Aurora B hyperactivation by Bub1 overexpression promotes chromosome missegregation

High expression of the mitotic kinase Bub1 is associated with a variety of human cancers and correlates with poor clinical prognosis, but whether Bub1 alone can drive tumorigenesis was unknown. We provided conclusive evidence that Bub1 has oncogenic properties by generating transgenic mice that overexpress Bub1 in a wide variety of tissues, resulting in aneuploidization. Consistently, Bub1 transgenic mice developed various kinds of spontaneous tumors as well as accelerated Mycinduced lymphomagenesis. While the mitotic checkpoint was robust in Bub1 overexpressing cells, misaligned and lagging chromosomes were observed. These defects originated from increased Aurora B activity and could be suppressed by inhibition of Aurora B. Taken together, this indicates that Bub1 has oncogenic properties and imply that aneuploidization and tumorigenesis result from Aurora B-dependent missegregation. Here, we focus on the complex relationship between Bub1 and Aurora B and discuss the broader implications of Bub1-dependent Aurora B activation in mediating error correction.Key words: Bub1, mitotic checkpoint, Aurora B, chromosome segregation, aneuploidy     

Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity

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.     

Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation

The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.     

Bub1 kinase activity drives error correction and mitotic checkpoint control but not tumor suppression

The mitotic checkpoint protein Bub1 is essential for embryogenesis and survival of proliferating cells, and bidirectional deviations from its normal level of expression cause chromosome missegregation, aneuploidy, and cancer predisposition in mice. To provide insight into the physiological significance of this critical mitotic regulator at a modular level, we generated Bub1 mutant mice that lack kinase activity using a knockin gene-targeting approach that preserves normal protein abundance. In this paper, we uncover that Bub1 kinase activity integrates attachment error correction and mitotic checkpoint signaling by controlling the localization and activity of Aurora B kinase through phosphorylation of histone H2A at threonine 121. Strikingly, despite substantial chromosome segregation errors and aneuploidization, mice deficient for Bub1 kinase activity do not exhibit increased susceptibility to spontaneous or carcinogen-induced tumorigenesis. These findings provide a unique example of a modular mitotic activity orchestrating two distinct networks that safeguard against whole chromosome instability and reveal the differential importance of distinct aneuploidy-causing Bub1 defects in tumor suppression.     

Early aging-associated phenotypes in Bub3/Rae1 haploinsufficient mice

Aging is a highly complex biological process that is believed to involve multiple mechanisms. Mice that have small amounts of the mitotic checkpoint protein BubR1 age much faster than normal mice, but whether other mitotic checkpoint genes function to prevent the early onset of aging is unknown. In this study, we show that several aging-associated phenotypes appear early in mice that are double haploinsufficient for the mitotic checkpoint genes Bub3 and Rae1 but not in mice that are single haploinsufficient for these genes. Mouse embryonic fibroblasts (MEFs) from Bub3/Rae1 haploinsufficient mice undergo premature senescence and accumulate high levels of p19, p53, p21, and p16, whereas MEFs from single haploinsufficient mice do not. Furthermore, although BubR1 hypomorphic mice have less aneuploidy than Bub3/Rae1 haploinsufficient mice, they age much faster. Our findings suggest that early onset of aging-associated phenotypes in mice with mitotic checkpoint gene defects is linked to cellular senescence and activation of the p53 and p16 pathways rather than to aneuploidy.     

Aurora B hyperactivation by Bub1 overexpression promotes chromosome missegregation

High expression of the mitotic kinase Bub1 is associated with a variety of human cancers and correlates with poor clinical prognosis, but whether Bub1 alone can drive tumorigenesis was unknown. We provided conclusive evidence that Bub1 has oncogenic properties by generating transgenic mice that overexpress Bub1 in a wide variety of tissues, resulting in aneuploidization. Consistently, Bub1 transgenic mice developed various kinds of spontaneous tumors as well as accelerated Myc-induced lymphomagenesis. While the mitotic checkpoint was robust in Bub1 overexpressing cells, misaligned and lagging chromosomes were observed. These defects originated from increased Aurora B activity and could be suppressed by inhibition of Aurora B. Taken together, this indicates that Bub1 has oncogenic properties and imply that aneuploidization and tumorigenesis result from Aurora B-dependent missegregation. Here, we focus on the complex relationship between Bub1 and Aurora B and discuss the broader implications of Bub1-dependent Aurora B activation in mediating error correction.     

Bub1: Escapades in a Cellular World

The spindle assembly checkpoint is an important surveillance mechanism that ensures high fidelity mitotic chromosome segregation. This is accomplished by monitoring whether sister chromatids lack tension or attachment to spindle microtubules. It is mediated by checkpoint complexes or individual proteins that inhibit the ubiquitin ligase activity of the anaphase-promoting complex/ cyclosome (APC/C) via targeting of the Cdc20 regulatory subunit. The Bub1 kinase is a key spindle checkpoint regulatory protein. Bub1 also plays more pleiotropic roles. Thus, Bub1 is required for assembly of a functional inner centromere, sister chromatid cohesion via targeting of the Shugoshin protein, and metaphase congression. Evidence based on Bub1 mutations in colorectal cancers suggests it might be a driving force in tumorigenesis via generation of chromosomal instability (CIN) and aneuploidy. Recently we reported a surveillance mechanism linking loss of Bub1 to activation of the p53 pathway, specifically premature cell senescence in normal human fibroblasts. Interestingly, SV40 large T antigen (LT) targets Bub1 and this is correlated with oncogenic transformation and compromise of the spindle checkpoint. Future studies on Bub1 combining genetic approaches with analysis of LT perturbations are likely to yield further insight.     

Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod

We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways. Received: 6 August 1998 / Accepted: 28 August 1998     

Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function

The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus (see [1] [2] [3] [4] for reviews). The Mad and Bub proteins were identified as key components of the spindle checkpoint through budding yeast genetics [5] [6] and are highly conserved [3]. Most of the spindle checkpoint proteins have been localised to kinetochores, yet almost nothing is known about the molecular events which take place there. Mad1p forms a tight complex with Mad2p [7], and has been shown to recruit Mad2p to kinetochores [8]. Similarly, Bub3p binds to Bub1p [9] and may target it to kinetochores [10]. Here, we show that budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and that this complex is found at significantly higher levels once the spindle checkpoint is activated. We find that formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, we identify a conserved motif 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.     

Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1

The spindle checkpoint controls mitotic progression. Checkpoint proteins are temporally recruited to kinetochores, but their docking site is unknown. We show that 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.     

Changes in cell morphology and cytoskeletal organization are induced by human mitotic checkpoint gene, Bub1

The mitotic spindle checkpoint prevents the onset of anaphase and subsequent cell division until chromosomes are properly aligned on a bipolar spindle. Thus, it regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. The budding uninhibited by benzimidazole (Bub1) is a key component of mitotic checkpoint. Bub1 encodes a serine/threonine kinase required for mitotic spindle checkpoint function. The regulation of cell morphology in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). Here we show that Bub1 directly affects the structural integrity of IFs. Constitutive expression of Bub1 caused disappearance of filamentous vimentin, a type III IF, and consequently changed cell morphology. Expression of kinase domain—deleted Bub1 induced neither morphological change nor disappearance of vimentin. These observations suggest that Bub1 not only regulates the cell cycle, but also may be involved in the cytoskeletal control in interphase cells.     

Bub1, a gatekeeper for Cdc20-dependent mitotic exit

The mitotic spindle assembly checkpoint arrests cells at metaphase by suppressing Cdc20, a protein required to trigger ubiquitination and consequent degradation of cyclin B. New evidence from Tang et al. appearing in the November 5th issue of Molecular Cell finds that one of the checkpoint proteins, Bub1, specifically phosphorylates Cdc20 to suppress APC/C activation.     

Budding yeast Bub2 is localized at spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2.

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.     

Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint

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. We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed 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 did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was 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.     

Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint

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. Checkpoint proteins are recruited to kinetochores when individual kinetochores are not bound to spindle microtubules or not under tension. 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. 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. 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.     

Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint

Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast. We have found that 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. Finally, we show that there is a Bub1p-dependent meiotic checkpoint, which is activated in recombination mutants.     

Phosphorylation of Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the spindle checkpoint

To ensure the fidelity of chromosome segregation, the spindle checkpoint blocks the ubiquitin ligase activity of APC/C(Cdc20) in response to a single chromatid not properly attached to the mitotic spindle. Here we show that HeLa cells depleted for Bub1 by RNA interference are defective in checkpoint signaling. Bub1 directly phosphorylates Cdc20 in vitro and inhibits the ubiquitin ligase activity of APC/C(Cdc20) catalytically. A Cdc20 mutant with all six Bub1 phosphorylation sites removed is refractory to Bub1-mediated phosphorylation and inhibition in vitro. Upon checkpoint activation, Bub1 itself is hyperphosphorylated and its kinase activity toward Cdc20 is stimulated. Ectopic expression of the nonphosphorylatable Cdc20 mutant allows HeLa cells to escape from mitosis in the presence of spindle damage. Therefore, Bub1-mediated phosphorylation of Cdc20 is required for proper checkpoint signaling. We speculate that inhibition of APC/C(Cdc20) by Bub1 in a catalytic fashion may partly account for the exquisite sensitivity of the spindle checkpoint.     

TAp73α binds the kinetochore proteins Bub1 and Bub3 resulting in polyploidy

Aneuploidy is a characteristic of most solid tumors, often associated with negative prognosis. It can arise from two principal mechanisms: from a tetraploid intermediate state, or directly from errors at cell division. The control of cell division, crucial to maintain genomic stability, is still poorly understood in its relationship to aneuploidy. Here we show that the TAp73α isoform induces polyploidy when over-expressed. This is possibly due to the interaction of TAp73α with kinetochore-related proteins leading to the alteration of mitotic checkpoint abilities. TAp73α but not p53 or any of the other p73 isoforms binds Bub1 and Bub3. Since TAp73α is frequently over-expressed in cancer, this interaction may contribute to the aneuploidy observed in cancer progression. Our results suggest a novel molecular mechanism leading to aneuploidy involving interference of TAp73α with Bub1 and Bub3 resulting in an altered mitotic checkpoint.     

Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.     

Bub1 up-regulation and hyperphosphorylation promote malignant transformation in SV40 tag-induced transgenic mouse models

Rodents do not naturally develop prostate cancer. Currently, most widely used genetically engineered mouse prostate cancer models use SV40 T/tag oncogene. To understand the mechanism underlying prostate cancer development in transgenic and knock-in SV40 Tag mouse models, we did cDNA microarray analyses, comparing gene expression profiles of prostate cancer tissues from early-, late-, and advance-stage androgen-independent prostate cancers. Of the 67 genes that were up-regulated by > or = 10-fold, 40 are known to be required for chromosome stability. In particular, the spindle checkpoint component Bub1 was persistently up-regulated from early to advanced androgen-independent prostate cancer lesions. Significantly, Bub1, which is required for accurate chromosome segregation during mitosis, has recently been reported to bind SV40 Tag. Consistent with a spindle checkpoint defect, flow cytometry experiments indicate that advanced androgen-independent prostate cancer tumors exhibit aneuploidy, along with up-regulation of levels of both Bub1 mRNA and Bub1 protein or hyperphosphorylation. Importantly, up-regulation and hyperphosphorylation of Bub1 were also observed in established human prostate cancer cell lines and in clinical studies. Furthermore, analysis of human prostate cancer lines showed impaired spindle checkpoint function and endoreduplication following exposure to spindle toxins. Small interfering RNA-mediated repression of Bub1 in the human prostate cancer line PC-3 restrained cell proliferation, an effect mimicked by inhibition of mitogen-activated protein kinase, an upstream activator of Bub1. Thus, by perturbing Bub1 function, our observations suggest a new mechanism whereby the SV40 Tag oncoprotein promotes chromosomal instability and aneuploidy in transgenic mouse prostate cancer models. Whereas the exact details of this mechanism remain unclear, our novel findings raise the possibility of exploiting Bub1 as a new therapeutic target in the treatment of prostate cancer, the most common cancer in adult men in North America.