.
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Fission Yeast BUB1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis
The Journal of cell biology, 1998Co-Authors: Pascal Bernard, Kevin G. Hardwick, Jean-paul JaverzatAbstract:The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast BUB1 gene in spindle checkpoint function and in unperturbed mitoses. We find that BUB1+ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of BUB1 to centromeres and a delay in the completion of mitosis. We show that BUB1 also has a crucial role in normal, unperturbed mitoses. Loss of BUB1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in ΔBUB1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that BUB1+ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, BUB1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of BUB1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the BUB1 kinase after the metaphase–anaphase transition.
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mad1p a phosphoprotein component of the spindle assembly checkpoint in budding yeast
Journal of Cell Biology, 1995Co-Authors: Kevin G. Hardwick, Andrew W MurrayAbstract:The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1-arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule-disrupting agents is defective in some (mad2, BUB1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.
Rey-huei Chen - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation and activation of BUB1 on unattached chromosomes facilitate the spindle checkpoint.
The EMBO journal, 2004Co-Authors: Rey-huei ChenAbstract:The spindle checkpoint inhibits anaphase until all kinetochores have attached properly to spindle microtubules. The protein kinase BUB1 is an essential checkpoint component that resides at kinetochores during mitosis. It is shown herein that Xenopus BUB1 becomes hyperphosphorylated and the kinase is activated on unattached chromosomes. MAP kinase (MAPK) contributes to this phosphorylation, as inhibiting MAPK or altering MAPK consensus sites in BUB1 to alanine or valine (BUB15AV) abolishes the phosphorylation and activation on chromosomes. Both BUB1 and BUB15AV support the checkpoint under an optimal condition for spindle checkpoint activation. However, BUB1, but not BUB15AV, supports the checkpoint at a relatively low concentration of nuclei or the microtubule inhibitor nocodazole. Similar to BUB15AV, BUB1 without the kinase domain (BUB1ΔKD) is also partially compromised in its checkpoint function and in its ability to recruit other checkpoint proteins to kinetochores. This study suggests that activation of BUB1 at kinetochores enhances the efficiency of the spindle checkpoint and is probably important in maintaining the checkpoint toward late prometaphase when the cell contains only a few or a single unattached kinetochore.
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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 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.
David Pellman - One of the best experts on this subject based on the ideXlab platform.
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BUB1 sgo1 and mps1 mediate a distinct pathway for chromosome biorientation in budding yeast
Molecular Biology of the Cell, 2011Co-Authors: Zuzana Storchova, Justin S Becker, Nicolas Talarek, Sandra Kogelsberger, David PellmanAbstract:The conserved mitotic kinase BUB1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, BUB1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both BUB1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of BUB1Δ haploid cells and restores viability to BUB1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both BUB1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid BUB1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither BUB1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B–independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that BUB1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B–mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.
