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 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.     

The yin and yang of the Cdkn2a locus in senescence and aging

Senescence of cultured cells involves activation of the p19Arf-p53 and the p16Ink4a-Rb tumor suppressor pathways. This, together with the observation that p19Arf and p16Ink4a expression increases with age in many tissues of humans and rodents, led to the speculation that these pathways drive in vivo senescence and natural aging. However, it has been difficult to test this hypothesis using a mammalian model system because inactivation of either of these pathways results in early death from tumors. One approach to bypass this problem would be to inactivate these pathways in a murine segmental progeria model such as mice that express low amounts of the mitotic checkpoint protein BubR1 (BubR1 hypomorphic mice). These mice have a five-fold reduced lifespan and develop a variety of early-aging associated phenotypes including cachetic dwarfism, skeletal muscle degeneration, cataracts, arterial stiffening, (subcutaneous) fat loss, reduced stress tolerance and impaired wound healing. Importantly, BubR1 hypomorphism elevates both p16Ink4a and p19Arf expression in skeletal muscle and fat. Inactivation of p16Ink4a in BubR1 mutant mice delays both cellular senescence and aging specifically in these tissues. Surprisingly, however, inactivation of p19Arf has the opposite effect; it exacerbates in vivo senescence and aging in skeletal muscle and fat. These mouse studies suggest that p16Ink4a is indeed an effector of aging and in vivo senescence, but p19Arf an attenuator. Thus, the role of the p19Arf-p53 pathway in aging and in vivo senescence seems far more complex than previously anticipated.     

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.     

BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.     

Timing and checkpoints in the regulation of mitotic progression

Accurate chromosome segregation relies on the precise regulation of mitotic progression. Regulation involves control over the timing of mitosis and a spindle assembly checkpoint that links anaphase onset to the completion of chromosome-microtubule attachment. In this paper, we combine live-cell imaging of HeLa cells and protein depletion by RNA interference to examine the functions of the Mad, Bub, and kinetochore proteins in mitotic timing and checkpoint control. We show that the depletion of any one of these proteins abolishes the mitotic arrest provoked by depolymerizing microtubules or blocking chromosome-microtubule attachment with RNAi. However, the normal progress of mitosis is accelerated only when Mad2 or BubR1, but not other Mad and Bub proteins, are inactivated. Moreover, whereas checkpoint control requires kinetochores, the regulation of mitotic timing by Mad2 and BubR1 is kinetochore-independent in fashion. We propose that cytosolic Mad2-BubR1 is essential to restrain anaphase onset early in mitosis when kinetochores are still assembling.     

A Highlights from MBoC Selection: The ARF tumor suppressor prevents chromosomal instability and ensures mitotic checkpoint fidelity through regulation of Aurora B

The ARF tumor suppressor is part of the CDKN2A locus and is mutated or undetectable in numerous cancers. The best-characterized role for ARF is in stabilizing p53 in response to cellular stress. However, ARF has tumor suppressive functions outside this pathway that have not been fully defined. Primary mouse embryonic fibroblasts (MEFs) lacking the ARF tumor suppressor contain abnormal numbers of chromosomes. However, no role for ARF in cell division has previously been proposed. Here we demonstrate a novel, p53-independent role for ARF in the mitotic checkpoint. Consistent with this, loss of ARF results in aneuploidy in vitro and in vivo. ARF^−/− MEFs exhibit mitotic defects including misaligned and lagging chromosomes, multipolar spindles, and increased tetraploidy. ARF^−/− cells exhibit overexpression of Mad2, BubR1, and Aurora B, but only overexpression of Aurora B phenocopies mitotic defects observed in ARF^−/− MEFs. Restoring Aurora B to near-normal levels rescues mitotic phenotypes in cells lacking ARF. Our results define an unexpected role for ARF in chromosome segregation and mitotic checkpoint function. They further establish maintenance of chromosomal stability as one of the additional tumor-suppressive functions of ARF and offer a molecular explanation for the common up-regulation of Aurora B in human cancers.     

A dominant interfering Bub1 mutant is insufficient to induce or alter thymic tumorigenesis in vivo, even in a sensitized genetic background

Aneuploidy is a common feature of human tumors, often correlating with poor prognosis. The mitotic spindle checkpoint is thought to play a major role in aneuploidy suppression. To investigate the role of the spindle checkpoint in tumor suppression in vivo, we developed transgenic mice in which thymocytes express a dominant interfering fragment of Bub1, a kinase regulator of the spindle checkpoint. We report that, despite high-level expression of dominant-negative Bub1 (Bub1DN), a protein known to inhibit spindle checkpoint activity in cultured cells, thymocytes show no evidence of spindle checkpoint impairment. Transgenic animals also failed to show an increased predisposition to spontaneous tumors. Moreover, the Bub1DN transgene failed to alter the timing or characteristics of thymic lymphoma development in p53 heterozygous or homozygous null backgrounds, indicating that the lack of tumorigenesis is not due to suppression by p53-dependent checkpoints. These results indicate that overexpression of a Bub1 N-terminal fragment is insufficient to impair the spindle checkpoint in vivo or to drive tumorigenesis in the highly susceptible murine thymocyte system, either alone or in combination with G(1) checkpoint disruption.     

有丝分裂检验点基因BubR1的研究进展

BubR1是存在于哺乳动物中的有丝分裂检查点基因家族Mad3的同源基因,其编码蛋白BubR1是一个多结构域蛋白,在监测细胞有丝分裂前中期向后期转化的过程中扮演重要的角色。BubR1可以通过自身或作为MCC的成分抑制APC的活性,从APC隔离Cdc20或者通过连接到微管驱动蛋白cENP—E,激活有丝分裂检查点信号级联放大。近年来关于BubR1的结构、功能及作用机理等研究工作颇为引人关注。这些研究表明:人BUBR11基因定位于人类染色体15q14-21,其编码蛋白BubR1在整个有丝分裂中聚集在外层动粒板;BubR1缺陷导致对DNA损伤的妥协反应,它的完全切除导致大量细胞凋亡甚至胚胎致死;BubR1单基因剔除可增强剔除基因组不稳定性,并导致肿瘤发生;BubR1表达减少至10%的导致一系列早老相关的表型出现;BubR1 /-Apcmin/ 复合突变提示BubR1和Apc相互作用调节中期一后期转化,这一反常现象可能在结直肠癌的基因组稳定性、发生和进展中发挥作用。本文将对BubR1蛋白就以上内容做一综述。     

Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31(comet)

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase-anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31(comet) delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31(comet) and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.     

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.     

Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex

Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) that is required for recruitment of dynein/dynactin to kinetochores but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences. Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores.     

Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes

The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold.     

Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR-KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.     

Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency

Expression of p16(Ink4a) and p19(Arf) increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16(Ink4a) or p19(Arf) die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.     

The Cdc20-binding Phe Box of the Spindle Checkpoint Protein BubR1 Maintains the Mitotic Checkpoint Complex During Mitosis

The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome, a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and anaphase-promoting complex/cyclosome inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms.     

Loss of BubR1 acetylation causes defects in spindle assembly checkpoint signaling and promotes tumor formation

BubR1 acetylation is essential in mitosis. Mice heterozygous for the acetylation-deficient BubR1 allele (K243R/+) spontaneously developed tumors with massive chromosome missegregations. K243R/+ mouse embryonic fibroblasts (MEFs) exhibited a weakened spindle assembly checkpoint (SAC) with shortened mitotic timing. The generation of the SAC signal was intact, as Mad2 localization to the unattached kinetochore (KT) was unaltered; however, because of the premature degradation of K243R-BubR1, the mitotic checkpoint complex disassociated prematurely in the nocodazole-treated condition, suggesting that maintenance of the SAC is compromised. BubR1 acetylation was also required to counteract excessive Aurora B activity at the KT for stable chromosome–spindle attachments. The association of acetylation-deficient BubR1 with PP2A-B56α phosphatase was reduced, and the phosphorylated Ndc80 at the KT was elevated in K243R/+ MEFs. In relation, there was a marked increase of micronuclei and p53 mutation was frequently detected in primary tumors of K243R/+ mice. Collectively, the combined effects of failure in chromosome–spindle attachment and weakened SAC cause genetic instability and cancer in K243R/+ mice.     

Bub1 and BubR1: at the interface between chromosome attachment and the spindle checkpoint

The spindle checkpoint ensures genome fidelity by temporarily halting chromosome segregation and the ensuing mitotic exit until the last kinetochore is productively attached to the mitotic spindle. At the interface between proper chromosome attachment and the metaphase-to-anaphase transition are the mammalian spindle checkpoint kinases. Compelling evidence indicates that the checkpoint kinases Bub1 and BubR1 have the added task of regulating kinetochore-microtubule attachments. However, the debate on the requirement of kinase activity is in full swing. This minireview summarizes recent advances in our understanding of the core spindle checkpoint kinases Bub1 and BubR1 and considers evidence that supports and opposes the role of kinase activity in regulating their functions during mitosis.     

Crystal structure of the spindle assembly checkpoint protein Bub3

Bub3 is one of at least six proteins that transmit the spindle assembly checkpoint signal. These proteins delay cell cycle progression from metaphase to anaphase in response to attachment defects between kinetochores and spindle microtubules and to tension defects between sister chromatids. To explore the molecular interactions mediated by Bub3, we have determined the crystal structure of the Saccharomyces cerevisiae protein Bub3p at 2.35 A resolution. Bub3p is a seven-blade beta-propeller, although its sequence diverges from that of other WD40 family members. Several loops are substantially elongated, but extra domains or insertions are not present at the termini. In particular, two extended loops project from the top face of the propeller, forming a cleft. Amino acid residues across the top face and one aspect of the lateral surface (spanning blades 5-6) are highly conserved among Bub3 proteins. We propose that these conserved surfaces are the loci for key interactions with conserved motifs in spindle checkpoint proteins Bub1 and Mad3/BubR1. Comparison of the Bub3 sequence to the WD40 protein, Rae1, shows high sequence conservation along the same surfaces. Rae1 interaction with Bub1 is, therefore, likely to involve a similar mode of binding.     

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.