The Aurora B kinase inhibitor AZD1152 sensitizes cancer cells to fractionated irradiation and induces mitotic catastrophe

AZD1152, an Aurora kinase inhibitor with selectivity for Aurora B kinase, can enhance the effect of ionizing radiation (IR). The aim of this study was to evaluate and to mechanistically explore scheduling effects of AZD1152 on tumor responses to IR, in three different settings: neoadjuvant (AZD1152 before IR), adjuvant (IR before AZD1152), or concomitant treatments (AZD1152 plus one single IR dose). A more pronounced tumor growth delay was observed in the neoadjuvant and adjuvant schedules as compared to the concomitant schedule. However, AZD1152 enhanced the efficacy of IR when concomitant IR was fractionated over several days. Histopathological examination revealed that AZD1152 + IR induced polyploidy, multinucleation and micronuclei in vivo. Time-lapse videomicroscopy confirmed that cell death induced by AZD1152 + IR was preceded by multinucleation and the formation of micronuclei, which both are hallmarks of mitotic catastrophe. Caspase inhibition or removal of the pro-apoptotic protein Bax did not ameliorate the long-term cell survival of AZD1152-treated cancer cells. In contrast, a chemical inhibitor of CHK1, Chir124, sensitized cancer cells to the lethal effect of AZD1152. Altogether, these data support the contention that AZD1152 mediates radiosensitization in vivo by enhancing mitotic catastrophe, which can be used as a biomarker of treatment efficacy.     

Aurora B kinase inhibition in mitosis: Strategies for optimizing the use of Aurora kinase inhibitors such as AT9283

Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.     

Preclinical evaluation of the Aurora kinase inhibitors AMG 900, AZD1152-HQPA,and MK-5108 on SW-872 and 93T449 human liposarcoma cells

Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue and is one of the most frequently diagnosed soft tissue sarcomas in humans. There is great interest in identifying novel chemotherapeutic options for treating liposarcoma based upon molecular alterations in the cancer cells. The Aurora kinases have been identified as promising chemotherapeutic targets based on their altered expression in many human cancers and cellular roles in mitosis and cytokinesis. In this study, we investigated the effects of an Aurora kinase A inhibitor (MK-5108), an Aurora kinase B inhibitor (AZD1152-HQPA), and a pan-Aurora kinase inhibitor (AMG 900) on undifferentiated SW-872 and well-differentiated 93T449 human liposarcoma cells. Treatment of the SW-872 and 93T449 cells with MK-5108 (0–1000 nM), AZD1152-HQPA (0–1000 nM), and AMG 900 (0–1000 nM) for 72 h resulted in a dose-dependent decrease in the total viable cell number. Based upon the EC₅₀ values, the potency of the three Aurora kinase inhibitors in the SW-872 cells was as follows: AMG 900 (EC₅₀ = 3.7 nM) > AZD1152-HQPA (EC₅₀ = 43.4 nM) > MK-5108 (EC₅₀ = 309.0 nM), while the potency in the 93T449 cells was as follows: AMG 900 (EC₅₀ = 6.5 nM) > AZD1152-HQPA (EC₅₀ = 74.5 nM) > MK-5108 (EC₅₀ = 283.6 nM). The percentage of polyploidy after 72 h of drug treatment (0–1000 nM) was determined by propidium iodide staining and flow cytometric analysis. AMG 900 caused a significant increase in polyploidy starting at 25 nM in the SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant increase starting at 100 nM in the SW-872 cells and 250 nM in the 93T449 cells. The Aurora kinase A inhibitor MK-5108 did not significantly increase the percentage of polyploid cells at any of the doses tested in either cell line. The expression of Aurora kinase A and B was evaluated in the SW-872 cells versus differentiated adipocytes and human mesenchymal stem cells by real-time RT-PCR and Western blot analysis. Aurora kinase A and B mRNA expression was significantly increased in the SW-872 cells versus the differentiated adipocytes and human mesenchymal stem cells. Western blot analysis revealed a ~ 48 kDa immunoreactive band for Aurora kinase A that was not present in the differentiated adipocytes or the human mesenchymal stem cells. A ~ 39 kDa immunoreactive band for Aurora kinase B was detected in the SW-872 cells, differentiated adipocytes, and human mesenchymal stem cells. A smaller immunoreactive band for Aurora kinase B was detected in the SW-872 cells but not in the differentiated adipocytes and human mesenchymal stem cells, and this may reflect the expression of a truncated splice variant of Aurora kinase B that has been associated with poor patient prognosis. The 93T449 cells demonstrated decreased expression of Aurora kinase A and B mRNA and protein compared to the SW-872 cells, and also expressed the truncated form of Aurora kinase B. The results of these in vitro studies indicate that Aurora kinase inhibitors should be further investigated as possible chemotherapeutic agents for human liposarcoma.     

Inhibition of aurora B kinase blocks chromosome segregation,overrides the spindle checkpoint,and perturbs microtubule dynamics in mitosis

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.     

Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.     

The inhibition of Aurora A abrogates the mitotic delay induced by microtubule perturbing agents

The spindle assembly checkpoint functions during mitosis to ensure that chromosomes are properly aligned in mitotic cells prior to the onset of anaphase, thereby ensuring an equal segregation of genetic material to each daughter cell. Defects in the function of this checkpoint lead to aneuploidy, and eventually to cell death or senescence. The Aurora-related kinases, and in particular Aurora B, have been shown to play a role in regulating the spindle assembly checkpoint. In this study, we demonstrate that Aurora A activity is required for maintainance of the spindle assembly checkpoint mediated-mitotic delay induced by microtubule perturbing agents. Inhibition of Aurora A using MLN8054, a selective small-molecule inhibitor of Aurora A, in paclitaxel- or nocodazole-treated cells induces cells to become multinucleated. Using time-lapse microscopy, we demonstrate that the multinucleation phenotype arises via mitotic slippage, which is significantly accelerated upon Aurora A inhibition. Under these conditions, the spindle assembly checkpoint protein BubR1 remains localized to kinetochores prior to mitotic slippage. Moreover, we demonstrate that Aurora B remains active in these mitotic cells, indicating that the mitotic slippage induced by MLN8054 is most likely due to the inhibition of Aurora A. This finding was corroborated by demonstrating that Aurora A depletion using RNA interference in paclitaxel-treated cells also induces multinucleation. Taken together, these results suggest that Aurora A is necessary for the maintenance of the mitotic delay induced in response to microtubule-perturbing agents.     

Small-molecule inducer of cancer cell polyploidy promotes apoptosis or senescence: Implications for therapy

Polyploidy results from deregulated cell division and has been considered an undesirable event leading to increased mutation rate and cancer development. However, polyploidy may also render cancer cells more vulnerable to chemotherapy. Here, we identify a small-molecule inducer of polyploidy, R1530, which interferes with tubulin polymerization and mitotic checkpoint function in cancer cells, leading to abortive mitosis, endoreduplication and polyploidy. In the presence of R1530, polyploid cancer cells underwent apoptosis or became senescent which translated into potent in vitro and in vivo efficacy. Normal proliferating cells were resistant to R1530-induced polyploidy thus supporting the rationale for cancer therapy by induced polyploidy. Mitotic checkpoint kinase BubR1 was found downregulated during R1530-induced exit from mitosis, a likely consequence of PLK4 inhibition. BubR1 knockdown in the presence of nocodazole induced an R1530-like phenotype, suggesting that BubR1 plays a key role in polyploidy induction by R1530 and could be exploited as a target for designing more specific polyploidy inducers.     

Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction

Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.     

Enhanced radiosensitivity of androgen-resistant prostate cancer: AZD1152-mediated Aurora kinase B inhibition

Aurora kinase B (AURKB) is critical to the process of mitosis, aiding in chromosome condensation by phosphorylating histone H3. We investigated the effects of AZD1152, an AURKB inhibitor, on radiosensitivity of androgen-insensitive prostate cancer cells. The goal of this study was to test whether AZD1152 increases the susceptibility of hormone-refractory prostate cancer cells to radiation-induced DNA damage and to determine the conditions of AZD1152 treatment that maximize radiosensitization. PC3 and DU145 cells were treated with various AZD1152 doses for various durations to elucidate the conditions that yielded maximal increases in G(2)/M-phase and polyploid cells. To assess DNA damage, γ-H2AX phosphorylation was quantified for cells grown under radiosensitizing conditions and subjected to either no radiation or 5 Gy radiation. Radiosensitivity was determined by clonogenic assays. Cell cycle effects in both cell lines were maximized by treatment with 60 nM AZD1152 for 48 h. AZD1152-treated cells exhibited significantly increased DNA damage 30 min postirradiation (PC3: 100% compared to 68%, P = 0.035; DU145: 100% compared to 69%, P = 0.034), with additional DNA damage 6 h postirradiation (PC3: 85% compared to 15%, P = 0.002; DU145: 67% compared to 21%, P = 0.012). Radiosensitivity was increased in both cell lines, with dose enhancement ratios of 1.53 for PC3 cells (P = 0.017) and 1.71 for DU145 cells (P = 0.02). This study identifies the optimal AZD1152 treatment conditions to maximize the radiosensitization of PC3 and DU145 cells. These results suggest a major role for DNA damage and impairment of DNA repair mechanisms in AZD1152-induced radiosensitization of prostate cancer cells.     

Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint

The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.     

p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G₁. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21^Waf1/Cip1 enter S phase with a ≥4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G₁/S and G₂/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21^+/+ and p21^−/− cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21^−/− cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G₁-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21^+/+ cells paralleled the onset of endoreduplication in HCT116 p21^−/− cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

FBXW7 is involved in Aurora B degradation

FBXW7, a component of E3 ubiquitin ligase, plays an important role in mitotic checkpoint, but its role remains unclear. Aurora B is a mitotic checkpoint kinase that plays a pivotal role in mitosis by ensuring correct chromosome segregation and normal progression through mitosis. Whether Aurora B and FBXW7 are coordinately regulated during mitosis is not known. Here, we show that FBXW7 is a negative regulator for Aurora B. Ectopic expression of FBXW7 can suppress the expression of Aurora B. Accordingly, FBXW7 deficiency leads to Aurora B elevation. Mechanistic studies show that all FBXW7 isoforms are negative regulators of Aurora B expression through ubiquitination-mediated protein degradation. Aurora B interacts with R465 and R505 residues of WD 40 domain of FBXW7. Significantly, inverse correlation between FBXW7 and Aurora B elevation is translated into the deregulation of mitosis. FBWX7 expression mitigates Aurora B-mediated cell growth and mitotic deregulation. In addition, FBXW7 reduces the percentage of multinucleated cells caused by Aurora B overexpression. These data suggest that FBXW7 is an important negative regulator of Aurora B, and that the loss or mutation of FBXW7 as seen in many types of cancer could lead to an abnormal elevation of Aurora B and result in deregulated mitosis, which accelerates cancer cell growth.     

Chk2 prevents mitotic exit when the majority of kinetochores are unattached

The spindle checkpoint delays exit from mitosis in cells with spindle defects. In this paper, we show that Chk2 is required to delay anaphase onset when microtubules are completely depolymerized but not in the presence of relatively few unattached kinetochores. Mitotic exit in Chk2-deficient cells correlates with reduced levels of Mps1 protein and increased Cdk1–tyrosine 15 inhibitory phosphorylation. Chk2 localizes to kinetochores and is also required for Aurora B–serine 331 phosphorylation in nocodazole or unperturbed early prometaphase. Serine 331 phosphorylation contributed to prometaphase accumulation in nocodazole after partial Mps1 inhibition and was required for spindle checkpoint establishment at the beginning of mitosis. In addition, expression of a phosphomimetic S331E mutant Aurora B rescued chromosome alignment or segregation in Chk2-deficient cells. We propose that Chk2 stabilizes Mps1 and phosphorylates Aurora B–serine 331 to prevent mitotic exit when most kinetochores are unattached. These results highlight mechanisms of an essential function of Chk2 in mitosis.     

FBXW7 is involved in Aurora B degradation

FBXW7, a component of E3 ubiquitin ligase, plays an important role in mitotic checkpoint, but its role remains unclear. Aurora B is a mitotic checkpoint kinase that plays a pivotal role in mitosis by ensuring correct chromosome segregation and normal progression through mitosis. Whether Aurora B and FBXW7 are coordinately regulated during mitosis is not known. Here, we show that FBXW7 is a negative regulator for Aurora B. Ectopic expression of FBXW7 can suppress the expression of Aurora B. Accordingly, FBXW7 deficiency leads to Aurora B elevation. Mechanistic studies show that all FBXW7 isoforms are negative regulators of Aurora B expression through ubiquitination-mediated protein degradation. Aurora B interacts with R465 and R505 residues of WD 40 domain of FBXW7. Significantly, inverse correlation between FBXW7 and Aurora B elevation is translated into the deregulation of mitosis. FBWX7 expression mitigates Aurora B-mediated cell growth and mitotic deregulation. In addition, FBXW7 reduces the percentage of multinucleated cells caused by Aurora B overexpression. These data suggest that FBXW7 is an important negative regulator of Aurora B, and that the loss or mutation of FBXW7 as seen in many types of cancer could lead to an abnormal elevation of Aurora B and result in deregulated mitosis, which accelerates cancer cell growth.     

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53^−/− cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53-dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.Key words: Aurora A kinase, polyploidy, apoptosis, p53, cell cycle     

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53-/- cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53- dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.     

Calmodulin protects Aurora B on the midbody to regulate the fidelity of cytokinesis

Aurora B kinase is an integral regulator of cytokinesis as it stabilizes the intercellular canal within the midbody to ensure proper chromosomal segregation during cell division. Here we identified an E3 ligase subunit, F box protein FBXL2, that by recognizing a calmodulin binding signature within Aurora B, ubiquitinates and removes the kinase from the midbody. Calmodulin, by competing with the F box protein for access to the calmodulin binding signature, protected Aurora B from FBXL2. Calmodulin co-localized with Aurora B on the midbody, preserved Aurora B levels in cells, and stabilized intercellular canals during delayed abscission. Genetic or pharmaceutical depletion of endogenous calmodulin significantly reduced Aurora B protein levels at the midbody resulting in tetraploidy and multi-spindle formation. The calmodulin inhibitor, calmidazolium, reduced Aurora B protein levels resulting in tetraploidy, mitotic arrest, and apoptosis of tumorigenic cells and profoundly inhibiting tumor formation in athymic nude mice. These observations indicate molecular interplay between Aurora B and calmodulin in telophase and suggest that calmodulin acts as a checkpoint sensor for chromosomal segregation errors during mitosis.     

Usp39 is essential for mitotic spindle checkpoint integrity and controls mRNA-levels of Aurora B

Accurate chromosome segregation relies on the mitotic spindle checkpoint. This checkpoint acts to restrict ubiquitin ligase activity of the Anaphase-promoting complex (APC/C) in mitosis until all chromosomes are bipolarly attached to the mitotic spindle. We performed a functional RNAi-based screen to identify De-ubiquitinating enzymes (Dubs) involved in mitotic progression. We identified Usp39 as a new factor required to maintain the spindle checkpoint and support successful cytokinesis. Strikingly, although Usp39 clearly contains an ubiquitin-protease domain, we show that Usp39 is entirely deprived of Dub activity. However, consistent with a previously described role for Usp39 in mRNA processing, we observed specific reduction in Aurora B-mRNA levels after depletion of Usp39. Although we find that exogenously expressed Aurora B cDNA is not sufficient to rescue the checkpoint defect of Usp39-depleted cells, Aurora B expression is restored. Our observations suggest Usp39 to be involved in splicing of Aurora B and other mRNAs that are essential for proper spindle checkpoint function.     

Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.