BUB1 and CENP-U, Primed by CDK1, Are the Main PLK1 Kinetochore Receptors in Mitosis.

Reflecting its pleiotropic functions, Polo-like kinase 1 (PLK1) localizes to various sub-cellular structures during mitosis. At kinetochores, PLK1 contributes to microtubule attachments and mitotic checkpoint signaling. Previous studies identified a wealth of potential PLK1 receptors at kinetochores, as well as requirements for various mitotic kinases, including BUB1, Aurora B, and PLK1 itself. Here, we combine ectopic localization, in vitro reconstitution, and kinetochore localization studies to demonstrate that most and likely all of the PLK1 is recruited through BUB1 in the outer kinetochore and centromeric protein U (CENP-U) in the inner kinetochore. BUB1 and CENP-U share a constellation of sequence motifs consisting of a putative PP2A-docking motif and two neighboring PLK1-docking sites, which, contingent on priming phosphorylation by cyclin-dependent kinase 1 and PLK1 itself, bind PLK1 and promote its dimerization. Our results rationalize previous observations and describe a unifying mechanism for recruitment of PLK1 to human kinetochores.

The BUB3-BUB1 Complex Promotes Telomere DNA Replication.

Telomeres and telomere-binding proteins form complex secondary nucleoprotein structures that are critical for genome integrity but can present serious challenges during telomere DNA replication. It remains unclear how telomere replication stress is resolved during S phase. Here, we show that the BUB3-BUB1 complex, a component in spindle assembly checkpoint, binds to telomeres during S phase and promotes telomere DNA replication. Loss of the BUB3-BUB1 complex results in telomere replication defects, including fragile and shortened telomeres. We also demonstrate that the telomere-binding ability of BUB3 and kinase activity of BUB1 are indispensable to BUB3-BUB1 function at telomeres. TRF2 targets BUB1-BUB3 to telomeres, and BUB1 can directly phosphorylate TRF1 and promote TRF1 recruitment of BLM helicase to overcome replication stress. Our findings have uncovered previously unknown roles for the BUB3-BUB1 complex in S phase and shed light on how proteins from diverse pathways function coordinately to ensure proper telomere replication and maintenance.

Phosphorylation of Bub1 by Mph1 promotes Bub1 signaling at the kinetochore to ensure accurate chromosome segregation.

Bub1 is a conserved mitotic kinase involved in signaling of the spindle assembly checkpoint. Multiple phosphorylation sites on Bub1 have been characterized, yet it is challenging to understand the interplay between the multiple phosphorylation sites due to the limited availability of phosphospecific antibodies. In addition, phosphoregulation of Bub1 in Schizosaccharomyces pombe is poorly understood. Here we report the identification of a new Mph1/Mps1-mediated phosphorylation site, i.e., Ser532, of Bub1 in Schizosaccharomyces pombe. A phosphospecific antibody against phosphorylated Bub1-Ser532 was developed. Using the phosphospecific antibody, we demonstrated that phosphorylation of Bub1-Ser352 was mediated specifically by Mph1/Mps1 and took place during early mitosis. Moreover, live-cell microscopy showed that inhibition of the phosphorylation of Bub1 at Ser532 impaired the localization of Bub1, Mad1, and Mad2 to the kinetochore. In addition, inhibition of the phosphorylation of Bub1 at Ser532 caused anaphase B lagging chromosomes. Hence, our study constitutes a model in which Mph1/Mps1-mediated phosphorylation of fission yeast Bub1 promotes proper kinetochore localization of Bub1 and faithful chromosome segregation.

Low tension recruits the yeast Aurora B protein Ipl1 to centromeres in metaphase.

Accurate genome segregation in mitosis requires that all chromosomes are bioriented on the spindle. Cells monitor biorientation by sensing tension across sister centromeres. Chromosomes that are not bioriented have low centromere tension, which allows Aurora B (yeast Ipl1) to perform error correction that locally loosens kinetochore-microtubule attachments to allow detachment of microtubules and fresh attempts at achieving biorientation. However, it is not known whether low tension recruits Aurora B to centromeres or, alternatively, whether low tension directly activates Aurora B already localized at centromeres. In this work, we experimentally induced low tension in metaphase Saccharomyces cerevisiae yeast cells, then monitored Ipl1 localization. We find low tension recruits Ipl1 to centromeres. Furthermore, low tension-induced Ipl1 recruitment depended on Bub1, which is known to provide a binding site for Ipl1. In contrast, Top2, which can also recruit Ipl1 to centromeres, was not required. Our results demonstrate cells are sensitive to low tension at centromeres and respond by actively recruiting Ip1l for error correction.

Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

An Attachment-Independent Biochemical Timer of the Spindle Assembly Checkpoint.

The spindle assembly checkpoint (SAC) generates a diffusible protein complex that prevents anaphase until all chromosomes are properly attached to spindle microtubules. A key step in SAC initiation is the recruitment of MAD1 to kinetochores, which is generally thought to be governed by the microtubule-kinetochore (MT-KT) attachment status. However, we demonstrate that the recruitment of MAD1 via BUB1, a conserved kinetochore receptor, is not affected by MT-KT interactions in human cells. Instead, BUB1:MAD1 interaction depends on BUB1 phosphorylation, which is controlled by a biochemical timer that integrates counteracting kinase and phosphatase effects on BUB1 into a pulse-generating incoherent feedforward loop. We propose that this attachment-independent timer serves to rapidly activate the SAC at mitotic entry, before the attachment-sensing MAD1 receptors have become fully operational. The BUB1-centered timer is largely impervious to conventional anti-mitotic drugs, and it is, therefore, a promising therapeutic target to induce cell death through permanent SAC activation.

BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability.

Chromosome instability (CIN) is a common contributor driving the formation and progression of anaplastic thyroid cancer (ATC), but its mechanism remains unclear. The BUB1 mitotic checkpoint serine/threonine kinase (BUB1) is responsible for the alignment of mitotic chromosomes, which has not been thoroughly studied in ATC. Our research demonstrated that BUB1 was remarkably upregulated and closely related to worse progression-free survival. Knockdown of BUB1 attenuated cell viability, invasion, migration and induced cell cycle arrests, whereas overexpression of BUB1 promoted the cell cycle progression of papillary thyroid cancer cells. BUB1 knockdown remarkably repressed tumour growth and tumour formation of nude mice with ATC xenografts and suppressed tumour metastasis in a zebrafish xenograft model. Inhibition of BUB1 by its inhibitor BAY-1816032 also exhibited considerable anti-tumour activity. Further studies showed that enforced expression of BUB1 evoked CIN in ATC cells. BUB1 induced CIN through phosphorylation of KIF14 at serine1292 (Ser1292 ). Overexpression of the KIF14ΔSer1292 mutant was unable to facilitate the aggressiveness of ATC cells when compared with that of the wild type. Collectively, these findings demonstrate that the BUB1/KIF14 complex drives the aggressiveness of ATC by inducing CIN.

Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability.

Chromosome segregation in mitosis requires the removal of catenation between sister chromatids. Timely decatenation of sister DNAs at mitotic centromeres by topoisomerase IIα (TOP2A) is crucial to maintain genomic stability. The chromatin factors that recruit TOP2A to centromeres during mitosis remain unknown. Here, we show that histone H2A Thr-120 phosphorylation (H2ApT120), a modification generated by the mitotic kinase Bub1, is necessary and sufficient for the centromeric localization of TOP2A. Phosphorylation at residue-120 enhances histone H2A binding to TOP2A in vitro. The C-gate and the extreme C-terminal region are important for H2ApT120-dependent localization of TOP2A at centromeres. Preventing H2ApT120-mediated accumulation of TOP2A at mitotic centromeres interferes with sister chromatid disjunction, as evidenced by increased frequency of anaphase ultra-fine bridges (UFBs) that contain catenated DNA. Tethering TOP2A to centromeres bypasses the requirement for H2ApT120 in suppressing anaphase UFBs. These results demonstrate that H2ApT120 acts as a landmark that recruits TOP2A to mitotic centromeres to decatenate sister DNAs. Our study reveals a fundamental role for histone phosphorylation in resolving centromere DNA entanglements and safeguarding genomic stability during mitosis.

BUB1B monoallelic germline variants contribute to prostate cancer predisposition by triggering chromosomal instability.

BACKGROUND: Prostate cancer (PrCa) is the most frequently diagnosed cancer in men. Variants in known moderate- to high-penetrance genes explain less than 5% of the cases arising at early-onset (< 56 years) and/or with familial aggregation of the disease. Considering that BubR1 is an essential component of the mitotic spindle assembly checkpoint, we hypothesized that monoallelic BUB1B variants could be sufficient to fuel chromosomal instability (CIN), potentially triggering (prostate) carcinogenesis. METHODS: To unveil BUB1B as a new PrCa predisposing gene, we performed targeted next-generation sequencing in germline DNA from 462 early-onset/familial PrCa patients and 1,416 cancer patients fulfilling criteria for genetic testing for other hereditary cancer syndromes. To explore the pan-cancer role of BUB1B, we used in silico BubR1 molecular modeling, in vitro gene-editing, and ex vivo patients' tumors and peripheral blood lymphocytes. RESULTS: Rare BUB1B variants were found in ~ 1.9% of the early-onset/familial PrCa cases and in ~ 0.6% of other cancer patients fulfilling criteria for hereditary disease. We further show that BUB1B variants lead to decreased BubR1 expression and/or stability, which promotes increased premature chromatid separation and, consequently, triggers CIN, driving resistance to Taxol-based therapies. CONCLUSIONS: Our study shows that different BUB1B variants may uncover a trigger for CIN-driven carcinogenesis, supporting the role of BUB1B as a (pan)-cancer predisposing gene with potential impact on genetic counseling and treatment decision-making.

Sudden unexpected postnatal collapse and BUB1B mutation: first forensic case report.

Sudden unexpected postnatal collapse (SUPC) is a sudden collapse of the clinical conditions of a full-term or near-term newborn, within the first 7 days of life, that requires resuscitation with positive ventilation and who either dies, has hypoxic-ischemic encephalopathy, or requires intensive care. The incidence of SUPC is very low, and most often presents a negative prognosis. The BUB1B gene is a mitotic checkpoint of serine/threonine kinase B that encodes a protein crucial for maintaining the correct number of chromosomes during cell division. Mutations in the BUB1B gene are linked to mosaic variegated aneuploidy syndrome 1 (MVA1), a rare autosomal recessive disorder characterized by diffuse mosaic aneuploidies involving several chromosomes and tissues. This paper discusses a case of a newborn who had a spontaneous delivery. After 2 h and 10 min, the infant showed generalized hypotonia and cyanosis, and his doctors performed orotracheal intubation, cardiac massage, pharmacological hemodynamic therapy, mechanical ventilation, antibiotic therapy, and hypothermic treatment. The newborn was discharged after 5 months with the diagnosis of hypoxic-ischemic encephalopathy. Suspecting an SUPC, a complete genetic analysis was performed demonstrating a compound heterozygous mutations in the BUB1B gene. The newborn died at 6 months of life, 1 month after discharge. A complete autopsy was performed, determining that the cause of death was due to sepsis starting from a brocopneumonic process, with outcomes of hypoxic-ischemic encephalopathy (HIE). In this scenario, it is not possible to demonstrate the causal effect of this mutation, considering that it could play a causal or concausal role in the onset of SUPC. Further research based on multicenter studies, as well as on animal models, could be very useful to clarify the pathological effect of this mutation.

Design and evaluation of novel inhibitors for the treatment of clear cell renal cell carcinoma.

The efficacy of conventional chemotherapies in treating clear cell renal cell carcinoma (ccRCC) is often limited due to its high molecular diversity, generally low response rates to standard treatments, and prevalent drug resistance. Recent advancements in the molecular understanding of ccRCC, alongside the discovery of novel therapeutic agents targeting specific proteins, have significantly altered the treatment landscape for ccRCC. Here, we synthesized 27 new compounds that are derivatives of TG-101209 to modulate BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B). BUB1B has been recently identified as a drug target for the development of effective ccRCC treatment based on global transcriptomics profiling of ccRCC tumours and gene co-expression network analysis. We characterized the molecular structures of these 27 compounds by 1H and 13C NMR and Mass spectrometry. We evaluated the effect of these 27 compounds by analysing the modulation of the BUB1B expression. Our primary objective was to design and assess the efficacy of these new compounds in reducing the viability of Caki-1 cells, a ccRCC cell line. We performed the computational docking studies by the Schrödinger Maestro software and demonstrated that three of these compounds (13a, 5i, and 5j) effectively downregulated BUB1B expression and eventually triggered necrosis and apoptosis in the Caki-1 cell line based on the structure-activity relationship (SAR) analysis. The IC50 values for compounds 13a, 5i, and 5j were calculated as 2.047 µM, 10.046 µM, and 6.985 µM, respectively, indicating their potent inhibitory effects on cell viability. Our study suggests that these compounds targeting BUB1B could offer a more effective and promising approach for ccRCC treatment compared to the conventionally used tyrosine kinase inhibitors. Our study underscores the potential of leveraging targeted therapies against specific molecular pathways in ccRCC may open new avenues for the development of effective treatment strategies against ccRCC.

Identification of MAD2L1 and BUB1B as Potential Biomarkers Associated with Progression and Prognosis of Ovarian Cancer.

Ovarian cancer develops insidiously and is frequently diagnosed at advanced stages. Screening for ovarian cancer is an effective strategy for reducing mortality. This study aimed to investigate the molecular mechanisms underlying the development of ovarian cancer and identify novel tumor biomarkers for the diagnosis and prognosis of ovarian cancer. Three databases containing gene expression profiles specific to serous ovarian cancer (GSE18520, GSE12470, and GSE26712) were acquired. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were analyzed for the differentially expressed gene (DEGs). The protein-protein interaction (PPI) network was constructed using the STRING database. The pivotal genes in the PPI network were screened using the Cytoscape software. Survival curve analysis was performed using a Kaplan-Meier Plotter. The cancer genome atlas and Gene Expression Omnibus databases were used to find the relationship between Hub gene and serous ovarian cancer. PCR and immunohistochemistry were used to detect the expression of Hub gene in serous ovarian cancer tissues and cells. Downstream pathways of the candidate tumor marker genes were predicted using Gene Set Enrichment Analysis. In this study, 252 DEGs were screened for pathway enrichment. 20 Hub genes were identified. Survival analysis suggested that Aurka, Bub1b, Cenpf, Cks1b, Kif20a, Mad2l1, Racgap1, and Ube2c were associated with the survival of patients with serous ovarian cancer. MAD2L1 and BUB1B levels were significantly different in serous ovarian cancer at different stages. Finally, Mad2l1 was found to play a role in the cell cycle, oocyte meiosis, and ubiquitin-mediated proteolysis. Meanwhile, Bub1b may play a role in the cell cycle, ubiquitin-mediated proteolysis, and spliceosome processes. Mad2l1 and Bub1b could be used as markers to predict ovarian carcinogenesis and prognosis, providing candidate targets for the diagnosis and treatment of serous ovarian cancer.

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex.

Kinetochore localized Mad1 is essential for generating a "wait anaphase" signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod-Zw10-Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1-Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1-Bub1 complex.

Kinetochore-catalyzed MCC formation: A structural perspective.

The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that functions to ensure accurate chromosome segregation during mitosis. Macromolecular complexes known as kinetochores, act as the interface of sister chromatid attachment to spindle microtubules. In response to unattached kinetochores, the SAC activates its effector, the mitotic checkpoint complex (MCC), which delays mitotic exit until all sister chromatid pairs have achieved successful attachment to the bipolar mitotic spindle. Formation of the MCC (composed of Mad2, BubR1, Bub3 and Cdc20) is regulated by an Mps1 kinase-dependent phosphorylation signaling cascade which assembles and repositions components of the MCC onto a catalytic scaffold. This scaffold functions to catalyze the conversion of the HORMA-domain protein Mad2 from an "inactive" open-state (O-Mad2) into an "active" closed-Mad2 (C-Mad2), and simultaneous Cdc20 binding. Here, our current understanding of the molecular mechanisms underlying the kinetic barrier to C-Mad2:Cdc20 formation will be reviewed. Recent progress in elucidating the precise molecular choreography orchestrated by the catalytic scaffold to rapidly assemble the MCC will be examined, and unresolved questions will be highlighted. Ultimately, understanding how the SAC rapidly activates the checkpoint not only provides insights into how cells maintain genomic integrity during mitosis, but also provides a paradigm for how cells can utilize molecular switches, including other HORMA domain-containing proteins, to make rapid changes to a cell's physiological state.

Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1.

During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD ) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.

Budding uninhibited by benzimidazoles 1 might be a poor prognosis biomarker promoting the progression of papillary thyroid cancer.

BACKGROUND: Papillary thyroid carcinoma (PTC) is one of the most widespread malignant tumors of the endocrine system, with a high incidence. Budding uninhibited by benzimidazoles 1 (BUB1), one of the spindle assembly checkpoint (SAC) genes, is a multitask protein kinase required for eukaryotic chromosome segregation. Although BUB1 has been explored in several types of cancer, its biological role and molecular mechanisms in PTC remain unclear. METHODS: In this study, we performed an examination of four public datasets along with local PTC cohorts and discovered that BUB1 was elevated in PTC compared to non-cancer tissues. High BUB1 expression was linked with the status of BRAFV600E , RAS, and TERT after statistical analysis. RESULTS: Clinically, BUB1 is associated with a variety of clinicopathological features in PTC patients. Interestingly, analysis of the TCGA database showed that BUB1 was closely associated with poor prognosis of PTC and significantly correlated with PFS. As determined by regression analysis, BUB1, and T stage were independent predictors of PTC and were related to BRAFV600E and lymph node metastatic status. By RT-qPCR, BUB1 was considerably overexpressed in PTC cell lines in comparison with normal thyroid epithelial cells. CONCLUSION: We confirmed that the knockdown of BUB1 in BCPAP and TPC1 cell lines significantly inhibited cell proliferation, cloning, and migration in vitro experiments. These results imply that BUB1 may be a significant oncogenic gene that is directly associated with the prognosis of PTC and may represent a future target for therapeutic intervention.

Ubiquitin chromatin remodelling after DNA damage is associated with the expression of key cancer genes and pathways.

Modification of the cancer-associated chromatin landscape in response to therapeutic DNA damage influences gene expression and contributes to cell fate. The central histone mark H2Bub1 results from addition of a single ubiquitin on lysine 120 of histone H2B and is an important regulator of gene expression. Following treatment with a platinum-based chemotherapeutic, there is a reduction in global levels of H2Bub1 accompanied by an increase in levels of the tumor suppressor p53. Although total H2Bub1 decreases following DNA damage, H2Bub1 is enriched downstream of transcription start sites of specific genes. Gene-specific H2Bub1 enrichment was observed at a defined group of genes that clustered into cancer-related pathways and correlated with increased gene expression. H2Bub1-enriched genes encompassed fifteen p53 target genes including PPM1D, BTG2, PLK2, MDM2, CDKN1A and BBC3, genes related to ERK/MAPK signalling, those participating in nucleotide excision repair including XPC, and genes involved in the immune response and platinum drug resistance including POLH. Enrichment of H2Bub1 at key cancer-related genes may function to regulate gene expression and influence the cellular response to therapeutic DNA damage.

Pathogenic correlation between mosaic variegated aneuploidy 1 (MVA1) and a novel BUB1B variant: a reappraisal of a severe syndrome.

BACKGROUND: The BUB 1 mitotic checkpoint serine/threonine kinase B (BUB1B) gene encodes a key protein in the mitotic spindle checkpoint, which acts as a surveillance mechanism, crucial for the maintenance of the correct chromosome number during cell deviation. Mutations of BUB1B gene are linked to mosaic variegated aneuploidy 1 (MVA1) syndrome, a rare autosomal recessive disorder characterized by widespread mosaic aneuploidies, involving different chromosomes and tissues. MVA1 is clinically characterized by intrauterine growth restriction, post-natal growth retardation, and severe neurologic impairment including microcephaly, developmental delay/intellectual disability, epileptic seizures, and generalized hypotonia. Malignancies are also serious sequelae associated with the disorder. We reported on a case of two-year-old Italian girl with MVA1 who shows severe neurologic impairment, microcephaly and epileptic seizures. MATERIALS AND METHODS: Clinical data collection and genetic diagnosis of the patient were assessed. Mutational analysis covers the chromosomal microarray analysis, the gene methylation pattern studied using the methylation-specific multiplex ligation-dependent probe amplification, and the family-based Whole Exome Sequencing (WES). A literature research based on reported cases of MVA and premature chromatid separation was also included. RESULTS: Karyotyping has revealed 12% of mosaics in the patient who carries a novel variant in BUB1B gene (c.2679A > T, p.Arg893Ser) detected by WES. Thirty-one cases of MVA1 including the present report, and four prenatally diagnosed cases with MVA1 were selected and inspected. CONCLUSION: Clinical and genetic findings reported in the girl strongly suggest a new MVA1 genotype-phenotype correlation and lead to a reappraisal of a severe syndrome. Diagnosis and in-depth follow-up provided worthwhile data.

Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint.

The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint.

Inhibition of BUB1 suppresses tumorigenesis of osteosarcoma via blocking of PI3K/Akt and ERK pathways.

Osteosarcoma (OS) is a primary malignant bone tumour that mainly affects teenagers, with patients displaying poor prognosis. Budding uninhibited by benzimidazoles 1 (BUB1), a type of serine/threonine kinase that is linked to pro-tumorigenic phenomena, has not been well studied in OS. Hence, this study aimed to explore the role of BUB1 in OS. The expression of BUB1 in OS specimens and cell lines was assessed using immunohistochemistry and Western blot analysis. Univariate and multivariate analyses were applied to evaluate the impact of BUB1 on patient survival. Cell counting kit-8, wound-healing and Transwell assays, as well as flow cytometry, were used to investigate the influence of BUB1 inhibition on OS in vitro. Moreover, a tumour xenograft model was established to investigate the in vivo effect of BUB1 inhibition on OS tumour growth. Results showed that BUB1 was overexpressed in OS specimens and cell lines. Furthermore, BUB1 overexpression was closely associated with the poor clinical outcomes of patients with OS. Inhibition of BUB1 markedly suppressed cell proliferation and tumour growth, cell migration, invasion and induced cell apoptosis of OS by blocking the PI3K/Akt and ERK signalling pathways. Thus, our study suggested that overexpression of BUB1 protein contributed to poor survival of OS patients and that inhibition of BUB1 resulted in considerable anti-tumour activity associated with proliferation, migration, invasion and apoptosis of OS.