We describe the cloning of a human homologue of Bub3, which, like murine Bub1, localizes to kinetochores during prometaphase (Taylor and McKeon, 1997
). In addition, we show that mBub1 and hBub3 can interact in mammalian cells. We have also identified the Bub3-binding site in Bub1. This same domain is required for kinetochore localization of Bub1, which we have previously shown is required for checkpoint function (Taylor and McKeon, 1997
). Therefore, these observations suggest that the role of Bub3 is to facilitate kinetochore localization of Bub1, thereby activating the checkpoint in response to unattached kinetochores. ScBub3 was identified as a high copy suppressor of the bub1-1
allele, suggesting that ScBub1 and ScBub3 interact in yeast (Hoyt et al., 1991
). Further support for this interaction came from genetic experiments, which showed that bub3Δ
strains have a similar phenotype to bub1Δ
strains (Roberts et al., 1994
). In addition, bub1Δ/bub3Δ
double mutants are phenotypically similar to strains with deletions of either gene, and Bub1p and Bub3p have been shown to coimmunoprecipitate when overexpressed in S. cerevisiae
(Roberts et al., 1994
). Our observations with Bub1 and Bub3 in mammalian cells are consistent with those in S. cerevisiae
and suggest the following model: in the absence of Bub3, Bub1 cannot localize to the kinetochore and hence the checkpoint is not activated in response to unattached kinetochores.
hBub3 also appears to be required for kinetochore localization of hBubR1. Whether Bub3 plays any roles in addition to kinetochore localization of Bub1 and BubR1 remains to be seen. Interestingly, hBub3 contains four WD repeats, a 40-amino acid motif found in many proteins involved in a diverse array of cellular processes, including G protein–linked receptor signaling, RNA processing and export, and cell cycle control (Neer et al., 1994
). The exact function of WD repeats is not clear, but it seems likely that they play a role in mediating protein–protein interactions. Indeed, WD repeat proteins are often part of multiprotein complexes (Neer et al., 1994
). Whether Bub3 is part of a large multiprotein complex remains to be seen. Thus far, Bub3 has been shown to interact with Bub1 and the human Mad3/Bub1-related protein, hBubR1 (Roberts et al., 1994
; this work). In addition, Bub3 and Mad3 have been shown to interact in S. cerevisiae
(Hardwick, K.G., and A.W. Murray, personal communication). Recently, Mad1, Mad2, and Mad3 have been shown to interact with cdc20 (Hwang et al., 1998
; Kim et al., 1998
), which in turn has been shown to interact with the APC (Visintin et al., 1997
). Significantly, the vertebrate homologues of Mad2, Bub1, Bub3 and the Mad3-related kinase, BubR1, have been shown to localize to kinetochores before chromosome alignment (Chen et al., 1996
; Li and Benezra, 1996
; Taylor and McKeon, 1997
; this work). It is therefore tempting to speculate that the checkpoint components, including Bub1, BubR1, Bub3, Mad1, Mad2, and Mad3, may be part of a large protein complex that is recruited to unattached kinetochores. This kinetochore-bound form of the checkpoint complex may bind and inhibit cdc20, thereby preventing activation of APC, and hence delaying the onset of anaphase. Based on the observations that Mad2, Bub1, Bub3, and BubR1 are not present at the kinetochores of metaphase chromosomes (Chen et al., 1996
; Li and Benezra, 1996
; Taylor and McKeon, 1997
; this work), it appears likely that the checkpoint complex dissociates from kinetochores upon achieving correct bipolar attachments. Upon dissociation, perhaps the composition or activity of the checkpoint complex is altered, rendering cdc20 active and hence allowing the onset of anaphase. However, although there is evidence that many of these checkpoint components can interact with each other (see above), the existence of such a complex has not yet been demonstrated to exist in vivo. It is therefore possible that some of these proteins interact only transiently as cells progress through mitosis.
hBub3 shares significant homology with the Rae1 family of proteins, both within the WD repeats and in the central, non-WD repeat region (refer to Fig. ). The S. pombe rae1
gene was identified as a poly(A)+
RNA export mutant (Brown et al., 1995
). SpRae1 has recently been shown to interact with the S. pombe
homologue of the Nic96 nucleoporin (Yoon et al., 1997
). In addition, the S. cerevisiae
homologue of Rae1, gle2p, interacts with the Nup100p nucleoporin and other proteins involved in nucleocytoplasmic transport (Murphy et al., 1996
). Furthermore, gle2
strains also show defects in poly(A)+
RNA export and have grossly perturbed nuclear pores. These observations suggest that Rae1 is required for nucleocytoplasmic transport and probably does not play a direct role in cell cycle control.
Despite the significant similarity between the Rae1 proteins and the human Bub3 homologue described here (39% identity), several lines of evidence suggest that hBub3 is not a member of the Rae1 family. First, a human cDNA encoding a protein with 49% identity to SpRae1 has recently been identified (Bharathi et al., 1997
). This cDNA complements the temperature sensitivity of the rae1-1
allele and is therefore likely to be the human Rae1 homologue. Second, an alignment of the Rae1 and Bub3 proteins identifies several amino acid sequences in the central domain, including the VATAER sequence (amino acids 174–179 in SpRae1), which are conserved in all the Rae1 proteins, but not in the Bub3 homologues. Conversely, there are several sequences, including the SSI(E/ D)GRVAVE sequence (amino acids 211–220 in hBub3), which are conserved in the Bub3 proteins, but are not conserved in the Rae1 homologues. Significantly, deletion of the VAVE sequence abolishes the ability of hBub3 to interact with mBub1 and hBubR1. Note that the S. pombe rae1-1
loss of function allele results in a substitution of the glycine at position 219 with a glutamic acid (Brown et al., 1995
). Significantly, this glycine is conserved in the Rae1 homologues, but is replaced by a serine in the Bub3 homologues.
In addition to the sequence analysis, two observations indicate that hBub3 is indeed the homologue of ScBub3. First, hBub3 interacts with mBub1, as do Bub3 and Bub1 in S. cerevisiae
(Roberts et al., 1994
). Second, hBub3 localizes to kinetochores during prometaphase, a property that one might predict based on the localizations of mBub1 (Taylor and McKeon, 1997
) and the vertebrate homologues of Mad2 (Chen et al., 1996
; Li and Benezra, 1996
). The significance, if any, of the similarity between the Bub3 and Rae1 proteins remains to be determined.
This work also describes the identification of a novel human Mad3/Bub1-related protein, hBubR1. hBubR1 shares homology with Mad3 from S. cerevisiae and yet it is significantly larger (1,050 amino acids) than ScMad3 (515 amino acids), due to a COOH-terminal extension which contains a kinase domain. This suggests that perhaps the human Mad3/Bub1-related kinase is a second Bub1 homologue. Indeed, within the NH2-terminal domains, hBubR1 is more similar to ScBub1 (26% identity) than mBub1 is to ScBub1 (23% identity). In addition, a BLAST search of the yeast genome shows that the kinase most closely related to hBubR1 is ScBub1. However, an alignment of the COOH-terminal kinase domains shows that hBubR1 is clearly distinct from the Bub1 family (refer to Fig. B). Within the kinase domains, there are 85 amino acids conserved among the Bub1 homologues from S. cerevisiae, mouse and human. However, 54 of these are not conserved in hBubR1. Indeed, within the kinase domain, mBub1 is more closely related to ScBub1 (33% identity) than it is to hBubR1 (26% identity). Within the NH2-terminal domain, hBubR1 is significantly more similar to ScMad3 (35% identity) than it is to ScBub1 (26% identity). These observations suggest that perhaps hBubR1 is a Mad3-related protein kinase. Whether hBubR1 and ScMad3 have indeed evolved from a common ancestor will remain uncertain until Mad3-related proteins from other organisms have been identified, or until we have a better understanding of the functions of these two proteins.
The functional analysis presented here illustrates similarities between mBub1 and hBubR1, as well as a significant difference. Like mBub1, hBubR1 can bind hBub3 in mammalian cells, and this binding requires a domain that is conserved between the Bub1 and Mad3-related proteins. In addition, hBubR1 can localize to kinetochores during prometaphase and the ability to bind Bub3 is required for this localization. However, unlike mBub1, ectopically expressed hBubR1 only localizes to kinetochores when hBub3 is overexpressed. In contrast, ectopically expressed mBub1 localizes to the kinetochore without coexpression of hBub3 (Taylor and McKeon, 1997
). One possible explanation for this observation is that hBubR1 cannot bind hamster Bub3, and hence kinetochore localization is not observed in transfected BHK cells unless exogenous human Bub3 is present. However, significant kinetochore staining was also not observed when hBubR1 was transfected into human cells (data not shown). Until the localization of endogenous hBubR1 has been determined, these observations have to be treated with caution. However, it does suggest that the amount of endogenous Bub3 is limiting with respect to ectopically expressed hBubR1. One possibility is that the endogenous Bub3 is complexed with Bub1 and hence there is no Bub3 available for binding to, and hence kinetochore localization of, transfected hBubR1. This suggests that perhaps the majority of BubR1 may not play a role at the kinetochore. Alternatively, perhaps hBubR1 does play a role at the kinetochore, but that overexpression of hBubR1 somehow prevents the formation of complexes capable of kinetochore localization. The generation of anti-hBubR1 antibodies will hopefully resolve this issue.
The role of hBubR1 remains to be determined. It is possible that Bub1 and BubR1 have similar or partially overlapping roles. Although there is only a single Bub1 kinase encoded in the S. cerevisiae
genome, functional redundancy may be beneficial in mammalian cells. Recently, mutant BUB1
alleles have been identified in colorectal tumors displaying a chromosome instability phenotype (Cahill et al., 1998
), suggesting that mitotic checkpoint defects may contribute to tumorigenesis. Therefore, overlapping or partially redundant roles for Bub1 and BubR1 may provide a selective advantage to multicellular organisms: increasing the fidelity of chromosome segregation may reduce the possibility of generating potentially tumorigenic aneuploid cells.
Redundancy may allow multiple spindle events to be monitored by the mitotic checkpoint. At present, there is evidence implicating both tension and microtubule attachment as the events which regulate anaphase onset (Li and Nicklas, 1995
; Rieder et al., 1995
). Perhaps in the quest for enhanced genome stability, both tension and attachment are monitored by the checkpoint. Perhaps, therefore, Bub1 and BubR1 respond to different types of spindle events. Tension and microtubule attachment may also be differentially monitored in mitosis relative to meiosis (Nicklas, 1997
). If Bub1 and BubR1 respond differentially to tension and microtubule attachment, perhaps they play differential roles in mitosis and meiosis.
The EMBL/GenBank/DDBJ accession numbers for the cDNA sequences reported here are AF053304 (hBub3); AF053305 (hBub1); and AF053306 (hBubR1).