Bioinformatic search for plant homologs of the protein kinase Bub1—a key component of the mitotic spindle assembly checkpoint

The bioinformatic search identified 14 plant homologs of the mitotic spindle assembly checkpoint protein kinase Bub1 of animals, yeasts, and myxomycetes. It was demonstrated that the closest plant homologs of the protein kinase Bub1 were proteins with previously unknown features: XP_002274770.1 (CBI21878.1) from Vitis vinifera, EEC82122.1 from Oryza sativa Indica, EEE67244.1 from O. sativa Japonica, EEF44403.1 from Ricinus communis, and CAL57156.1 from Ostreococcus tauri. The simulation and analysis of spatial structures of the catalytic domains of the EEC82122.1, EEE67244.1, and XP_002274770.1 (CBI21878.1) proteins confirmed their conformity with the spindle assembly checkpoint protein kinases Bub1.     

Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint

Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G₂ DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.     

Spindle Assembly Checkpoint Protein Dynamics Reveal Conserved and Unsuspected Roles in Plant Cell Division

Background

In eukaryotes, the spindle assembly checkpoint (SAC) ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the spindle.

Methodology/Principal Findings

We investigated the mechanism underlying this surveillance mechanism in plants, by characterising the orthogolous SAC proteins BUBR1, BUB3 and MAD2 from Arabidopsis. We showed that the cell cycle-regulated BUBR1, BUB3.1 and MAD2 proteins interacted physically with each other. Furthermore, BUBR1 and MAD2 interacted specifically at chromocenters. Following SAC activation by global defects in spindle assembly, these three interacting partners localised to unattached kinetochores. In addition, in cases of ‘wait anaphase’, plant SAC proteins were associated with both kinetochores and kinetochore microtubules. Unexpectedly, BUB3.1 was also found in the phragmoplast midline during the final step of cell division in plants.

Conclusions/Significance

We conclude that plant BUBR1, BUB3.1 and MAD2 proteins may have the SAC protein functions conserved from yeast to humans. The association of BUB3.1 with both unattached kinetochore and phragmoplast suggests that in plant, BUB3.1 may have other roles beyond the spindle assembly checkpoint itself. Finally, this study of the SAC dynamics pinpoints uncharacterised roles of this surveillance mechanism in plant cell division.     

BUB1 and BUBR1: multifaceted kinases of the cell cycle

The multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central components of the mitotic checkpoint for spindle assembly (SAC). This evolutionarily conserved and essential self-monitoring system of the eukaryotic cell cycle ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. Despite their amino acid sequence conservation and similar domain organization, BUB1 and BUBR1 perform different functions in the SAC. Recent structural information provides crucial molecular insights into the regulation and recognition of BUB1 and BUBR1, and a solid foundation to dissect the roles of these proteins in the control of chromosome segregation in normal and oncogenic cells.     

Mammalian BUB1 Protein Kinases: Map Positions andin VivoExpression

The spindle assembly checkpoint modulates the timing of anaphase initiation in mitotic cells containing improperly aligned chromosomes and increases the probability of successful delivery of a euploid chromosome set to each daughter cell. We have characterized cDNA sequences from several organisms with highly significant predicted protein sequence homologies toSaccharomyces cerevisiaeBub1p, a protein required for function of the spindle assembly checkpoint in budding yeast. The localization of mouse and human orthologs is in agreement with known conservation of synteny. Mouse backcross mapping data indicate that the murine gene resides on chromosome 2 near IL1A, 73 cM from the mouse centromere. Radiation hybrid mapping data indicate that the human locus exhibits linkage to microsatellite marker D2S176, which is located within 10 cM of human IL1A. Multiple-tissue Northern analysis indicates conservation of expression pattern in mouse and human with markedly high mRNA levels in testis. Northern analysis of two different spindle assembly checkpoint protein gene products from human, BUB1 and MAD2, reveals an expression pattern with common tissue distribution consistent with roles in a common pathway. In addition, we demonstrate that an mRNA found to accumulate in a rat fibroblast cell transformation system encodes rat BUB1, and we find that rat BUB1 mRNA accumulation correlates with the proliferation status of cells in culture.     

Saccharomyces cerevisiae BUB2 prevents mitotic exit in response to both spindle and kinetochore damage

The spindle assembly checkpoint-mediated mitotic arrest depends on proteins that signal the presence of one or more unattached kinetochores and prevents the onset of anaphase in the presence of kinetochore or spindle damage. In the presence of either damage, bub2 cells initiate a preanaphase delay but do not maintain it. Inappropriate sister chromatid separation in nocodazole-treated bub2 cells is prevented when mitotic exit is blocked using a conditional tem1(c) mutant, indicating that the preanaphase failure in bub2 cells is a consequence of events downstream of TEM1 in the mitotic exit pathway. Using a conditional bub2(tsd) mutant, we demonstrate that the continuous presence of Bub2 protein is required for maintaining spindle damage-induced arrest. BUB2 is not required to maintain a DNA damage checkpoint arrest, revealing a specificity for spindle assembly checkpoint function. In a yeast two-hybrid assay and in vitro, Bub2 protein interacts with the septin protein Cdc3, which is essential for cytokinesis. These data support the view that the spindle assembly checkpoint encompasses regulation of distinct mitotic steps, including a MAD2-directed block to anaphase initiation and a BUB2-directed block to TEM1-dependent exit.     

Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast

Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways.     

Evolutionary analysis of WD40 super family proteins involved in spindle checkpoint and RNA export: molecular evolution of spindle checkpoint

The spindle checkpoint delays sister chromatid separation until all chromosomes have undergone bipolar spindle attachment. Previous studies have revealed BUB3, as an essential spindle checkpoint protein and its extensive sequence similarity with Rae1 (Gle2), a highly conserved member of WD40 repeat protein family throughout their length which was first shown to be involved in mRNA export. However, the recent discovery of Rae1 as an essential mitotic checkpoint protein, based on the studies from mouse and drosophila, has renewed the interest in its function during cell division. Study of evolution of proteins involved in checkpoint might throw light on evolution of eukaryotic cell cycle regulation. Here we report the evolutionary relationships between these two WD40 repeat family proteins. Amino acid sequences of BUB3 and Rae1 homologs were retrieved from various databases and phylogenetic analysis was performed with the MEGA program. Multiple sequence alignments of these two protein homologues with the ClustalX software revealed specific amino acid signatures corresponding to the protein function and also few amino acids, which are conserved in BUB3 and Rae1 indicating some common overlapping function. Data indicated a common ancestral origin of these two important proteins and further suggest that, BUB3 mediated cell cycle checkpoint might have evolved with compartmentalization of genetic material into the nucleus in eukaryotes.     

Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast.

Saccharomyces cerevisiae cells containing one or more abnormal kinetochores delay anaphase entry. The delay can be produced by using centromere DNA mutations present in single-copy or kinetochore protein mutations. This observation is strikingly similar to the preanaphase delay or arrest exhibited in animal cells that experience spontaneous or induced failures in bipolar attachment of one or more chromosomes and may reveal the existence of a conserved surveillance pathway that monitors the state of chromosome attachment to the spindle before anaphase. We find that three genes (MAD2, BUB1, and BUB2) that are required for the spindle assembly checkpoint in budding yeast (defined by antimicrotubule drug-induced arrest or delay) are also required in the establishment and/or maintenance of kinetochore-induced delays. This was tested in strains in which the delays were generated by limited function of a mutant kinetochore protein (ctf13-30) or by the presence of a single-copy centromere DNA mutation (CDEII delta 31). Whereas the MAD2 and BUB1 genes were absolutely required for delay, loss of BUB2 function resulted in a partial delay defect, and we suggest that BUB2 is required for delay maintenance. The inability of mad2-1 and bub1 delta mutants to execute kinetochore-induced delay is correlated with striking increases in chromosome missegregation, indicating that the delay does indeed have a role in chromosome transmission fidelity. Our results also indicated that the yeast RAD9 gene, necessary for DNA damage-induced arrest, had no role in the kinetochore-induced delays. We conclude that abnormal kinetochore structures induce preanaphase delay by activating the same functions that have defined the spindle assembly checkpoint in budding yeast.     

The spindle checkpoint of Saccharomyces cerevisiae responds to separable microtubule-dependent events

The spindle checkpoint regulates microtubule-based chromosome segregation and helps to maintain genomic stability [1,2]. Mutational inactivation of spindle checkpoint genes has been implicated in the progression of several types of human cancer. Recent evidence from budding yeast suggests that the spindle checkpoint is complex. Order-of-function experiments have defined two separable pathways within the checkpoint. One pathway, defined by MAD2, controls the metaphase-to-anaphase transition and the other, defined by BUB2, controls the exit from mitosis [3-6]. The relationships between the separate branches of the checkpoint, and especially the events that trigger the pathways, have not been defined. We localized a Bub2p-GFP fusion protein to the cytoplasmic side of the spindle pole body and used a kar9 mutant to show that cells with misoriented spindles are arrested in anaphase of mitosis. We used a kar9 bub2 double mutant to show that the arrest is BUB2 dependent. We conclude that the separate pathways of the spindle checkpoint respond to different classes of microtubules. The MAD2 branch of the pathway responds to kinetochore microtubule interactions and the BUB2 branch of the pathway operates within the cytoplasm, responding to spindle misorientation.     

IBD2 encodes a novel component of the Bub2p-dependent spindle checkpoint in the budding yeast Saccharomyces cerevisiae

During mitosis, genomic integrity is maintained by the proper coordination of mitotic events through the spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the incorrect orientation of the mitotic spindle. Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for proper mitotic exit. We have isolated a novel Bfa1p interacting protein named Ibd2p in the budding yeast Saccharomyces cerevisiae. We found that IBD2 (Inhibition of Bud Division 2) is not an essential gene but its deletion mutant proceeded through the cell cycle in the presence of microtubule-destabilizing drugs, thereby inducing a sharp decrease in viability. In addition, overexpression of Mps1p caused partial mitotic arrest in ibd2Delta as well as in bub2Delta, suggesting that IBD2 encodes a novel component of the spindle checkpoint downstream of MPS1. Overexpression of Ibd2p induced mitotic arrest with increased levels of Clb2p in wild type and mad2Delta, but not in deletion mutants of BUB2 and BFA1. Pds1p was also stabilized by the overexpression of Ibd2p in wild-type cells. The mitotic arrest defects observed in ibd2Delta in the presence of nocodazole were restored by additional copies of BUB2, BFA1, and CDC5, whereas an extra copy of IBD2 could not rescue the mitotic arrest defects of bub2Delta and bfa1Delta. The mitotic arrest defects of ibd2Delta were not recovered by MAD2, or vice versa. Analysis of the double mutant combinations ibd2Deltamad2Delta, ibd2Deltabub2Delta, and ibd2Deltadyn1Delta showed that IBD2 belongs to the BUB2 epistasis group. Taken together, these data demonstrate that IBD2 encodes a novel component of the BUB2-dependent spindle checkpoint pathway that functions upstream of BUB2 and BFA1.     

Structure of a Blinkin-BUBR1 complex reveals an interaction crucial for kinetochore-mitotic checkpoint regulation via an unanticipated binding Site

The maintenance of genomic stability relies on the spindle assembly checkpoint (SAC), which ensures accurate chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bioriented and attached to the mitotic spindle. BUB1 and BUBR1 kinases are central for this process and by interacting with Blinkin, link the SAC with the kinetochore, the macromolecular assembly that connects microtubules with centromeric DNA. Here, we identify the Blinkin motif critical for interaction with BUBR1, define the stoichiometry and affinity of the interaction, and present a 2.2 ? resolution crystal structure of the complex. The structure defines an unanticipated BUBR1 region responsible for the interaction and reveals a novel Blinkin motif that undergoes a disorder-to-order transition upon ligand binding. We also show that substitution of several BUBR1 residues engaged in binding Blinkin leads to defects in the SAC, thus providing the first molecular details of the recognition mechanism underlying kinetochore-SAC signaling.     

Components of the human spindle checkpoint control mechanism localize specifically to the active centromere on dicentric chromosomes

The spindle checkpoint control mechanism functions to ensure faithful chromosome segregation by delaying cell division until all chromosomes are correctly oriented on the mitotic spindle. Initially identified in budding yeast, several mammalian spindle checkpoint-associated proteins have recently been identified and partially characterized. These proteins associate with all active human centromeres, including neocentromeres, in the early stages of mitosis prior to the commencement of anaphase. We have examined the status of proteins associated with the checkpoint protein complex (BUB1, BUBR1, BUB3, MAD2), the anaphase-promoting complex (Tsg24, p55CDC), and other proteins associated with mitotic checkpoint control (ERK1, 3F3/2 epitope, hZW10), on a human dicentric chromosome. Each of these proteins was found to specifically associate with only the active centromere, suggesting that only active centromeres participate in the spindle checkpoint. This finding complements previous studies on multicentric chromosomes demonstrating specific association of structural and motor-related centromere proteins with active centromeres, and suggests that centromere inactivation is accompanied by loss of all functionally important centromere proteins.     

BUB1 mediation of caspase-independent mitotic death determines cell fate

The spindle checkpoint that monitors kinetochore-microtubule attachment has been implicated in tumorigenesis; however, the relation between the spindle checkpoint and cell death remains obscure. In BUB1-deficient (but not MAD2-deficient) cells, conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG) induced DNA fragmentation during early mitosis. This mitotic cell death was independent of caspase activation; therefore, we named it caspase-independent mitotic death (CIMD). CIMD depends on p73, a homologue of p53, but not on p53. CIMD also depends on apoptosis-inducing factor and endonuclease G, which are effectors of caspase-independent cell death. Treatment with nocodazole, paclitaxel, or 17-AAG induced CIMD in cell lines derived from colon tumors with chromosome instability, but not in cells from colon tumors with microsatellite instability. This result was due to low BUB1 expression in the former cell lines. When BUB1 is completely depleted, aneuploidy rather than CIMD occurs. These results suggest that cells prone to substantial chromosome missegregation might be eliminated via CIMD.     

Reduced level of the spindle checkpoint protein BUB1B is associated with aneuploidy in colorectal cancers

Abstract.   Objectives : The majority of solid human malignancies demonstrate DNA aneuploidy as a consequence of chromosomal instability. We wanted to investigate whether Aurora A, Aurora B, BUB1B and Mad2 were associated with the development of aneuploidy in colorectal adenocarcinomas as suggested by several in vitro studies, and if their protein levels were related to alterations at the corresponding chromosomal loci. Materials and methods : Expression levels of these spindle proteins were investigated by immunohistochemistry using tissue micro-arrays in a series of DNA aneuploid and diploid colorectal adenocarcinomas previously examined for genomic aberrations by comparative genomic hybridization. Results : All proteins were overexpressed in malignant tissues compared to controls ( P <  0.001 for all). BUB1B level was significantly reduced in aneuploid compared to diploid cancers ( P =  0.001), whereas expression of the other proteins was not associated with DNA ploidy status. High levels of Aurora A ( P =  0.049) and low levels of Aurora B ( P =  0.031) were associated with poor prognosis, but no associations were revealed between protein expression and genomic aberration. Conclusions : A significant reduction of BUB1B level was detected in aneuploid compared to diploid colorectal cancers, consistent with earlier studies showing that loss of spindle checkpoint function may be involved in development of DNA aneuploidy. Our data also show that spindle proteins are overexpressed in colorectal cancers, and that expression of the Aurora kinases is associated with prognosis in colorectal cancer.     

A field guide to the Mps1 family of protein kinases

Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability. The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity. The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis. Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint. More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis.     

A Field Guide to the Mps1 Family of Protein Kinases

Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability. The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity. The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis. Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint. More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis.