Gene name - Bub3
Cytological map position - 99B7
Function - mitotic checkpoint signaling
Symbol - Bub3
FlyBase ID: FBgn0025457
Genetic map position - 3R
Classification - WD domain protein
Cellular location - nuclear
During mitosis, a checkpoint mechanism delays metaphase-anaphase transition in the presence of unattached and/or unaligned chromosomes. This delay is achieved through inhibition of the anaphase promoting complex/cyclosome (APC/C) preventing sister chromatid separation and cyclin degradation. Bub3 is an essential protein required during normal mitotic progression to prevent premature sister chromatid separation, missegregation and aneuploidy. Bub3 is required during G2 and early stages of mitosis to promote normal mitotic entry. Loss of Bub3 function by mutation or RNAi depletion causes cells to progress slowly through prophase, a delay that appears to result from a failure to accumulate mitotic cyclins A and B. Defective accumulation of mitotic cyclins results from inappropriate APC/C activity, since mutations in the gene encoding the APC/C subunit Cdc27 (see Drosophila Cdc27) partially rescue this phenotype. Furthermore, analysis of mitotic progression in cells carrying mutations for cdc27 and bub3 suggests the existence of differentially activated APC/C complexes. Altogether, these data support the hypothesis that the mitotic checkpoint protein Bub3 is also required to regulate entry and progression through early stages of mitosis (Lopez, 2005).
During mitosis, a checkpoint mechanism delays entry into anaphase until all chromosomes are properly attached and aligned at the metaphase plate, thus preventing the unequal segregation of genetic material. Genetic screens in budding yeast for mutants that do not arrest in mitosis after the induction of spindle damage allowed the identification of several components of this checkpoint. These include the Mad1, Mad2 and Mad3 proteins, the Bub1, Bub2 and Bub3 proteins and the kinase Mps1. Soon after, homologues of most of these proteins were identified in higher eukaryotes, including Bub1 (see Drosophila Bub1), Bub3, Mad1, Mad2 (see Drosophila Mad2) and the human/mouse homologue of Mad3, BubR1; these proteins were further shown to localize preferentially to unattached kinetochores (Lopez, 2005).
A biochemical link between the checkpoint and known regulators of mitotic progression first emerged from studies showing that the Xenopus and human homologues of Mad2 are able to bind and sequester Cdc20/Fizzy, an activator of the APC/C. The APC/C is a multi-subunit E3 ubiquitin ligase that targets several mitotic regulators, including securin and mitotic cyclins, for degradation by the proteasome, thus triggering mitotic exit. The APC/C subunits and many of its target proteins are present throughout the cell cycle, but APC/C activity and specificity towards the substrates is modulated by its association with co-factors such as Cdc20/Fizzy (see Drosophila Fizzy) and Cdh1/Fizzy-related (see Drosophila Fizzy-related). APC/C association with Cdc20 occurs upon entry into mitosis and requires the phosphorylation of APC/C subunits. These phosphorylation events are thought to be mediated by cdc2/cyclin B (see Drosophila cdc2 and cyclin B) and Polo kinase (see Drosophila Polo), and enhance the activity of the APC/CCdc20 towards its substrates. The activity of APC/CCdc20 triggers the metaphase-anaphase transition both by inducing the ubiquitination of securin and by targeting cyclin B for degradation. Cdh1, another co-factor, mediates the ability of the APC/C to degrade mitotic regulators, like polo and aurora kinases, and to degrade cyclin B completely, thus promoting mitotic exit. The interaction of the APC/C with Cdh1 is inhibited by Cdh1 phosphorylation, which is mediated by Cdk1 and Cdk2. As a result of cyclin B destruction, Cdk activity drops, ensuring that Cdh1 remains dephosphorylated and active, thus preventing the accumulation of mitotic cyclins during the subsequent G1. However, the G1/S transition and the G2 stage of the cell cycle require accumulation of cyclins A and B and therefore APC/CCdh1 inactivation. The mechanism by which the APC/C is regulated during these stages of cell cycle is still poorly understood. Recently it was found that APC/C inactivation during the G1/S transition is achieved by Emi1, a newly identified inhibitor of the APC/C, as well as by phosphorylation of Cdh1 by Cdks (Lopez, 2005).
The downstream target of the mitotic checkpoint is APC/CCdc20, whose inhibition prevents sister chromatid separation. However, the role of the various checkpoint components in APC/C inhibition has been a matter of some controversy. Not only Mad2, but also Mad3/BubR1 can interact directly with Cdc20. An APC/CCdc20 inhibitory complex has been purified from interphase cells, called the mitotic checkpoint complex (MCC); it contains Mad2, BubR1, Bub3 and Cdc20 (Sudakin, 2001). A similar complex was found in budding yeast and shown to be independent of kinetochore assembly (Fraschini, 2001; Hardwick, 2000). Tang and colleagues obtained similar results showing that a BubR1-containing complex was a stronger inhibitor of the APC/CCdc20 than was Mad2, although this complex contained only BubR1 and Bub3 (Tang, 2001). Despite the discrepancies relative to the constitution of the complexes, these results suggest that checkpoint proteins may exist as APC/C inhibitory complexes already in interphase, however, the function of these complexes before mitosis is not yet known (Lopez, 2005).
Even though most checkpoint components are strongly conserved through evolution, the role of some checkpoint proteins, in the checkpoint response is still unknown, particularly in the case of Bub3. Besides its association with BubR1 and Mad2 during interphase, Bub3 was also found in two independent complexes with Bub1 and BubR1 in mitotic mammalian and Xenopus cells (Campbell, 2003; Taylor, 1998), and was shown to be required for the localization of the mammalian proteins to the kinetochores (Taylor, 1998). In yeast, Bub3 was also found in a complex with Mad1 and Bub1; the formation of this complex is dependent on Mad2 and seems essential for the checkpoint response (Brady, 2000) (Lopez, 2005).
In order to study the function of Bub3 further, mutant alleles were sought in Drosophila and depletion of the protein was carried out in S2 cells. The data show that Bub3 has an additional role besides its involvement in a checkpoint dependent mitotic arrest upon spindle damage. Bub3 is necessary to prevent APC/C-dependent degradation of mitotic cyclins during G2, thereby regulating both entry and transit through the initial stages of mitosis. Furthermore, the data suggest the existence of differentially activated APC/C complexes, which are inhibited by Bub3 to ensure accumulation of mitotic cyclins (Lopez, 2005).
Therefore, apart from its essential role in the checkpoint, Bub3 is also required during the G2/M transition and prophase to allow normal accumulation of mitotic cyclins, presumably by regulating the activity of the APC/C. In the absence of Bub3, cells are unable to arrest in response to spindle damage and also progression through early stages of mitosis is delayed owing to significantly lower levels of cyclins A and B (Lopez, 2005).
In order to study the role of Bub3 during mitosis bub31, the first mutant allele of the gene in Drosophila, was identified and characterized. Analysis of bub31 mutant cells indicates that as in other systems (Kalitsis, 2000), bub3 is an essential gene. The bub31 mutant allele appears to be hypomorphic since hemizygous neuroblasts and S2 cells depleted of Bub3 by RNAi show a more severe phenotype than bub31 homozygous mutant cells. Also, as previously shown for other organisms (Babu, 2003; Campbell, 2003; Kalitsis, 2000), in Drosophila, bub3 is required for checkpoint-dependent mitotic arrest, since its loss either by mutation or by RNAi causes PSCS, abnormal anaphase organization, significant aneuploidy and inability to arrest in mitosis after spindle damage (Lopez, 2005).
In vivo analysis of cell division in Bub3-depleted cells reveals that this checkpoint protein could be required for the normal timing of mitosis. Using S2 cells stably expressing GFP-tubulin it was found that after Bub3 depletion, mitosis (from NEBD to anaphase onset) is significantly faster than in control cells. This is at odds with recently published results where it was shown that in HeLa cells stably expressing H2B-GFP, Bub3-depleted cells enter anaphase with misaligned chromosomes but the timing between NEBD and anaphase onset is not altered (Meraldi, 2004). The reason for this discrepancy is not clear and may be due to a species-specific requirement for Bub3 during mitosis (Lopez, 2005).
More significantly, the data revealed that cell cycle progression after bub3 mutation or depletion is characterized by a high frequency of cells in prophase, suggesting a slower progression through the early stages of mitosis. Live analysis of Bub3-depleted cells confirmed these results. This delay in prophase appears to result from a defective accumulation of cyclins in both interphase and mitotic cells. Indeed, if cyclin B is stabilized in bub31 mutant cells by a mutation in the gene for the APC/C subunit cdc27 or through expression of a stable form of cyclin B, the mitotic index is increased and bub31 mutant cells transit normally through early stages of mitosis. These results also suggest that the defective accumulation of cyclins in bub31 mutant cells is likely to be APC/C dependent, suggesting that Bub3 is able to regulate APC/C activity well before its established role in the mitotic checkpoint response during prometaphase (Lopez, 2005).
Bub3 has never been shown to bind either the APC/C or cdc20 directly. Therefore, it is possible that Bub3 affects APC/C activity through one of its binding partners, for example BubR1. However, the analysis of mitotic progression of bub31 mutant cells causes a very different mitotic phenotype from that caused by mutations in bubR1 (Basu, 1999) (see also Logarinho, 2004). Cells mutant for bubR1 enter prophase normally but progress into anaphase as soon as the nuclear envelope breaks down, even before the completion of chromosome condensation, resulting in chromosome breakage and apoptosis. None of these phenotypes is observed after mutation of bub3 or depletion of the Bub3 protein in S2 cells. These cells show significant aneuploidy and undergo premature mitotic exit, but with properly condensed chromosomes and showing no signs of apoptosis. Furthermore, the analysis of Bub3-depleted cells shows that the majority of the cells are delayed in prophase because they display mitosis-specific phosphorylation of histone H3 and retain an intact nuclear envelope. Although classified as prophase, only half of these cells show matured asters, a cytoplasmic event that should have taken place as cells enter mitosis. These results suggest that after Bub3 depletion, nuclear and cytoplasmic events can uncouple, an observation that is in agreement with a reduced activity of the cyclin/cdk complexes in the cytoplasm. A correlation between cyclin A and cyclin B dependent kinases and formation of the mitotic spindle is well established. It has been shown that cyclin A-dependent kinase activity increases the microtubule nucleating activity of centrosomes. In contrast, the reorganization of microtubules that ultimately leads to mitotic spindle assembly seems to involve both cyclin A- and cyclin B-dependent kinase activities. Furthermore, it has been recently proposed that centrosome nucleated microtubules, at the prophase-prometaphase transition, promote tension when attached to the nuclear envelope and induce tearing of the nuclear lamin, thus promoting nuclear envelope invagination, permeabilization and eventually NEBD (Beaudouin, 2002). This model could explain how mitotic spindle formation and nuclear disassembly are two highly coordinated processes. Furthermore, in vivo studies in Drosophila embryos have shown that APC/C subunits, cdc27 and cdc16, accumulate at the nuclear envelope region during interphase and are only enriched in the nuclear area as cells enter prophase and NEBD takes place (Huang, 2002). Thus, the integrity of the nuclear envelope appears to establish a barrier between the nucleus and the cytoplasm during early stages of mitosis. Uncoupling of mitotic processes and delayed prophase may explain why bub31 mutant cells undergo mitosis with properly condensed chromosomes (Lopez, 2005).
It is widely accepted that during mitosis APC/C activity is suppressed by the checkpoint proteins as long as there is one kinetochore that is unattached to the spindle or that is not otherwise under tension. According to this model, checkpoint proteins including Bub1, BubR1, Bub3 and Mad2 are recruited to unattached kinetochores where specific protein complexes are produced that directly or indirectly inhibit APC/C activity. However, the results show that Bub3 appears to be required for inhibition of the APC/C independently of its localization to kinetochores since it only localizes to kinetochores during mitosis. Recently published results also suggest that multiprotein complexes, including Bub3-BubR1, Bub3-Mad2 or Bub3-BubR1-Mad2, that inhibit the APC/C, can form in the absence of kinetochores (Sudakin, 2001; Tang, 2001). These results establish a possible mechanistic basis for the action of checkpoint proteins as regulators of APC/C activity independent of their kinetochore localization. Tight regulation of the APC/C activity ensures sequential destruction of APC/C substrates and the correct timing of mitotic events. During interphase, APC/C activity is regulated by Emi1, most probably by preventing binding of substrates to the APC/C (Reimann, 2001b). During G1/S, Emi1 blocks APC/CCdh1 activity, allowing cyclin A accumulation and thus promoting G1/S transition (Reimann, 2001b). During G2 and early prophase, Emi1 is able to inhibit Cdc20, thereby promoting cyclin B accumulation and mitotic progression (Reimann, 2001a). Degradation of Emi1 occurs during prophase releasing APC/C inhibition (Margottin-Goguet, 2003). In this context, the data suggest that Bub3 could mediate APC/C inhibition before NEBD (Lopez, 2005).
Recent results have suggested that more than one APC/C complex may be responsible for either sister chromatid separation or cyclin B destruction during mitosis (Huang, 2002). The results on the mitotic behaviour and cyclin B accumulation in single (bub31 and cdc27) and double (bub31; cdc27) mutant cells support this data and suggest that Bub3 might affect the activity of the different APC/C complexes: (1) it was shown that incubation of cdc27 mutant neuroblasts with colchicine leads to a mitotic arrest with unseparated sister chromatids and normal cyclin B levels, revealing a classical checkpoint response; (2) sister chromatid cohesion in cdc27 mutants can be abolished by a mutation in bub3, showing that the APC/C-dependent separation of sister chromatids does not require cdc27; (3) cyclin B can be degraded during G2 or mitosis in the absence of cdc27 when Bub3 is depleted or mutated. These observations are fully in accordance with previously published results showing that the APC/C subunits cdc16 and cdc27 have distinct locations during mitosis and that individual depletion of cdc16 or cdc27 proteins by RNAi leads to distinct mitotic phenotypes (Huang, 2002). Similarly, in yeast it has been shown that the APC/C subunit cdc27 is not required for the degradation of securin since overexpression of the cdk inhibitor Sic1 is sufficient to rescue the viability of cdc27 mutants (Thornton, 2003). Furthermore, mutations in the Drosophila homologue of APC5, another APC/C subunit, result in a phenotype similar in all respects to mutations in cdc27 including high levels of cyclin B and sister chromatid separation (Bentley, 2002). These data suggest that the activity of the different APC/C subunits may be required at different times during mitosis and at different locations within a cell, and may help to determine the specificity of the APC/C towards the substrates, thus reflecting differentially activated APC/C complexes (Lopez, 2005).
Overall, the results suggest that checkpoint proteins might be required to restrain APC/C activity at multiple times during entry and progression through mitosis, revealing that what has been previously called the spindle assembly checkpoint is indeed a much broader regulatory mechanism that monitors events both before and during mitosis (Lopez, 2005).
See Larsen (2004) and Wilson (2004) for information regarding the structure of the highly conserved yeast Bub3 homolog, which, like the fly protein consists of WD domains.
date revised: 15 April 2005
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