Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, a screen was carried out for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. BUB1 gene and its product have been characterized. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrate a physical association between the two. Sequence analysis of BUB1 reveals a domain with similarity to protein kinases. In vitro experiments confirm that Bub1 possesses kinase activity; Bub1 is able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus (Roberts, 1994).
The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. The role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses has been investigated. bub1+ has been found to be 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. 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 Delta bub1 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. The possibility of a role for the Bub1 kinase after the metaphase-anaphase transition is discussed (Bernard, 1998).
Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. A dominantly acting BUB1 allele has been identified that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 does not cause detectable spindle damage, yet it delays yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possess a G2 DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein do not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay is dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. The mitotic delay caused by BUB1-5 or MPS1 overexpression is interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal (Farr, 1998).
The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus. The Mad and Bub proteins are key components of the spindle checkpoint through budding yeast genetics and are highly conserved. Most of the spindle checkpoint proteins have been localized to kinetochores, yet almost nothing is known about the molecular events which take place there. Mad1p forms a tight complex with Mad2p, and has been shown to recruit Mad2p to kinetochores. Similarly, Bub3p binds to Bub1p and may target it to kinetochores. Budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and this complex is found at significantly higher levels once the spindle checkpoint is activated. Formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, a conserved motif has been identified within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex formation. Mutation of this motif abolishes checkpoint function, indicating that formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle checkpoint mechanism (Brady, 2000).
The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. Known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Saccharomyces Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage (Warren, 2002).
The spindle checkpoint delays the metaphase-to-anaphase transition in response to spindle and kinetochore defects. Genetic screens in budding yeast identified the Mad and Bub proteins as key components of this conserved regulatory pathway. Fission yeast devoid of mad3(+) are unable to arrest their cell cycle in the presence of microtubule defects. Mad3p coimmunoprecipitates Bub3p, Mad2p, and the spindle checkpoint effector Slp1/Cdc20p. Mad3p function is required for the overexpression of Mad2p to result in a metaphase arrest. Mad1p, Bub1p, and Bub3p are not required for this arrest. Thus, Mad3p appears to have a crucial role in transducing the inhibitory 'wait anaphase' signal to the anaphase-promoting complex (APC). Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. For the first time, the dependency of Mad3/BubR1 protein recruitment to kinetochores has been systematically studied. Mad3-GFP kinetochore localization is dependent upon Bub1p, Bub3p, and the Mph1p kinase, but not upon Mad1p or Mad2p. The implications of these findings are discussed in the context of the current understanding of spindle checkpoint function (Millband, 2002).
The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or failed to complete steps during mitosis, including those involving kinetochore function. The molecular nature of the primary signal transmitted from defective kinetochores and how it is detected by the spindle checkpoint are unknown. Bub1, a component of the spindle checkpoint, associates with centromere (CEN) DNA via Skp1, a core kinetochore component in budding yeast. The Skp1's interaction with Bub1 is required for the mitotic delay induced by kinetochore tension defects, but not for the arrest induced by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression. It is proposed that the Skp1-Bub1 interaction is important for transmitting a signal to the spindle checkpoint pathway when insufficient tension is present at kinetochores (Kitagawa, 2003).
Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast. The spindle checkpoint kinase Bub1p becomes phosphorylated by Cdc2p during spindle damage in mitotic cells. Cdc2p directly phosphorylates Bub1p in vitro at the CDK consensus sites. A Bub1p mutant that cannot be phosphorylated by Cdc2p is checkpoint defective, indicating that Cdc2p-dependent Bub1p phosphorylation is required to activate the checkpoint after spindle damage. The kinase activity of Bub1p is required, but is not sufficient, for complete spindle checkpoint function. The role of Bub1p in maintaining centromeric localization of Rec8p during meiosis I is entirely dependent upon its kinase activity, suggesting that Bub1p kinase activity is essential for establishing proper kinetochore function. There is also a Bub1p-dependent meiotic checkpoint that is activated in recombination mutants (Yamaguchi, 2003).
Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells. To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), which requires that sister chromatid cohesion persists at centromeres. The factors that protect centromeric cohesion during meiosis I have remained elusive. This study identifies Sgo1 (shugoshin: Drosophila homolog mei-s332), a protector of the centromeric cohesin Rec8 in fission yeast. A homologue of Sgo1 was also identified in budding yeast. Evidence is provided that shugoshin is widely conserved among eukaryotes. Moreover, Sgo2, a paralogue of shugoshin, was identified in fission yeast that is required for faithful mitotic chromosome segregation. Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1. These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis (Kitajima, 2004).
Nerusheva, O. O., Galander, S., Fernius, J., Kelly, D. and Marston, A. L. (2014). Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation. Genes Dev 28: 1291-1309. PubMed ID: 24939933
During mitosis and meiosis, sister chromatid cohesion resists the pulling forces of microtubules, enabling the generation of tension at kinetochores upon chromosome biorientation. How tension is read to signal the bioriented state remains unclear. Shugoshins form a pericentromeric platform that integrates multiple functions to ensure proper chromosome biorientation. This study shows that budding yeast shugoshin Sgo1 (see Drosophila Mei-S332) dissociates from the pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1 kinase and the Sgo1-bound phosphatase protein phosphatase 2A (PP2A)-Rts1 (see Drosophila Twins) underlie a tension-dependent circuitry that enables Sgo1 removal upon sister kinetochore biorientation. Sgo1 dissociation from the pericentromere triggers dissociation of condensin and Aurora B (see Drosophila Aurora B) from the centromere, thereby stabilizing the bioriented state. Conversely, forcing sister kinetochores to be under tension during meiosis I leads to premature Sgo1 removal and precocious loss of pericentromeric cohesion. Overall, this study shows that the pivotal role of shugoshin is to build a platform at the pericentromere that attracts activities that respond to the absence of tension between sister kinetochores. Disassembly of this platform in response to intersister kinetochore tension signals the bioriented state. Therefore, tension sensing by shugoshin is a central mechanism by which the bioriented state is read (Nerusheva, 2014).
The mitotic checkpoint ensures proper chromosome segregation by delaying anaphase until chromosomes are aligned on the spindle. Following prolonged spindle damage, however, cells eventually exit mitosis and undergo apoptosis. A murine homolog of the yeast mitotic checkpoint gene BUB1 localizes to the kinetochore during mitosis. By expressing a dominant-negative mutant, it has been shown that mBub1 is not only required for the checkpoint response to spindle damage, but acts in the timing of a normal mitosis. In addition, when mBub1 function is compromised, cells escape apoptosis and continue cell cycle progression, despite leaving mitosis with a disrupted spindle. These data demonstrate a role for kinetochore-associated mBub1 in regulating exit from mitosis, and suggest functional links between the mitotic checkpoint and subsequent apoptotic events in G1 (Taylor, 1997).
A feedback control mechanism, or cell cycle checkpoint, delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. The murine homolog of Bub1 is not only required for checkpoint response to spindle damage, but also restrains progression through a normal mitosis. A human homolog of Bub3 has been characterized. It consists of a 37-kD protein with four WD repeats. Like Bub1, Bub3 localizes to kinetochores before chromosome alignment. In addition, Bub3 and Bub1 interact in mammalian cells. Deletion mapping was used to identify the domain of Bub1 required for binding Bub3. Significantly, this same domain is required for kinetochore localization of Bub1, suggesting that the role of Bub3 is to localize Bub1 to the kinetochore, thereby activating the checkpoint in response to unattached kinetochores. The identification of a human Mad3/Bub1-related protein kinase, hBubR1, which can also bind Bub3 in mammalian cells, is described. Ectopically expressed hBubR1 also localizes to kinetochores during prometaphase, but only when hBub3 is overexpressed. The implications of the common interaction between Bub1 and hBubR1 with hBub3 for checkpoint control are discussed (Taylor, 1998).
The kinetochore binds an evolutionarily conserved set of checkpoint proteins that function to monitor whether chromosomes have aligned properly at the spindle equator. Human cells contain two related protein kinases, hBUB1 and hBUBR1, that appear to have evolved from a single ancestral BUB1 gene. hBUB1- and hBUBR1-specific antibodies were generated so that the localization patterns of these kinases could be directly compared. In the human U2OS osteosarcoma cell line, hBUB1 first appears at kinetochores during early prophase before all kinetochores are occupied by hBUBR1 or CENP-F. Both proteins remain at kinetochores throughout mitosis but their staining intensity is reduced from anaphase onward. Kinetochores of unaligned chromosomes exhibit stronger hBUB1 and hBUBR1 staining. Immunoelectron microscopy shows that hBUBR1 appears to be concentrated in the outer kinetochore plate and in some instances the inner plate as well. When chromosome spreads were examined by light microscopy, hBUB1 and hBUBR1 were coincident with CENP-E. This suggests that both kinases are concentrated near the surface of the kinetochore where they can monitor kinetochore-microtubule interactions (Jablonski, 1998).
A 350-amino acid domain has been identified in the kinetochore motor CENP-E that specifies kinetochore binding in mitosis but not during interphase. The kinetochore binding domain was used in a yeast two-hybrid screen to isolate interacting proteins that include the kinetochore proteins CENP-E, CENP-F, and hBUBR1, a BUB1-related kinase that was found to be mutated in some colorectal carcinomas. CENP-F, hBUBR1, and CENP-E assemble onto kinetochores in sequential order during late stages of the cell cycle. These proteins therefore define discrete steps along the kinetochore assembly pathway. Kinetochores of unaligned chromosome exhibit stronger hBUBR1 and CENP-E staining than those of aligned chromosomes. CENP-E and hBUBR1 remain colocalized at kinetochores until mid-anaphase when hBUBR1 localizes to portions of the spindle midzone that do not overlap with CENP-E. Since CENP-E and hBUBR1 can coimmunoprecipitate with each other from HeLa cells, they may function as a motor-kinase complex at kinetochores. However, the complex distribution pattern of hBUBR1 suggests that it may regulate multiple functions that include the kinetochore and the spindle midzone (Chan, 1998).
Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator. In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint. hBUBR1 is also an essential component of the mitotic checkpoint. hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis. Additionally, hBUBR1 is essential for normal mitotic progression, since it prevents cells from prematurely entering anaphase. One of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E. hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis. hBUBR1 kinase activity is rapidly stimulated when the spindle is disrupted in mitotic cells. Finally, hBUBR1 is associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells. The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E-dependent activities at the kinetochore and regulating cyclosome/APC activity (Chan, 1999).
Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle. Enforced expression of human BUBR1, but not a BUBR1 mutant allele, enhances accumulation of mitotic cells. Yeast two-hybrid system and GST-pulldown analyses show that p55CDC/hCdc20, a protein known to link spindle checkpoint components such as MAD2 to anaphase promoting complex (APC), interacts with BUBR1. In addition, p55CDC is capable of pulling down BUBR1 in sf-9 cells infected with both p55CDC and His6-BUBR1 recombinant baculoviruses but not in the cells infected with p55CDC baculoviruses or with the baculoviral vector alone. Moreover, immunoprecipitation followed by Western blot analyses confirmed that native p55CDC is associated with BUBR1 in HeLa cells. Spindle checkpoint activation by nocodazole treatment enhances the association between p55CDC and His6-BUBR1. In nocodazole-arrested mitotic cells, both CDC16 and hyperphosphorylated CDC27 (see Drosophila Cdc27), two APC components, preferentially associate with His6-BUBR1 resins, but not the control resins. Furthermore, BUBR1 phosphorylates p55CDC in vitro, and the phosphorylation of p55CDC by BUBR1 appears to be correlated with spindle checkpoint activation. Together, these studies strongly suggest that BUBR1 may target APC via p55CDC (Wu, 2000).
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 localize 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, relative localizations during mitosis were characterized in the presence and absence of microtubule toxins. In prometaphase cells, both kinases colocalize to the same domain of the kinetochore. However, whereas the localization of BubR1 at sister kinetochores is symmetrical, localization 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 localizing 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 localization is sensitive to changes in both tension and microtubule attachment. Consistent with this, Bub1 is shown to be 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. It is suggested 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 (Taylor, 2001).
Metaphase checkpoint controls sense abnormalities of chromosome alignment during mitosis and prevent progression to anaphase until proper alignment has been attained. A number of proteins, including mad2, bub1, and bubR1, have been implicated in the metaphase checkpoint control in mammalian cells. Metaphase checkpoints have been shown, in various systems, to read loss of either spindle tension or microtubule attachment at the kinetochore. Characteristically, HeLa cells arrest in metaphase in response to low levels of microtubule inhibitors that leave an intact spindle and a metaphase plate. The arrest induced by nanomolar vinblastine is shown to correlate with loss of tension at the kinetochore, and, in response, the checkpoint proteins bub1 and bubR1 are recruited to the kinetochore but mad2 is not. mad2 remains competent to respond and is recruited at higher drug doses that disrupt spindle association with the kinetochores. Further, although mad2 forms a complex with cdc20, it does not associate with bub1 or bubR1. It is concluded that mammalian bub1/bubR1 and mad2 operate as elements of distinct pathways sensing tension and attachment, respectively (Skoufias, 2001).
The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. The mechanism by which the checkpoint inhibits the APC/C has been examined by purifying an APC/C inhibitory factor from HeLa cells. This factor has been called the mitotic checkpoint complex (MCC) since it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, since it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells is sensitive to inhibition by MCC. The majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes do not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. It is proposed that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC (Sudakin, 2001).
The mitotic checkpoint blocks the activation of the anaphase-promoting complex (APC) until all sister chromatids have achieved bipolar attachment to the spindle. A checkpoint complex containing BubR1 and Bub3 has been purified from mitotic human cells. Upon checkpoint activation, the BubR1-Bub3 complex interacts with Cdc20. In the absence of Mad2, BubR1 inhibits the activity of APC by blocking the binding of Cdc20 to APC. Surprisingly, the kinase activity of BubR1 is not required for the inhibition of APCCdc20. BubR1 also prevents the activation of APCCdc20 in Xenopus egg extracts, and restores the mitotic arrest in Cdc20-overexpressing cells treated with nocodazole. Because BubR1 also interacts with the mitotic motor CENP-E, the ability of BubR1 to inhibit APC may be regulated by kinetochore tension or occupancy (Tang, 2001).
The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. A Xenopus homolog of the spindle checkpoint component Bub1 has been isolated, and its role in the spindle checkpoint has been investigated 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. These 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 (Sharp-Baker, 2001).
The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk; see Drosophila RSK) involves components of the spindle assembly checkpoint. This study shows that xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. It is concluded that Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk) (Schwab, 2001).
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. 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 (Chen, 2002).
The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. In checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC (Fang, 2002).
The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. PtK1 cells in hypothermic conditions (23°C) have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23°C, PtK1 cells remain in metaphase for an average of 101 min, compared with 21 min for cells at 37°C. The metaphase delay at 23°C is abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint are responsible for the prolonged metaphase. Live cell imaging shows that kinetochore Mad2 becomes undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23°C, and metaphase kinetochores at 23°C exhibit higher levels of 3F3/2, Bub1, and BubR1 compared with 37°C. Microinjection of anti-BubR1 antibody abolishes the metaphase delay at 23°C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induces anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. It is concluded that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling (Shannon, 2002).
The Ran GTPase is required for nuclear assembly, nuclear transport, spindle assembly, and mitotic regulation. While the first three processes are relatively well understood, details of Ran's role in mitotic progression remain obscure. Elevated levels of Ran's exchange factor (RCC1) have been found to abrogate the spindle assembly checkpoint in Xenopus egg extracts, restore APC/C activity, and disrupt the kinetochore localization of checkpoint regulators, including Mad2, CENP-E, Bub1, and Bub3. Depletion of Ran's GTPase activating protein (RanGAP1) and its accessory factor (RanBP1) similarly abrogates checkpoint arrest. By contrast, the addition of RanGAP1 and RanBP1 to extracts with exogenous RCC1 restores the spindle checkpoint. Together, these observations suggest that the spindle checkpoint is directly responsive to Ran-GTP levels. Finally, a clear wave of RCC1 association to mitotic chromosomes has been observed at the metaphase-anaphase transition in normal cycling extracts, suggesting that this mechanism has an important role in unperturbed cell cycles (Arnaoutov, 2003).
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. ZM447439, a novel selective Aurora kinase inhibitor, is described. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, it has been shown that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset (Ditchfield, 2003).
The abnormal expression of breast cancer-specific gene 1 (BCSG1) in malignant mammary epithelial cells is highly associated with the development and progression of breast cancer. BCSG1 expression has been found to significantly stimulates proliferation, invasion, and metastasis of breast cancer cells. However, little is known about how BCSG1 exerts its oncogenic functions. To elucidate the cellular mechanisms underlying the effects of BCSG1 in breast cancer cells, a yeast two-hybrid system was used to discover proteins that could associate with BCSG1. Through this screening, the mitotic checkpoint protein BubR1 was identified as a novel binding partner of BCSG1. The specific association of BCSG1 with BubR1 in breast cancer cells was demonstrated by immunoprecipitation and GST pull-down assays. Intriguingly, experiments conducted in four different cell lines all showed that exogenous expression of BCSG1 consistently reduces the cellular levels of the BubR1 protein without affecting BubR1 mRNA expression. The tendency of endogenous BCSG1 expression coinciding with lower BubR1 protein levels was also observed in seven out of eight breast cancer cell lines. The reducing effect of BCSG1 on BubR1 protein expression can be prevented by treating BCSG1-transfected cells with MG-132, a selective 26S proteasome inhibitor, implying that the proteasome machinery may be involved in the BCSG1-induced reduction of the BubR1 protein. Accompanied with a reduction of BubR1 protein level, BCSG1 expression results in multinucleation of breast cancer cells upon treatment with spindle inhibitor nocodazole, indicating an impaired mitotic checkpoint. Taken together, these novel findings suggest that BCSG1 may accelerate the progression of breast cancer at least in part by compromising the mitotic checkpoint control through inactivation of BubR1 (Gupta, 2003).
The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. The small molecule Hesperadin has been identified as an inhibitor of chromosome alignment and segregation. The data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, these data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling (Hauf, 2003).
The stabilization of improper microtubule attachments is sufficient to explain the precocious exit from mitosis that Hesperadin induces in monastrol- and taxol-treated cells. However, even under conditions where none of the kinetochores are attached, Aurora B function is required to maintain checkpoint signaling over prolonged periods of time, indicating that it might also have a direct role in the spindle assembly checkpoint. Consistent with this notion, kinetochore localization of the checkpoint kinases BubR1 and Bub1 was found to be impaired in Hesperadin-treated cells. It is conceivable that Mad2, which is still present at kinetochores in cells treated with nocodazole and Hesperadin, is sufficient to sustain the transient mitotic delay that is observed. In contrast, the low levels of Mad2 at kinetochores in taxol-arrested cells might not be sufficient to delay cells in mitosis when BubR1 is depleted from kinetochores by Hesperadin (Hauf, 2003).
In summary, the data suggest that Aurora B has a dual role. It acts in the destabilization of improper microtubule attachments, which indirectly keeps checkpoint signaling active, but it also could have a more direct role in the spindle assembly checkpoint. This is consistent with data from budding yeast, where it was found that Ipl1 is required for the spindle assembly checkpoint in a kinetochore-dependent, but probably also in a kinetochore-independent, manner. Likewise, Aurora B antibodies have been shown to overcome a nocodazole-induced arrest both in Xenopus egg extracts and cultured cells, also suggesting a direct role of Aurora B in the spindle assembly checkpoint (Hauf, 2003).
BubR1 and Bub1 could be Aurora B substrates that play a role in either of these pathways, or in both. It is conceivable that BubR1 and Bub1 themselves have dual roles. Both proteins have been shown to be required for checkpoint signaling in the presence of unattached kinetochores. Interestingly, in some experimental settings, their kinase activity is not required for checkpoint function, but Bub1's kinase activity is essential for a genetically separable function that may be required for microtubule-kinetochore attachments. It will therefore be interesting to test if the kinase activity of both Bub1 and BubR1 and their Aurora B-dependent recruitment to kinetochores is required to regulate kinetochore attachments (Hauf, 2003).
The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway was studied. The results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other (Vigneron, 2004).
Sister chromatids in mammalian cells remain attached mostly at their centromeres at metaphase because of the loss of cohesion along chromosome arms in prophase. Bub1 retains centromeric cohesion in mitosis of human cells. Depletion of Bub1 or Shugoshin (Sgo1: Drosophila homolog Mei-s332) in HeLa cells by RNA interference causes massive missegregation of sister chromatids that originates at centromeres. Surprisingly, loss of chromatid cohesion in Bub1 and Sgo1 RNA-interference cells does not appear to require the full activation of separase but, instead, triggers a mitotic arrest that depends on Mad2 and Aurora B. Bub1 maintains the steady-state levels and centromeric localization of Sgo1. Therefore, Bub1 protects centromeric cohesion through Shugoshin in mitosis (Tang, 2004).
The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other components to generate a diffusible 'stop anaphase' inhibitor. Not only does direct binding of BubR1 to the centromere-associated kinesin family member CENP-E activate its essential kinase, binding of a motorless fragment of CENP-E is shown here to constitutively activate BubR1 bound at kinetochores, producing checkpoint signaling that is not silenced either by spindle microtubule capture or the tension developed at those kinetochores by other components. Using purified BubR1, microtubules, and CENP-E, microtubule capture by the CENP-E motor domain is shown to silence BubR1 kinase activity in a ternary complex of BubR1-CENP-E-microtubule. Together, this reveals that CENP-E is the signal transducing linker responsible for silencing BubR1-dependent mitotic checkpoint signaling through its capture at kinetochores of spindle microtubules (Mao, 2005).
Shugoshin (Sgo) proteins constitute a conserved protein family defined as centromeric protectors of Rec8-containing cohesin complexes in meiosis. In vertebrate mitosis, Scc1/Rad21-containing cohesin complexes (see Drosophila Rad21) are also protected at centromeres because arm cohesin, but not centromeric cohesin, is largely dissociated in prophase and prometaphase. The dissociation process is dependent on the activity of polo-like kinase (Plk1) and partly dependent on Aurora B. Recently, it has been demonstrated that vertebrate shugoshin is required for preserving centromeric cohesion during mitosis; however, whether human shugoshin protects cohesin itself was not addressed. The persistence of human Scc1 at centromeres in mitosis is indeed dependent on human Sgo1. In fission yeast, Sgo localization depends on Bub1, a conserved spindle checkpoint protein, which is enigmatically also required for chromosome congression during prometaphase in vertebrate cells. Human Sgo1 fails to localize at centromeres in Bub1-repressed cells, and centromeric cohesion is significantly loosened. Remarkably, in these cells, Sgo1 relocates to chromosomes all along their length and provokes ectopic protection from dissociation of Scc1 on chromosome arms. These results reveal a hitherto concealed role for human Bub1 in defining the persistent cohesion site of mitotic chromosomes (Kitajima, 2005).
The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore-microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. Live-cell imaging coupled with RNA interference was used to investigate the functions of human Bub1. Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore-microtubule attachments (Meraldi, 2005).
The spindle checkpoint is a cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a protein serine-threonine kinase that plays multiple roles in chromosome segregation and the spindle checkpoint. In response to misaligned chromosomes, Bub1 directly inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator Cdc20. The protein level and the kinase activity of Bub1 are regulated during the cell cycle; they peak in mitosis and are low in G1/S phase. Bub1 is degraded during mitotic exit and degradation of Bub1 is mediated by APC/C in complex with its activator Cdh1 [APC/C(Cdh1)]. Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1, whereas depletion of Cdh1 by RNA interference increases the level of the endogenous Bub1 protein. Bub1 is ubiquitinated by immunopurified APC/C(Cdh1) in vitro. Two KEN-box motifs on Bub1 were identified that are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells but fails to elicit a cell cycle phenotype, indicating that degradation of Bub1 by APC/C(Cdh1) is not required for mitotic exit. Nevertheless, this study clearly demonstrates that Bub1, an APC/C inhibitor, is also an APC/C substrate. The antagonistic relationship between Bub1 and APC/C may help to prevent the premature accumulation of Bub1 during G1 (Qi, 2007).
The accurate segregation of chromosomes in mitosis requires the stable attachment of microtubules to kinetochores. The details of this complex and dynamic process are poorly understood. This study reports the interaction of a kinetochore-associated mitotic checkpoint kinase, BubR1, with two microtubule plus end-associated proteins, adenomatous polyposis coli (APC) and EB1, providing a potential link in stable kinetochore microtubule attachment. Using immunodepletion from and antibody addition to Xenopus laevis egg extracts, it was shown that BubR1 and its kinase activity are essential for positioning chromosomes at the metaphase plate. BubR1 associates with APC and EB1 in egg extracts, and the complex formation is necessary for metaphase chromosome alignment. Using purified components, BubR1 directly phosphorylates APC and forms a ternary complex with APC and microtubules. These findings support a model in which BubR1 kinase may directly regulate APC function involved in stable kinetochore microtubule attachment (Zhang, 2007).
The spindle checkpoint controls mitotic progression. Checkpoint proteins are temporally recruited to kinetochores, but their docking site is unknown. A human kinetochore oncoprotein, AF15q14/blinkin, a member of the Spc105/Spc7/KNL-1 family, directly links spindle checkpoint proteins BubR1 and Bub1 to kinetochores and is required for spindle checkpoint and chromosome alignment. Blinkin RNAi causes accelerated mitosis due to a checkpoint failure and chromosome misalignment resulting from the lack of kinetochore and microtubule attachment. Blinkin RNAi phenotypes resemble the double RNAi phenotypes of Bub1 and BubR1 in living cells. While the carboxy domain associates with the c20orf172/hMis13 and DC8/hMis14 subunits of the hMis12 complex in the inner kinetochore, association of the amino and middle domain of blinkin with the TPR domains in the amino termini of BubR1 and Bub1 is essential for BubR1 and Bub1 to execute their distinct mitotic functions. Blinkin may be the center of the network for generating kinetochore-based checkpoint signaling (Kiyomitsu, 2007).
The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. This study demonstrated that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1 (see Drosophila Mps1). Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint (London, 2014).
The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. It is proposed that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling (Zachos, 2007).
Mitotic phosphorylation of the spindle checkpoint component BubR1 is highly conserved throughout evolution. This study demonstrates that BubR1 is phosphorylated on the Cdk1 site T620, which triggers the recruitment of Plk1 and phosphorylation of BubR1 by Plk1 both in vitro and in vivo. Phosphorylation does not appear to be required for spindle checkpoint function but instead is important for the stability of kinetochore-microtubule (KT-MT) interactions, timely mitotic progression, and chromosome alignment onto the metaphase plate. By quantitative mass spectrometry, S676 was identified as a Plk1-specific phosphorylation site on BubR1. Furthermore, using a phospho-specific antibody, this site was shown to be phosphorylated during prometaphase, but dephosphorylated at metaphase upon establishment of tension between sister chromatids. These findings describe the first in vivo verified phosphorylation site for human BubR1, identify Plk1 as the kinase responsible for causing the characteristic mitotic BubR1 upshift, and attribute a KT-specific function to the hyperphosphorylated form of BubR1 in the stabilization of KT-MT interactions (Elowe, 2007).
This study describes the identification of S676 as a highly conserved Plk1-specific target site on BubR1. Several lines of evidence indicate that phosphorylation of this site correlates with lack of tension. (1) KTs stained with anti-pS676 antibody preferentially during prometaphase. Staining was gradually lost as sister chromatids reached metaphase, concomitant with the generation of tension in response to bipolar attachment. (2) Prometaphase cells were equally phosphorylated on S676 in the presence of Taxol or nocodazole, indicating that loss of tension rather than lack of attachment efficiently induced phosphorylation. (3) BubR1 phosphorylation was enhanced at KTs of misaligned chromosomes in CenpE-depleted cells, presumed to be attached but not under tension. (4) Taxol treatment of metaphase cells led to rapid reacquisition of the anti-pS676 signal and concomitant loss of RanGAP1 binding to KTs, indicative of destabilized KT-MT interactions due to loss of tension. These results raise the question of how pS676 relates to the tension-related 3F3/2 phospho-epitope. A recent study in Xenopus suggested that loading of the 3F3/2 epitope onto KTs is dependent on prior assembly of checkpoint proteins and that simultaneous recruitment of BubR1 and Plk1 represents the final step in KT loading of the 3F3/2 antigen. Although these observations made it tempting to speculate that pS676 could make a major contribution to the 3F3/2 epitope, preliminary experiments have failed to substantiate this possibility (Elowe, 2007).
At present, the exact biochemical role of phosphorylation of BubR1 on S676 (and presumably other Plk1 sites) is not known, but it is emphasized that this phosphorylation is transient during M-phase progression. In prophase, before MTs approach KTs, there is virtually no phosphorylation at S676, which is in striking contrast to 3F3/2 staining and indicates that S676 phosphorylation requires KT-MT interactions. As cells progress through prometaphase, KTs begin to associate with spindle MTs, but as long as sister chromosomes have not undergone bipolar attachment, tension is absent and KT-MT interactions are unstable. At this stage, S676 on KT-associated BubR1 is maximally phosphorylated by Plk1, and it is postulated that the highly phosphorylated BubR1 contributes to the establishment of stable KT-MT interactions during chromosome congression. However, as soon as KT-MT interactions come under tension (due to bipolar attachment), phosphate is lost from S676. It is thus proposed that transient phosphorylation of BubR1 on S676 (and perhaps other Plk1 sites) is required for stabilizing KT-MT interactions. Whether this involves a conformational change in BubR1 or the recruitment of additional proteins remains to be determined in the future. Finally, it will be interesting to explore whether this tension-sensitive phosphorylation is regulated primarily at the level of the substrate, the upstream kinase (Plk1), or an as-yet-unidentified antagonistic phosphatase (Elowe, 2007).
BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control, as an active kinase at unattached kinetochores, and as a cytosolic inhibitor of APC/C(Cdc20) activity, as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, it was demonstrated that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. The BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G(2) phase prior to mitosis onset. Together, these results suggest that kinetochore-bound BubR1 is nonessential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/C(Cdc20) during interphase to prevent unscheduled degradation of specific APC/C substrates (Malureanu, 2009).
Premature anaphase onset is prevented by the mitotic checkpoint through production of a 'wait anaphase' inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, this study demonstrates that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. Purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. These results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1 (Kulukian, 2009).
While unattached kinetochores have been widely inferred to be the source of a 'wait anaphase' mitotic checkpoint inhibitor, this study has now demonstrated that kinetochores can, in fact, catalyze production of an initial Mad2-Cdc20 inhibitor, significantly accelerating the initial rate of its production. Unattached kinetochores did not affect inhibition by Bub3/BubR1 in the absence of Mad2. Production of at least two inhibitors can be enhanced by unattached kinetochores: one containing diffusible Cdc20 and another in which Cdc20 is already bound in a megadalton complex to APC/C, consistent with reports that Cdc20 and checkpoint proteins are present in two complexes with differing sizes during mitosis. Both inhibitors prevent recognition by APC/C of cyclin B as an ubiquitination substrate. Disruption of cyclin B ubiquitination by a kinetochore-derived inhibitor even while Cdc20 remains bound to APC/C provides a potential explanation for the differential timing of destruction of cyclins A and B. Instead of simple sequestration of Cdc20, a kinetochore-derived mitotic checkpoint inhibitor bound to APC/CCdc20 may block recognition of cyclin B as an ubiquitination substrate, while permitting APC/CCdc20-mediated ubiquitination and destruction of cyclin A, an event that is known to initiate immediately after mitotic entry (Kulukian, 2009).
Despite amplification of Cdc20 inhibition when equal molar levels of BubR1, Mad2, and Cdc20 were added, no evidence was found for assembly of a quaternary mitotic checkpoint- (MCC-) like complex as a bona fide inhibitor produced by unattached kinetochores. Rather, almost all Cdc20 shifted to a complex comigrating with the majority of BubR1 but containing very little Mad2. Also arguing against a contribution in kinetochore-derived checkpoint signaling, it is noted that MCC-like complexes in animal cells are present outside of mitosis, and their formation in yeast continues in the absence of a functional centromere/kinetochore. All of this supports an MCC-like, premade Cdc20 inhibitor produced in a kinetochore-independent manner in interphase that restrains APC/C ubiquitination activity for cyclin B just after mitotic entry, which has been referred to as a 'timer' (Kulukian, 2009).
More importantly, at physiologically relevant concentrations of unattached kinetochores and Mad2, chromosomes catalyzed production of Cdc20 inhibition of cyclin B recognition by APC/C by at least 8-fold relative to inhibitors formed spontaneously in the absence of chromosomes. The actual in vivo effect is likely to be much greater than observed in vitro, since chromosome purification resulted in partial loss of signaling molecules from kinetochores, including a proportion of Mad1 and kinases that include Bub1, BubR1, and Aurora B (Kulukian, 2009).
Chromosome amplification of Cdc20 inhibition required Mad1 recruitment of Mad2 to kinetochores and dimerization-competent Mad2, thereby providing a direct demonstration that a Mad1:Mad2 core complex recruits and converts soluble 'inactive' Mad2 into a more potent inhibitor of Cdc20. At least part of this is from action of kinetochores on Mad2. Although it has previously been argued that the kinetochore may sensitize the APC/C for checkpoint-mediated inhibition, direct contact of chromosomes with APC/C was not required to amplify inhibition. While a kinetochore-dependent function of BubR1 can by no means be excluded from roles in microtubule attachment and chromosome alignment or from further amplification of a kinetochore derived signal, kinetochore-mediated enhancement of Cdc20 inhibition did not require BubR1 localization to or contact with kinetochores. It is concluded that immobilized, kinetochore-bound Mad1/Mad2, but not BubR1, catalyzes conversion at the kinetochore of soluble, open Mad2 into a form with its seatbelt domain poised for Cdc20 capture. Further support for this conclusion includes evidence that kinetochore-bound BubR1 is nonessential (Kulukian, 2009).
Moreover, incubation of physiologically relevant concentrations of each component ultimately produced most Cdc20 bound to BubR1, not Mad2, whether or not chromosomes were present. In fact, amplification of Cdc20 inhibition by unattached kinetochores was accompanied by a shift to a more rapidly eluting Bub3/BubR1-Cdc20 complex, without a stable pool of Mad2-Cdc20. Evidence also demonstrated that most Cdc20 is complexed with BubR1 in vivo, rather than Mad2. A model from all of this is proposed in which Mad1/Mad2 immobilized at kinetochores templates conversion of an inactive, open Mad2 to one capable of transient capture of Cdc20 followed by relay to BubR1 as sequentially produced mitotic checkpoint inhibitors that may be soluble or APC/C bound. This evidence supports Mad2-Cdc20, and perhaps an MCC-like complex, as a transient intermediate in kinetochore-mediated checkpoint signaling and one that is a precursor to BubR1-Cdc20. Further, Bub3/BubR1 binds to APC/C, but only in a Mad2-dependent manner that is stimulated by unattached kinetochores, demonstrating that kinetochores facilitate loading of Bub3/BubR1 onto APC/C. That BubR1-APC/CCdc20 is produced indirectly by unattached kinetochores as the final Cdc20 inhibitor would also support suggestions that BubR1 acts as a nonproductive pseudosubstrate of the APC/C or mediates Cdc20 proteolytic turnover (Kulukian, 2009).
Combining kinetochore-derived Bub3/BubR1-Cdc20 with evidence for two Cdc20 binding sites on BubR1 further suggests that the spontaneous and kinetochore-derived Bub3/BubR1-Cdc20 complexes may represent generation of Cdc20 bound at the two different sites, respectively, a point now testable with the appropriate BubR1 mutants (Kulukian, 2009).
Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes. Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks. Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1(F) allele. Loss of Bub1 greatly accelerates resolution of chiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres. By using a quantitative assay for APC/C-mediated securin destruction, it as shown that the APC/C is activated in an exponential fashion, with activity peaking 12-13 hr after GVBD, and that this process is advanced by 5 hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/C's Apc2 subunit or separase. Finally, it was showm that Bub1's kinase domain is not required to delay APC/C activation. It is concluded that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr (McGuinness, 2009).
The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. This study found that PP2A in association with its B56gamma regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56gamma, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2A's role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. This study shows that inactivation of only the PP2A-B56gamma subunit can affect the efficiency of the SAC. Data is provided that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant (Varadkar, 2017).
The spindle assembly checkpoint (SAC) prevents premature sister chromatid separation during mitosis. Phosphorylation of unattached kinetochores by the Mps1 kinase (see Drosophila Mps1) promotes recruitment of SAC machinery that catalyzes assembly of the SAC effector mitotic checkpoint complex (MCC). The SAC protein Bub3 (see Drosophila Bub3) is a phospho-amino acid adaptor that forms structurally related stable complexes with functionally distinct paralogs named Bub1 (see Drosophila Bub1) and BubR1 (see Drosophila Bub1R). A short motif ("loop") of Bub1, but not the equivalent loop of BubR1, enhances binding of Bub3 to kinetochore phospho-targets. This study asked whether the BubR1 loop directs Bub3 to different phospho-targets. The BubR1 loop is essential for SAC function and cannot be removed or replaced with the Bub1 loop. BubR1 loop mutants bind Bub3 and are normally incorporated in MCC in vitro but have reduced ability to inhibit the MCC target anaphase-promoting complex (APC/C), suggesting that BubR1:Bub3 recognition and inhibition of APC/C requires phosphorylation. Thus, small sequence differences in Bub1 and BubR1 direct Bub3 to different phosphorylated targets in the SAC signaling cascade (Overlack, 2017).
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