Related Molecules in the Encyclopedia
Bub1 was originally discovered as a gene required for cell cycle arrest during mitosis in response to the microtubule depolymerizing drug benzimidazole in the model organism Saccharomyces cerevisiae (Hoyt et al. 1991). Mutant yeast were unable to arrest and inhibit the budding process at the end of mitosis, a marker for cell cycle progression, hence the name Budding Uninhibited by Benzimidazole 1 (BUB1). Through its capacity to contribute to mitotic arrest, BUB1 functions as an integral component of the spindle assembly checkpoint (SAC), a surveillance mechanism that delays mitotic progression until all kinetochores are properly attached to microtubules, and aligned at the spindle equator in metaphase. Since its original discovery in budding yeast, a SAC function for BUB1 has been verified in all model organisms studied to date (reviewed in (Elowe 2011). Soon after its discovery, hBUB1 was characterized as a Serine/Threonine kinase able to autophosphorylate and constitutively associate with another of the original BUB proteins, BUB3 (Budding Uninhibited by Benzimidazoles 3). It was recognized early on that BUB3 plays an important role in BUB1 recruitment to kinetochores (Roberts et al. 1994; Taylor et al. 1998) which are macromolecular structures that assemble onto centromeres at each mitosis and form both the signaling platform for the SAC as well as the major microtubule binding interface on dividing chromosomes (reviewed (Cheeseman 2014)). The human Bub1 gene maps to chromosome 2 at 2q14, includes 25 exons (NCBI gene ID 699) (Cahill et al. 1999), and was identified by virtue of its homology to budding yeast BUB1 (Cahill et al. 1998). Indeed mutants of this gene were identified in panel of chromosomally instable colorectal cancers. This led to an enormous body of work that aimed to explore mutations in SAC genes in various cancers. While it is now recognized that complete inactivation of the SAC is incompatible with cell survival, this early work was nevertheless influential in that it revived interest in the role of aneuploidy in the development of cancer, and set the pace for much of the more recent work exploring the relationship between mitotic deregulation, chromosome segregation defects, aneuploidy, and cancer (reviewed in (Gordon et al. 2012).
Structural Aspects of BUB1
The BUB1 N-Terminus: A Kinetochore Localization Module
The BUB1 Middle Region Contains the SAC Signaling Motifs
BUB1 contains two KEN (Lys-Glu-Asn) boxes (residues 535–537 and 625–627) required for the interaction with and phosphorylation of CDC20 (Cell Division Cycle 20), an activating and specificity-determining co-factor of APC/C (Kang et al. 2008; Jia et al. 2016). Earlier reports identified BUB1 KEN boxes as degradation motifs required for BUB1 destruction by the APC/C (Qi and Yu 2007). However, a later investigation reported a requirement for KEN boxes in hCDC20 binding and phosphorylation in synergy with PLK1, which was proposed to be essential for SAC activation (Jia et al. 2016). Recently, a motif termed as ABBA (Cyclin A, BUBR1, BUB1, and Acm1, also known as the Phe-box, A-box; BUB1 residues 527–532) was shown to contribute to the BUB1-CDC20 interaction (Di Fiore et al. 2015). Deletion of BUB1 residues encompassing this region caused a reduction in CDC20 kinetochore localization (Di Fiore et al. 2015; Vleugel et al. 2015), although a similar role has been proposed for the ABBA motif of the BUB1 paralog BUBR1 (Lischetti et al. 2014). Another important segment in the middle region is the conserved motif1(CD1) (hBUB1 residues 458–476) and is required for SAC function likely through mediating recruitment of MAD (Mitotic Arrest Deficient) 1 and 2 (Klebig et al. 2009).
The BUB1 C-Terminus Includes a Highly Conserved Serine/Threonine Kinase Domain
At the C-terminus, BUB1 has a kinase extension region (amino acids 724–783) required for the activation of BUB1 followed by the kinase domain (amino acids 784–1085), which contributes to chromosome congression and alignment (Kang et al. 2008; Klebig et al. 2009), and potentially the SAC (Tang et al. 2004; Ricke et al. 2011; Ricke et al. 2012; Jia et al. 2016). Two phospho-substrates of BUB1 have been identified so far in addition to its autophosphorylation (See below). BUB1 phosphorylates Histone H2A at S121 which then allows SGO protein binding and recruitment to kinetochores in fission yeast (Kawashima et al. 2010). Similarly mouse and human H2A phosphorylation by BUB1at the equivalent residue has also been reported (Ricke et al. 2012; Liu et al. 2013). A single mutation in Sgo1 (K492A) abolishes the interaction between SGO1 and H2ApT120 (Liu et al. 2013), suggesting that it is the direct site of interaction between these two proteins. On the other hand, CDK1 (cyclin dependent kinase 1) phosphorylates SGO1 at T346, which is required for SGO1 interaction with cohesin, a protein complex needed for sister chromatid cohesion (Liu et al. 2013). The mutation of both sites (K492A and T346A) abolishes SGO1 localization at chromosomes. Hence, both H2AT120 phosphorylation by BUB1 and cohesin binding promote SGO1 recruitment to inner centromeres (Liu et al. 2013). SGO proteins form a complex with PP2A (protein phosphatase 2A) that removes phosphorylation of cohesin subunits to prevent premature sister chromatid separation; thus, BUB1 kinase activity is essential for cohesion protection at centromeres through recruitment of SGO-PP2A complex (Kitajima et al. 2006; Tang et al. 2006). BUB1 phosphorylation of H2AT120 has also been suggested to contribute to SAC functioning through proper localization and activation of the AURORA B kinase at centromeres (Ricke et al. 2012). The second direct substrate of BUB1 kinase activity is CDC20 which contains, at least six sites in its N-terminus potentially phosphorylated by BUB1. Mutation of these sites to alanine causes inefficient APC/C inhibition in vitro and SAC defects in vivo, as measured by early mitotic exit compared control cells (Tang et al. 2004). Recent work suggest that PLK1 may cooperate with BUB1 to phosphorylate these sites, demonstrating yet another potential redundancy in SAC kinase signaling (Jia et al. 2016).
Regulation of BUB1 Kinetochore in Early and Late Mitosis
BUB1 is a stable kinetochore protein in fission yeast and mammalian cells as demonstrated by its relatively slow turnover and exchange at unattached kinetochores compared to other SAC proteins like MAD2, BUBR1, and MPS1 (Howell et al. 2004; Shah et al. 2004; Rischitor et al. 2007). Autophosphorylation has been implicated in restricting hBUB1 turnover at kinetochores (Asghar et al. 2015). Mutation of a single autophosphorylation site to alanine (T589A) increases hBUB1 kinetochore turnover, resulting in an increase in cytoplasmic BUB1 levels and ectopic phosphorylation of its substrate H2AT120, at chromosome arms, and consequently ectopic recruitment of the H2ApT120 binding partner SGO1, resulting in aberrant chromosome congression and sister chromatic cohesion. Artificially stabilizing this BUB1 mutant at kinetochores refocuses H2ApT120 and SGO1 levels back to centromeres (Asghar et al. 2015).
Activation of BUB1 Kinase
The crystal structure of the active form of BUB1 kinase domain (Kang et al. 2008) and, more recently, the structure of the active autophosphorylated (pS969) kinase have been reported (Lin et al. 2014). The BUB1 kinase domain deviates from canonical kinase domain found in other kinases such as PKA (protein kinase A) in certain aspects (Lin et al. 2014). For example, the canonical motifs at the catalytic and activation segments are slightly different. The canonical HRD is modified into HGD, the DFG into DLG, and the APE into CVE. Moreover, BUB1, as discussed above, has an extended kinase activation domain also known as an N-terminal extension domain which forms extensive interactions with N- and C-lobe of kinase domain to stabilize it. The mode of activation of BUB1 kinase domains by kinase extension domain is much like cyclins in activating CDKs, and mutations in this kinase extension domain severely attenuate kinase activity of BUB1(Kang et al. 2008). The structural comparison of unphosphorylated and phosphorylated BUB1 (BUB1pS969) showed that there are no major differences between the two structures except in P+1 loop of activation segment. Structural rearrangements in this region after autophosphorylation at S969 act as a molecular switch required for activation of BUB1 kinase (Lin et al. 2014). Autophosphorylation of S969 is needed for kinase activity towards H2A yet it is dispensable for CDC20 phosphorylation which could be due to differences in binding affinity of CDC20 and H2A phosphoresidues with the activation segment of BUB1 (Lin et al. 2014). In addition to localization, the TPR domain of BUB1 was proposed to induce long range activation of C-terminal kinase domain as mutagenesis of this region produced less effective BUB1 kinase activity (Krenn et al. 2012; Ricke et al. 2012). However, later studies using similar methods did not support this mode of activation (Lin et al. 2014; Asghar et al. 2015).
Regulation of SAC by BUB1
BUB1 is a genuine component of the SAC, and its role in SAC has been confirmed and studied in several model organisms including fission yeast, budding yeast, frog, worm, fruit fly, mouse, and humans (Roberts et al. 1994; Taylor and McKeon 1997; Basu et al. 1998; Bernard et al. 1998; Sharp-Baker and Chen 2001; Tang et al. 2004; Encalada et al. 2005). In these studies, depletion or structural mutations in BUB1 cause precocious exit from mitosis. For example, in humans, mutations in TPR domain and BUB3 binding domain caused SAC defects (Klebig et al. 2009). The role of BUB1 kinase activity in SAC function remains controversial. As mentioned above, one target of BUB1 kinase activity for SAC activation is CDC20 (Tang et al. 2004). CDC20 binding to KEN boxes allows for its phosphorylation by hBUB1 and hPLK1 for SAC activation (Jia et al. 2016). Thus, a nonkinase region of hBUB1 may be necessary for kinase activity during SAC activation. BUB1 kinase activity may also promote SAC activity through H2A-T120 phosphorylation and timely AURORA B localization and activation (Ricke et al. 2012). Nevertheless, others have found that kinase-inactivating mutations in the BUB1 catalytic domain do not affect the strength of the SAC (Klebig et al. 2009; Perera and Taylor 2010; Vleugel et al. 2015).
The major role of BUB1 kinase in SAC function may lie in its ability to function as a kinetochore scaffold for downstream proteins (Johnson et al. 2004; Rischitor et al. 2007; Klebig et al. 2009), including BUBR1, MAD1, MAD2, BUB3, SGO, CENP (centromere protein)-E and -F, CDC20 and RZZ (Rod–Zwilich–Zw10) complex (Johnson et al. 2004; Kang et al. 2008; Klebig et al. 2009; Kawashima et al. 2010; Zhang et al. 2015). Distinct structural regions on BUB1 have been implicated in its scaffolding functions. The role of BUB1 in kinetochore recruitment of BUBR1 has been reported in humans (Johnson et al. 2004; Klebig et al. 2009). A central region of BUB1 protein following the TPR domain (residues 266–311), termed as the R1LM (BUBR1 localization motif), is involved in direct pseudo-symmetrical BUBR1 binding and kinetochore recruitment (Overlack et al. 2015; Zhang et al. 2015). In addition to BUBR1 recruitment, a region containing amino acids 430–530 binds and recruits components of RZZ (Rod–Zwilich–ZW10), a complex required for MAD1 and MAD2 protein recruitment and SAC (Zhang et al. 2015). Furthermore, depletion of BUB1 severely reduces ZW10 and Zwilch recruitment to kinetochores, which suggests that BUB1 is required to recruit the entire RZZ complex (Zhang et al. 2015). BUB1 is also required for kinetochore recruitment of MAD1 and MAD2, likely through the CD1 region as mutation of CD1 causes reduction in MAD1 and MAD2 kinetochore localization (Klebig et al. 2009). Furthermore, a conserved RLK (Arg-Leu-Lys) motif of MAD1 is implicated in its interaction with BUB1 and kinetochore recruitment in humans (Kim et al. 2012).
BUB1 and Chromosome Congression
Chromosome congression is the process of chromosome alignment at the spindle equator during a symmetric mitosis. BUB1 is required for this as depletion of BUB1 or structural mutations that reduce BUB1 kinetochore localization cause defects in chromosome alignment (Johnson et al. 2004; Fernius and Hardwick 2007; Logarinho et al. 2008). However, the requirement of kinase activity for chromosome congression is controversial. Expression of BUB1 mutants devoid of kinase activity did not rescue chromosome congression defects caused by BUB1 depletion, demonstrating the importance of BUB1 kinase activity for chromosome congression (Vanoosthuyse et al. 2004; Klebig et al. 2009). However, this remains controversial and other studies in mice and humans did not support the above findings (Perera and Taylor 2010; Baron et al. 2016).
BUB1 and Cancer
Most solid tumors exhibit aneuploidy, a state defined by a number of chromosomes that deviates from the norm for a given species (Weaver and Cleveland 2006). Although aneuploidy may arise due to several contributing factors, in the context of cell division, chromosome cohesion, SAC, and microtubule attachment defects are often observed in aneuploid cells (Gordon et al. 2012). However, the SAC, a signaling cascade particularly essential for cell survival, is rarely fully defective in human tumors, and it has been suggested that an imbalance in SAC signaling in aneuploid tumors contributes to chromosomal instability (CIN), which reflects a higher rate of chromosome gain or loss (Schvartzman et al. 2010). In agreement with this, complete abrogation of SAC causes early development arrest in mouse models and lethality in several tumors; thus, a weakened SAC is detected in many tumors (Weaver and Cleveland 2006; Schvartzman et al. 2010). However, SAC overactivation manifested by abnormal delay in APC/C inhibition can also contribute to CIN due to accumulation of lagging chromosomes and merotelic attachments (Schvartzman et al. 2010). Indeed, BUB1 MAD2 overexpression has been reported in breast cancer patients (Wang et al. 2015), and this overexpression is associated with poor survival and tumor aggressiveness. Reduction of BUB1 and MAD2 expression was sufficient to reduce invasive nature of some tumor cells (Wang et al. 2015). hBub1 is also overexpressed in several human lymphomas, and Bub1 overexpression in mice causes increased chromosome segregation defects due to AURORA B kinase hyperactivation (Ricke et al. 2011).
Although mutations in the SAC genes are not very common (Gordon et al. 2012), one mutation identified in Bub1 results in an amino acid substitution (A130S, in the Bub1 kinetochore localization module) and leads to defects in SAC, chromosome congression and SGO1, BUBR1 and CENP-F recruitment (Klebig et al. 2009). Thus, both structural mutations and abnormal expression of BUB1 might contribute to cancer.
Recent evidence suggests that BUB1 kinase activity plays a role in TGF-β (transforming growth factor-β) signaling in lung and breast cancer cells (Nyati et al. 2015). TGF-β is ubiquitously expressed and involved in many cellular processes related to growth, cell proliferation, and differentiation and its deregulation is associated with cancer (Weiss and Attisano 2013). BUB1 binds to TGFBRs (transforming growth factor beta receptor) at cell membranes and mediates TGF-β signaling through its kinase activity (Nyati et al. 2015). These results show a novel pathway that requires BUB1 kinase activity, which might contribute to cell migration and invasion of tumor cells. In this context, inhibition of BUB1 activity could provide a therapeutic strategy against tumor metastasis. Efforts to date have yielded 2 classes of BUB1 kinase inhibitors: an adenine analog 2OH-BNPP1 and the benzylpyrazole compounds, BAY-320, and BAY-524 (Kang et al. 2008; Baron et al. 2016). Interestingly, 2OH-BNPP1-mediated BUB1 inhibition attenuated TGFβ signaling, suggesting that this may be a viable therapeutic avenue in cancers with hyperactive TGFβ signaling (Nyati et al. 2015). BAY-320 and BAY-524 treatment presented antiproliferative effects in combination with the microtubule-stabilizing and chemotherapeutic drug Paclitaxel (Baron et al. 2016). These studies support further examining the potential use of BUB1 kinase inhibitors for cancer treatment.
The BUB1 kinase was initially discovered in yeast for its role in mitotic progression and the SAC. Later, it was also identified in other model organisms including fruit fly, frogs, worms, mice, and humans. BUB1 coordinates its activity with other SAC components to delay mitotic progression until correct kinetochore-microtubule attachments are established. Although most studies agree that BUB1 kinase activity is dispensable for its role in the SAC, this remains controversial and the role of the kinase domain may well be context dependent. BUB1’s scaffolding function however is clearly required for the SAC. BUB1 is one of the first SAC proteins to dock at kinetochores to recruit a number of other SAC proteins and mitotic regulators including (but not limited to) BUB3, BUBR1, MAD1, MAD2, SGO, and PP2A. BUB3, BUBR1, MAD, and RZZ are recruited as a result of direct interactions with BUB1(Elowe 2011), while SGO and PP2A are recruited indirectly via BUB1 phosphorylation of H2AT120 or through secondary interactions (e.g., a PP2A pool is recruited through BUBR1 (Kawashima et al. 2010; Suijkerbuijk et al. 2012)). BUB1 phosphorylation of H2AT120 is also required for proper chromosome congression likely, through promoting proper recruitment of AUROR B and SGO proteins. Finally, BUB1 expression is deregulated in several tumors and has a role in tumor progression and is being actively explored as a potential target for therapeutic intervention.
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