Bub1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Bub1-related kinase

Synonyms - CG7838, BubR1 (the proper term for this gene based on homology; Logarinho, 2004)

Cytological map position - 42A1--2

Function - signaling

Keywords - mitotic checkpoint control protein, response to DNA damage

Symbol Symbol - BubR1

FlyBase ID: FBgn0025458

Genetic map position - 2R

Classification - protein serine/threonine kinase

Cellular location - kinetochore associated protein

NCBI link: Entrez Gene

BubR1 orthologs: Biolitmine
Recent literature
Duranteau, M., Montagne, J. J. and Rahmani, Z. (2016). A novel mutation in the N-terminal domain of Drosophila BubR1 affects the spindle assembly checkpoint function of BubR1. Biol Open [Epub ahead of print]. PubMed ID: 27742609
The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism that ensures accurate segregation of chromosomes into two daughter cells. BubR1, a key component of the SAC, play also a role in the mitotic timing since depletion of BubR1 leads to an accelerated mitosis. Unlike what was reported in mammalian cells, mutation of the KEN1-box domain of Drosophila BubR1 (bubR1-KEN1 mutant) that affects the binding of BubR1 to Cdc20, the activating co-factor of the APC/C, did not accelerate the mitotic timing despite resulting in a defective SAC. This study shows that a mutation in a novel Drosophila short sequence (bubR1-KAN mutant) leads to an accelerated mitotic timing as well as SAC failure. Moreover, the data indicate that the level of Fzy, the Drosophila homolog of Cdc20, recruited to kinetochores is diminished in bubR1-KEN1 mutant cells and further diminished in bubR1-KAN mutant cells. Altogether, these data show that this newly identified Drosophila BubR1 KAN motif is required for a functional SAC and suggest that it may play an important role on Cdc20/Fzy kinetochore recruitment.
Huang, Y., Lin, L., Liu, X., Ye, S., Yao, P. Y., Wang, W., Yang, F., Gao, X., Li, J., Zhang, Y., Zhang, J., Yang, Z., Liu, X., Yang, Z., Zang, J., Teng, M., Wang, Z., Ruan, K., Ding, X., Li, L., Cleveland, D. W., Zhang, R. and Yao, X. (2019). BubR1 phosphorylates CENP-E as a switch enabling the transition from lateral association to end-on capture of spindle microtubules. Cell Res. PubMed ID: 31201382
Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, powered congression of those chromosomes, their segregation in anaphase, and assembly of a spindle midzone at mitotic exit. The centromere-associated kinesin motor CENP-E, whose binding partner is BubR1, has been implicated in congression of misaligned chromosomes and the transition from lateral kinetochore-microtubule association to end-on capture. Although previously proposed to be a pseudokinase, this study reports the structure of the kinase domain of Drosophila melanogaster BubR1, revealing its folding into a conformation predicted to be catalytically active. BubR1 is shown to be a bona fide kinase whose phosphorylation of CENP-E switches it from a laterally attached microtubule motor to a plus-end microtubule tip tracker. Computational modeling is used to identify bubristatin as a selective BubR1 kinase antagonist that targets the alphaN1 helix of N-terminal extension and alphaC helix of the BubR1 kinase domain. Inhibition of CENP-E phosphorylation is shown to prevent proper microtubule capture at kinetochores and, surprisingly, proper assembly of the central spindle at mitotic exit. Thus, BubR1-mediated CENP-E phosphorylation produces a temporal switch that enables transition from lateral to end-on microtubule capture and organization of microtubules into stable midzone arrays.
Tang, R., Jiang, Z., Chen, F., Yu, W., Fan, K., Tan, J., Zhang, Z., Liu, X., Li, P. and Yuan, K. (2020). The Kinase Activity of Drosophila BubR1 Is Required for Insulin Signaling-Dependent Stem Cell Maintenance. Cell Rep 31(12): 107794. PubMed ID: 32579921
As a core component of the mitotic checkpoint complex, BubR1 has a modular organization of molecular functions, with KEN box and other motifs at the N terminus inhibiting the anaphase-promoting complex/cyclosome, and a kinase domain at the C terminus, whose function remains unsettled, especially at organismal levels. This study generated knock-in BubR1 mutations in the Drosophila genome to separately disrupt the KEN box and the kinase domain. All of the mutants are homozygously viable and fertile and show no defects in mitotic progression. The mutants without kinase activity have an increased lifespan and phenotypic changes associated with attenuated insulin signaling, including reduced InR on the cell membrane, weakened PI3K and AKT activity, and elevated expression of dFoxO targets. The BubR1 kinase-dead mutants have a reduced cap cell number in female germaria, which can be rescued by expressing a constitutively active InR. It is concluded that one major physiological role of BubR1 kinase in Drosophila is to modulate insulin signaling.
Jang, J. K., Gladstein, A. C., Das, A., Shapiro, J. G., Sisco, Z. L. and McKim, K. S. (2021). Multiple pools of PP2A regulate spindle assembly, kinetochore attachments and cohesion in Drosophila oocytes. J Cell Sci 134(14). PubMed ID: 34297127
Meiosis in female oocytes lacks centrosomes, the microtubule-organizing centers. In Drosophila oocytes, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). To investigate the mechanisms that regulate Aurora B activity, this study examined the role of protein phosphatase 2A (PP2A) in Drosophila oocyte meiosis. Both forms of PP2A, B55 and B56, antagonize the Aurora B spindle assembly function, suggesting that a balance between Aurora B and PP2A activity maintains the oocyte spindle during meiosis I. PP2A-B56, which has a B subunit encoded by two partially redundant paralogs, wdb and wrd, is also required for maintenance of sister chromatid cohesion, establishment of end-on microtubule attachments, and metaphase I arrest in oocytes. WDB recruitment to the centromeres depends on BUBR1, MEI-S332 and kinetochore protein SPC105R. Although BUBR1 stabilizes microtubule attachments in Drosophila oocytes, it is not required for cohesion maintenance during meiosis I. At least three populations of PP2A-B56 regulate meiosis are proposed, two of which depend on SPC105R and a third that is associated with the spindle.

The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or have failed to complete steps during mitosis, including those involving kinetochore function. Bub1 is an evolutionarily conserved mitotic checkpoint control protein that is present in organisms as diverse as yeast and humans. Bub1 inhibits ubiquitin ligase activity of anaphase promoting complex (APC) preventing mitosis until all chromosomes are correctly attached to the mitotic spindle. Drosophila Bub1 (see below: The gene described in this section of the Interactive Fly is more properly termed BudR1) localizes strongly to the centromere/kinetochore of mitotic and meiotic chromosomes that have not yet reached the metaphase plate. Animals homozygous for P-element-induced, near-null mutations of bub1 die during late larval/pupal stages due to severe mitotic abnormalities indicative of a bypass of checkpoint function. These abnormalities include accelerated exit from metaphase and chromosome missegregation and fragmentation. Chromosome fragmentation possibly leads to the significantly elevated levels of apoptosis seen in mutants (Basu, 1999).

The relationship between Bub1 and other kinetochore components was investigated. Bub1 kinase activity is not required for phosphorylation of 3F3/2 epitopes (detected by anti 3F3/2 antibody) at prophase/prometaphase, but is needed for 3F3/2 dephosphorylation at metaphase. Neither 3F3/2 dephosphorylation nor loss of Bub1 from the kinetochore is a prerequisite for anaphase entry. Bub1's localization to the kinetochore does not depend on the products of the genes zw10, rod, polo, or fizzy, indicating that the kinetochore is constructed from several independent subassemblies (Basu, 1999).

Mammalian BubR1, a protein that is functionally similar, but not equivalent to mammalian Bud1 (Taylor, 2001), inhibits APC activity (Sudakin, 2001; Tang, 2001; Fang, 2002). BubR1 is a protein kinase that associates with Cdc20 (the homolog of Drosophila Fizzy in yeast and vertebrates) and the APC (Chan, 1999; Wu, 2000; Skoufias, 2001). Recombinant BubR1 has been shown to directly inhibit the ubiquitin ligase activity of the APC; the kinase activity of BubR1 is not required for this inhibition (Tang, 2001). In addition, a checkpoint complex has been purified (Tang, 2001) from HeLa cells that contains BubR1, Bub3 (see Drosophila Bub3) and substoichiometric amounts of Cdc20. Independently, Sudakin (2001) purified a mitotic checkpoint complex (MCC) that contains nearly stoichiometric amounts of BubR1, Bub3, Mad2 (see Drosophila ) and Cdc20 (Sudakin, 2001). The isolated MCC was shown to be about 3000-fold more potent than purified Mad2 alone at inhibiting the ubiquitin ligase activity of the APC. In experiments consistent with these studies, Fang found that BubR1 binds to Cdc20 with a high affinity and is more efficient than Mad2 as an inhibitor of Cdc20-APC in vitro (Fang, 2002). Moreover, BubR1 functions synergistically with Mad2 at physiological concentrations to inhibit APC activity. Interestingly, studies in fission yeast demonstrate that Mad3 also associates with Bub3, Cdc20 and Mad2 and that Mad3 is required for metaphase arrest caused by Mad2 overexpression (Millband, 2002). Collectively, these studies suggest that BubR1 and Mad2 cooperate in transducing the anaphase-delaying signal by inhibiting APC activity (Zhou, 2002 and references therein).

Both human Bub1 and Drosophila Bud1 associate strongly with the kinetochores of chromosomes unattached to the spindle prior to anaphase onset of normal mitosis, and with all the kinetochores in cells treated with microtubule depolymerizing drugs. Reduced amounts of both proteins are also found at the kinetochores of chromosomes either at the metaphase plate or being pulled toward the poles at anaphase. Near null mutations in the gene encoding this Drosophila protein cause phenotypes indicating an abrogation of the spindle checkpoint. These same mutations abolish the ability of another checkpoint component, Drosophila Bub3, to localize to the kinetochores (Basu, 1998). This latter finding fits well with a wealth of data substantiating an intimate relationship between Bub1 and Bub3. Taken together, these observations in Drosophila provide strong evidence for the conservation of Bub1 function throughout evolution (Basu, 1999).

In S. cerevisiae, bub and mad genes are nonessential in the absence of microtubule depolymerizing agents, though the growth of mutant cells is slowed (Roberts, 1994). In S. pombe, bub1 null mutants are viable, though some abnormalities in chromosome segregation are observable during mitosis (Bernard, 1998). In marked contrast, loss of bub1 function in Drosophila leads to lethality at the larval/pupal transition. Lethality at this stage has been observed for many mutations affecting essential cell cycle components, presumably because maternally supplied stores of protein obtained from a nonmutant mother are exhausted by this point in development. Examination of brain neuroblasts dissected from dying third instar bub1 homozygous mutant larvae has thus facilitated an analysis of how loss of checkpoint function affects cell division in a multicellular organism. In the description below, the possibility cannot be excluded that some aspects of the phenotype reported are indirect consequences of problems encountered in earlier cell divisions. However, it should be noted that all embryonic divisions appear to be normal, and essentially all bub1 mutant animals hatch into larvae that survive until the third instar. Since there is very little cell division in the larval brain before the third instar, the number of cell divisions that could take place between the exhaustion of maternal stores of Bub1 protein and the time of analysis is limited. Moreover, these phenotypes are quite specific to bub1 mutants, and have not been observed in analysis of many other mitotic mutants in Drosophila (Basu, 1999).

Treatment of bub1 mutant neuroblasts with colchicine causes precocious sister chromatid separation, instead of the prometaphase arrest with attached sister chromatids typical of wild-type neuroblasts. This phenotype is a predictable property of mutations affecting the operation of the spindle checkpoint, since it indicates that bub1 mutant neuroblasts attempt to enter anaphase despite the absence of a functional spindle (Basu, 1999).

More interesting are the effects of bub1 mutations on normal cell division in neuroblasts that have not been treated with microtubule depolymerizing drugs. Observations suggest that bub1 mutant neuroblasts enter anaphase prematurely even in these untreated cells. In particular, the ratio of metaphase figures to anaphase figures is decreased 5-10-fold in bub1 brains relative to wild-type brains. This result is consistent with studies showing that microinjection of Mad2 antibodies into mammalian cells causes premature sister chromatid separation and entry into anaphase. Interestingly, loss of Bub1 in Drosophila generates a sharp decrease in mitotic index. This finding could be explained by an accelerated transit through mitosis as has been suggested for mammalian cell cultures expressing dominant negative forms of mouse Bub1 (Taylor, 1997). However, it is also possible that the lowered mitotic index reflects the assumption of an apoptotic fate by many neuroblasts in the brain (Basu, 1999).

A high proportion of anaphases in untreated bub1 mutant brains show a variety of aberrations, including extensive chromatin bridging, lagging chromosomes (most likely leading to aneuploidy), and chromosome fragmentation. These aberrations are interpreted as further evidence for the precocious entry into anaphase. In this view, the proper synchronization of different aspects of sister chromatid separation at the metaphase/anaphase transition does not occurred. It is well known that the forces holding sister chromatids together along their arms are separable from the forces joining sister chromatids at their centromeres. For example, acentric chromosome fragments in irradiated grasshopper neuroblasts remain associated until the onset of anaphase. In addition, sister chromatid cohesion along the arms can also be disrupted independently of centromeric cohesion through treatment with hypotonic solutions. It is hypothesized that absence of bub1 function leads to loss of cohesive forces at the centromere before the separation of sister chromatids along their arms is completed. Thus, the chromatin bridging and fragmentation seen in bub1 mutant anaphases most likely reflect a failure to resolve concatenated sister chromatid DNAs along the arms at a time at which the centromeres have already separated and are being pulled toward the poles. In support of this interpretation, mutations in the Drosophila gene barren, which encodes a chromosome-associated protein that interacts with topoisomerase II, cause substantial chromatin bridging during anaphase of late embryonic divisions (Basu, 1999).

A striking feature of Drosophila bub1 mutants is the occurrence of significantly elevated frequencies of apoptotic nuclei in larval brains. This result was unexpected, since expression of a dominant negative form of mouse Bub1 has been reported to reduce the frequency of apoptotic nuclei in nocodazole-treated cells (Taylor, 1997), implying that loss of checkpoint function prevents the apoptotic response. The reasons for the apparent dichotomy between the results in Drosophila and those from mouse tissue culture cells are not clear. These effects could be organism or cell type-specific, or the differences could reflect unusual consequences of the dominant negative forms of Bub1 utilized in the mouse study (Basu, 1999).

A strong possibility for the high level of apoptotic cells seen in bub1 mutant brains emerges from findings that the chromosomes in many mutant anaphase figures are extensively fragmented. It has been well documented that chromosome breakage in Drosophila is normally a cell lethal event preventing entry into the next round of mitosis. The larval brains were examined of a number of new, relatively uncharacterized mitotic mutants that cause massive chromosome fragmentation, and these uniformly have high levels of apoptotic cells. Moreover, the induction of chromosome breakage with the FLP/FRT system is also associated with apoptosis. Apoptosis (or in fact any aspect of the bub1 mutant phenotype) cannot be an indirect consequence of aneuploidy, because brains from zw10 and rod mutants, which have many aneuploid cells, do not show the massive apoptotic response (or any of the cell cycle defects) generated by bub1 mutants. Regardless of the mechanism underlying the induction of apoptosis in bub1 mutant brains, it is clear that loss of spindle checkpoint function does not prevent a cell's entry into the apoptotic pathway (Basu, 1999).

In yeast, Bub1 acts as a kinase that can phosphorylate both itself and Bub3 (Roberts, 1994). Because Bub1's cell cycle distribution parallels that of 3F3/2 phosphoepitopes that appear to be intimately involved in the metaphase-anaphase transition, Bub1 has been suggested as a possible source of the kinase activity that generates these phosphoepitopes. This is not the case in Drosophila. F3/2 epitopes are strongly phosphorylated in a bub1 mutant, showing that Bub1 cannot be a significant source of 3F3/2 kinase activity in vivo. In addition, Bub3 fails to associate with the kinetochore in bub1 mutants, ruling out Bub3 as a major 3F3/2 phosphoepitope (Basu, 1999).

If Bub1 does not phosphorylate 3F3/2 phosphoepitopes, what kinase(s) can supply such an activity? A recent report indicates that ERK and MKK (extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase) localize to the kinetochore and can phosphorylate 3F3/2 phosphoepitopes. It is not clear whether this activity is direct or indirect; in any event, results indicate that Bub1 does not participate in the same 3F3/2 phosphorylation pathway (Basu, 1999 and references therein).

It was surprising to find that 3F3/2 epitopes at the kinetochores remain phosphorylated in anaphase figures from bub1 mutants. In contrast, 3F3/2 phosphoepitopes at the kinetochores are normally lost completely at the start of anaphase. The implications of this result are twofold. First, dephosphorylation of kinetochore-associated 3F3/2 epitopes is not required for the metaphase/anaphase transition, at least in a bub1 mutant background. One possibility is that 3F3/2 dephosphorylation is not, as commonly suggested, a part of the signaling pathway for anaphase onset, but is instead a downstream response to the signal. Alternatively, Bub1 may function downstream of 3F3/2 dephosphorylation in the pathway governing the metaphase–anaphase transition. A second implication of the observation is that Bub1 kinase activity is required, presumably indirectly, for the dephosphorylation of 3F3/2 epitopes at the metaphase-anaphase transition. A possible explanation for the continued phosphorylation of kinetochore-based 3F3/2 epitopes is that the accelerated transit through mitosis in bub1 mutants may not allow enough time for action of the relevant phosphatase(s) (Basu, 1999).

The localization of Bub1 to the kinetochore is not abolished by mutations in several genes encoding other kinetochore components, nor do mutations in bub1 affect the association of ZW10 or Polo proteins with the kinetochore. These findings suggest that the kinetochore may be assembled in at least two independent pathways. In one pathway, interaction between Bub1 and Bub3 is required for the kinetochore targeting of either protein (Roberts, 1994; Basu, 1998; Taylor, 1998). In a second subassembly, ZW10 and Rod proteins form a complex needed for the recruitment of the microtubule motor dynein to the kinetochore. The fact that polo mutations do not disrupt the kinetochore localization of Bub1, Bub3, or ZW10 suggests either a third independent pathway or that the kinetochore binding of Polo protein is subsequent to the recruitment of one of the two subassemblies (Basu, 1999 and references therein).

In colchicine-treated larval neuroblasts from zw10 mutants where the sister chromatids have separated prematurely, high levels of Bub1 protein remain at the kinetochores. This phenomenon is not restricted to a zw10 mutant background, since prolonged treatment of wild-type larval neuroblasts with hypotonic solution after colchicine incubation also generates precocious sister chromatid separation with continued strong Bub1 staining at the kinetochores. These observations indicate that it is possible to initiate anaphase despite the presence of the Bub1 'wait-anaphase' signal at the kinetochores. It is thus conceivable that the relative loss of Bub1 from kinetochores at metaphase and anaphase is not normally a prerequisite for anaphase onset (Basu, 1999).

Although the existence of a tension-dependent 'wait-anaphase' checkpoint in meiotic grasshopper spermatocytes has been well established, several observations suggest that such a checkpoint may not play a major role in Drosophila spermatogenesis. The presence of univalents (chromosomes without a pairing partner) does not obviously affect meiotic progression. Mutations in mei-S322 and ord, which lead to sister chromatid separation before the start of the second meiotic division, do not affect entry into anaphase II. Finally, colchicine-treated spermatocytes that cannot segregate their chromosome still exit meiosis and differentiate into spermatids (Basu, 1999).

Nevertheless, Drosophila Bub1 and Bub3 both associate strongly with the kinetochores of primary spermatocytes before metaphase of both meiotic divisions, and kinetochore staining cas be observed with antibodies against Xenopus Mad2 in prometaphase primary spermatocytes. Bub1 responds differentially to the presence and absence of tension across chromosomes during meiosis exactly as would be predicted were it acting as part of a functional spindle checkpoint. In addition, bub1 mutations have a dramatic effect on Drosophila spermatogenesis. Though it is difficult to distinguish aberrations introduced during mitotic germ line cell proliferation from those occurring during meiosis, the appearance of disrupted meiotic spindles and of multiple nuclei of uneven volume within 'onion-stage' spermatids are suggestive of defects specifically affecting meiosis (Basu, 1999).

On the basis of these observations, it is believed that a spindle checkpoint does exist in Drosophila meiotic spermatocytes, but that it operates with significantly reduced efficiency or according to different signals. The reasons underlying this apparent inefficiency remain unclear, but very likely involve part of the checkpoint pathway downstream of Bub1. One prediction of this viewpoint is that conditions that should enable the checkpoint would delay, but not completely block, cell cycle progression past the metaphase of either meiotic division. It will thus be of importance in the near future to verify this prediction through real-time observations of male meiosis in cultured spermatocytes (Basu, 1999).

Sister centromere fusion during meiosis I depends on maintaining cohesins and destabilizing microtubule attachments

Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. This study found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. This study has demonstrated that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, this study proposes that two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments (Wang, 2019).

The necessity of sister kinetochores to co-orient toward the same pole for co-segregation at anaphase I differentiates the first meiotic division from the second division. A meiosis-specific mechanism exists that ensures sister chromatid co-segregation by rearranging sister kinetochores, aligning them next to each other and facilitating microtubule attachments to the same pole]. This process is referred to as co-orientation, in contrast to mono-orientation, when homologous kinetochores orient to the same pole. Given the importance of co-orientation in meiosis the mechanism underlying this process is still poorly understood, maybe because many of the essential proteins are not conserved across phyla (Wang, 2019).

Most studies of co-orientation have focused on how fusion of the centromeres and kinetochores is established. In budding yeast, centromere fusion occurs independently of cohesins: Spo13 and the Polo kinase homolog Cdc5 recruit a meiosis-specific protein complex, monopolin (Csm1, Lrs4, Mam1, CK1) to the kinetochore. Lrs4 and Csm1 form a V-shaped structure that interacts with the N-terminal domain of Dsn1 in the Mis12 complex to fuse sister kinetochores. While the monopolin complex is not widely conserved, cohesin-independent mechanisms may exist in other organisms. A bridge between the kinetochore proteins MIS12 and NDC80 is required for co-orientation in maize. In contrast, cohesins are required for co-orientation in several organisms. The meiosis-specific cohesin Rec8 is indispensable for sister centromere fusion in fission yeast and Arabidopsis. Cohesin is localized to the core-centromere in fission yeast and mice. In Drosophila melanogaster oocytes, cohesins (SMC1/SMC3/SOLO/SUNN) establish cohesion in meiotic S-phase and show an enrichment that colocalizes with centromere protein CID/CENP-A. Like fission yeast and mouse, Drosophila may require high concentrations of cohesins to fuse sister centromeres together for co-orientation during meiosis (Wang, 2019).

In mice, a novel kinetochore protein, Meikin, recruits Plk1 to protect Rec8 at centromeres. Although poorly conserved, Meikin is proposed to be a functional homolog of Spo13 in budding yeast and Moa1 in fission yeast. They all contain Polo-box domains that recruit Polo kinase to centromeres. Loss of Polo in both fission yeast (Plo1) and mice results in kinetochore separation, suggesting a conserved role for Polo in co-orientation. In fission yeast, Moa1-Plo1 phosphorylates Spc7 (KNL1) to recruit Bub1 and Sgo1 for the protection of centromere cohesion in meiosis I. These results suggest the mechanism for maintaining sister centromere fusion involves kinetochore proteins recruiting proteins that protect cohesion. However, how centromere cohesion is established prior to metaphase I, and how sister centromere fusion is released during meiosis II, still needs to be investigated (Wang, 2019).

Previous work has found that depletion of the kinetochore protein SPC105R (KNL1) in Drosophila oocytes results in separated centromeres at metaphase I, suggesting a defect in sister centromere fusion. Thus, Drosophila SPC105R and fission yeast Spc7 may have conserved functions in co-orientation (Radford, 2015). This study has identified a second Drosophila protein required for sister-centromere fusion, Protein Phosphatase 1 isoform 87B (PP1-87B). However, sister centromere separation in SPC105R and PP1-87B depleted Drosophila oocytes occurs by different mechanisms, the former is Separase dependent and the latter is Separase independent. Based on these results, a model is proposed for the establishment, protection and release of co-orientation. Sister centromere fusion necessary for co-orientation is established through cohesins that are protected by SPC105R. Subsequently, PP1-87B maintains co-orientation in a cohesin-independent manner by antagonizing stable kinetochore-microtubule (KT-MT) interactions. The implication is that the release of co-orientation during meiosis II is cohesin-independent and MT dependent. A surprising interaction was found between PP1-87B and C(3)G, the transverse element of the synaptonemal complex (SC), in regulating sister centromere separation. Overall, these results suggest a new mechanism where KT-MT interactions and centromeric SC regulate sister kinetochore co-orientation during female meiosis (Wang, 2019).


cDNA clone length - 4615 bp

Bases in 5' UTR - 56

Exons - 5

Bases in 3' UTR - 159


Amino Acids - 1460

Structural Domains

Two Drosophila EST sequences were identified through a BLAST search of the Berkeley Drosophila Genome Project (BDGP) database with mammalian Bub1. The cDNA sequence contains an open reading frame predicting a 165-kD protein closely related to Bub1. This protein shows 24.6% identity to human Bub1, 23.8% identity to mouse Bub1, and 14.5% identity to budding yeast Bub1p; it also displays 17.2% amino acid sequence identity to the human Bub1-related protein BubR1. The size of this Drosophila protein is somewhat larger than that of previously characterized human, mouse, and yeast members of the Bub1 family, whose predicted sizes range from 117-122 kD. The COOH-terminal third of the fly protein contains the strongly conserved kinase domain characteristic of Bub1. The NH2-terminal third of the fly protein, in common with the other members of the Bub1 family, shares significant sequence similarity with the yeast checkpoint control component Mad3p. Most of the additional residues resulting in the relatively larger size of the fly protein are located in its middle third (Basu, 1999).

The gene described in this section of the Interactive Fly is more properly termed BudR1

Previous molecular and genetic analysis in Drosophila identified a gene (CG7838) that encodes a 165 kDa protein with significant homology to Bub1 proteins from other organisms and was reported as the Drosophila Bub1 homologue (Basu, 1999). However, completion of the Drosophila genome sequence revealed the existence of another gene (CG14030) that encodes a 125 kDa protein closely related to Bub1 proteins. Higher eukaryotes also have two genes that encode proteins closely related to Bub1, one of which has retained the name Bub1, while the other is called BubR1. The N-terminal domains of both Bub1 and BubR1 are highly homologous to yeast Mad3, but both Bub1 and BubR1 contain a C-terminal Ser/Thr kinase domain not found in Mad3. Neither Drosophila nor vertebrate genomes appear to encode a Mad3-like protein without a kinase domain. Since Bub1 and BubR1 appear to respond to different checkpoint signals, it became essential to determine which of the two Drosophila genes corresponds to Bub1 and BubR1. Phylogenetic analysis, including both Bub1-like Drosophila proteins, as well as Bub1 and BubR1 proteins from other species, did not provide a simple solution to this problem. The results show that the proteins encoded by the CG7838 and CG14030 genes are more closely related to each other and to the Anopheles homologues than to either Bub1 or BubR1 from other species, whereas the human, mouse and Xenopus proteins do fall into defined clusters. More recently, it has been suggested that a distinctive feature of BubR1 proteins is the presence of a conserved sequence, the KEN motif, in the N-terminal domain. The KEN motif is also present in yeast Mad3 and other Mad3 homologues, but not in Bub1. Sequence comparisons indicated that the previously described CG7838-encoded protein (165 kDa) contains a N-terminal KEN motif, while the new Bub1-like CG14030 protein (125 kDa) does not. This suggests that CG7838 encodes the Drosophila homologue of BubR1 while CG14030 encodes the bona fide Drosophila homologue of Bub1. However, if the sequence alignment is restricted to the C-terminal kinase domain, CG7838 is found to encode a protein, which is significantly more similar to Bub1 than to BubR1 proteins. In vertebrates, BubR1 is clearly distinct from Bub1 because the kinase domain of the former is significantly less conserved. Surprisingly, in Drosophila, the kinase domain of the KEN-box-containing protein (CG7838: 165 kDa) has almost as many conserved amino acids as the newly identified Bub1 protein (CG14030: 125 kDa) explaining why the protein encoded by the CG7838 gene was originally classified as Bub1. Even though from sequence analysis it seems difficult to determine unequivocally which Drosophila gene codes for Bub1 and BubR1, functional analysis of these proteins presented in this study clearly suggests that the newly identified CG14030 gene encodes the Drosophila homologue of bub1. Therefore, in contrast with previous usage (Basu, 1998; Basu, 1999), CG7838 is more accurately described as the Drosophila bubR1 gene, so the previously described mutant alleles l(2)K06109 and l(2)K03113 of CG7838 should be renamed bubR11 and bubR12. The use of this classification is suggested for future investigations and it will be adopted in this study (Logarinho, 2004).

Bub1: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 December 2019

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