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1.
Science ; 371(6535)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33542149

RESUMO

The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) detects microbial and self-DNA in the cytosol to activate immune and inflammatory programs. cGAS also associates with chromatin, especially after nuclear envelope breakdown when cells enter mitosis. How cGAS is regulated during cell cycle transition is not clear. Here, we found direct biochemical evidence that cGAS activity was selectively suppressed during mitosis in human cell lines and uncovered two parallel mechanisms underlying this suppression. First, cGAS was hyperphosphorylated at the N terminus by mitotic kinases, including Aurora kinase B. The N terminus of cGAS was critical for sensing nuclear chromatin but not mitochondrial DNA. Chromatin sensing was blocked by hyperphosphorylation. Second, oligomerization of chromatin-bound cGAS, which is required for its activation, was prevented. Together, these mechanisms ensure that cGAS is inactive when associated with chromatin during mitosis, which may help to prevent autoimmune reaction.


Assuntos
Cromatina/metabolismo , Mitose , Nucleotidiltransferases/metabolismo , Aurora Quinase B/metabolismo , Ciclo Celular , Linhagem Celular , DNA/metabolismo , DNA Mitocondrial/metabolismo , Ativação Enzimática , Humanos , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/química , Fosforilação , Multimerização Proteica
2.
J Cancer Res Clin Oncol ; 147(3): 703-712, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33386469

RESUMO

OBJECTIVE: The malignant transformation of normal bladder cells (SV-HUC-1) was induced by arsenite to explore the possible mechanism of circRNA-100284 influencing bladder cancer cell proliferation. METHODS: Normal bladder SV-HUC-1 cells were cultured with 2 µM arsenite to induce malignant transformation. After 0, 3, 6, 12, and 24 h of culture, the expression level of circRNA-100284 in cells was detected by quantitative real-time PCR. Western blotting assays were used to detect the expression levels of EZH2 and cyclin-D1 proteins in cells treated with different media. Cell cycle was analyzed by flow cytometry. In addition, through cell transfection and CCK-8 experiments, the effect and mechanism of circRNA-100284 targeting microRNA-217 on proliferation was determined. The interaction between HSP70 methylation and Aurora-B was determined by Western blotting and immunoprecipitation experiments. RESULTS: With prolonged contact time with arsenite, the expression level of circRNA-100284 in cells increased continuously (P < 0.05). Western blotting assays showed that the expression levels of EZH2 and cyclin-D1 proteins in arsenite-transformed cells increased. Flow cytometry and CCK-8 showed that circRNA-100284 accelerated cell cycle transition and cell proliferation through miR-217. Finally, after culturing human bladder cancer T24 cells, combined with immunoprecipitation and in vitro kinase experiments, it was found that K561- dimethyl HSP70 activated Aurora-B, thus promoting the proliferation of bladder cancer cells. CONCLUSION: CircRNA-100284 activates aurora kinase B by inducing methylation of HSP70 via microRNA-217 to promote the proliferation of bladder cancer cells.


Assuntos
Aurora Quinase B/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Animais , Arsenitos/farmacologia , Aurora Quinase B/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Transformação Celular Neoplásica/induzido quimicamente , Ciclina D2/genética , Ciclina D2/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Ativação Enzimática , Feminino , Proteínas de Choque Térmico HSP70/genética , Xenoenxertos , Humanos , Metilação , Camundongos , MicroRNAs/genética , RNA Circular/genética , Regulação para Cima , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
3.
Cell Prolif ; 53(10): e12895, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32914523

RESUMO

OBJECTIVES: DNA damage and errors of accurate chromosome segregation lead to aneuploidy and foetal defects. DNA repair and the spindle assembly checkpoint (SAC) are the mechanisms developed to protect from these defects. Checkpoint kinase 1 (CHK1) is reported to be an important DNA damage response protein in multiple models, but its functions remain unclear in early mouse embryos. MATERIALS AND METHODS: Immunofluorescence staining, immunoblotting and real-time reverse transcription polymerase chain reaction were used to perform the analyses. Reactive oxygen species levels and Annexin-V were also detected. RESULTS: Loss of CHK1 activity accelerated progress of the cell cycle at the first cleavage; however, it disturbed the development of early embryos to the morula/blastocyst stages. Further analysis indicated that CHK1 participated in spindle assembly and chromosome alignment, possibly due to its regulation of kinetochore-microtubule attachment and recruitment of BubR1 and p-Aurora B to the kinetochores, indicating its role in SAC activity. Loss of CHK1 activity led to embryonic DNA damage and oxidative stress, which further induced early apoptosis and autophagy, indicating that CHK1 is responsible for interphase DNA damage repair. CONCLUSIONS: Our results indicate that CHK1 is a key regulator of the SAC and DNA damage repair during early embryonic development in mice.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Reparo do DNA , Pontos de Checagem da Fase M do Ciclo Celular , Animais , Apoptose/efeitos dos fármacos , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Segregação de Cromossomos/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Camundongos , Microtúbulos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo
4.
Oncogene ; 39(37): 6009-6023, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32772043

RESUMO

Aurora B kinase plays essential roles in mitosis. Its protein levels increase before the onset of mitosis and sharply decrease during mitosis exit. The latter decrease is due to a balance between the actions of the E3 ubiquitin ligase anaphase-promoting complex or cyclosome (activated by the Cdh1 adapter), and the deubiquitinating enzyme USP35. Aurora B also executes important functions in interphase. Abnormal modulation of Aurora B in interphase leads to cell cycle defects often linked to aberrant chromosomal condensation and segregation. Very little is however known about how Aurora B levels are regulated in interphase. Here we found that USP13-associates with and stabilizes Aurora B in cells, especially before their entry into mitosis. In order for USP13 to exert its stabilizing effect on Aurora B, their association is promoted by the Aurora B-mediated phosphorylation of USP13 at Serine 114. We also present evidence that USP13 instigates Aurora B deubiquitination and/or protect it from degradation in a non-catalytic manner. In addition, we report that genetic or chemical modulation of the cellular levels/activity of USP13 affects unperturbed cell-cycle progression. Overall our study unveils the molecular and cellular connections of the USP13-Aurora B axis, which potentially participates in the rewiring of the cell cycle happening in cancer cells.


Assuntos
Aurora Quinase B/metabolismo , Ciclo Celular/genética , Endopeptidases/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Progressão da Doença , Endopeptidases/metabolismo , Estabilidade Enzimática , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fosforilação , Ligação Proteica , Serina/metabolismo
5.
Nucleic Acids Res ; 48(12): 6583-6596, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479628

RESUMO

The nucleolus is a membrane-less nuclear structure that disassembles when cells undergo mitosis. During mitosis, nucleolar factors are thus released from the nucleolus and dynamically change their subcellular localization; however, their functions remain largely uncharacterised. Here, we found that a nucleolar factor called nucleolar protein 11 (NOL11) forms a protein complex with two tryptophan-aspartic acid (WD) repeat proteins named WD-repeat protein 43 (WDR43) and Cirhin in mitotic cells. This complex, referred to here as the NWC (NOL11-WDR43-Cirhin) complex, exists in nucleoli during interphase and translocates to the periphery of mitotic chromosomes, i.e., perichromosomal regions. During mitotic progression, both the congression of chromosomes to the metaphase plate and sister chromatid cohesion are impaired in the absence of the NWC complex, as it is required for the centromeric enrichment of Aurora B and the associating phosphorylation of histone H3 at threonine 3. These results reveal the characteristics of a novel protein complex consisting of nucleolar proteins, which is required for regulating kinetochores and centromeres to ensure faithful chromosome segregation.


Assuntos
Aurora Quinase B/metabolismo , Segregação de Cromossomos , Mitose , Proteínas Nucleares/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/genética , Ligação Proteica
6.
Oncogene ; 39(21): 4312-4322, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32300176

RESUMO

Aurora kinases are a family of serine/threonine kinases vital for cell division. Because of the overexpression of Aurora kinases in a broad range of cancers and their important roles in mitosis, inhibitors targeting Aurora kinases have attracted attention in cancer therapy. VX-680 is an effective pan-Aurora kinase inhibitor; however, its clinical efficacy was not satisfying. In this study, we performed CRISPR/Cas9 screens to identify genes whose depletion shows synthetic lethality with VX-680. The top hit from these screens was GSG2 (also known as Haspin), a serine/threonine kinase that phosphorylates histone H3 at Thr-3 during mitosis. Moreover, both Haspin knockout and Haspin inhibitor-treated HCT116 cells were hypersensitive to VX-680. Furthermore, we showed that the synthetic lethal interaction between Haspin depletion and VX-680 was mediated by the inhibition of Haspin with Aurora kinase B (AURKB), but not with Aurora kinase A (AURKA). Strikingly, combined inhibition of Haspin and AURKB had a better efficacy than single-agent treatment in both head and neck squamous cell carcinoma and non-small cell lung cancer. Taken together, our findings have uncovered a synthetic lethal interaction between AURKB and Haspin, which provides a strong rationale for this combination therapy for cancer patients.


Assuntos
Aurora Quinase B , Sistemas CRISPR-Cas , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Neoplasias , Neoplasias , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases , Células A549 , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Estudo de Associação Genômica Ampla , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
7.
Nat Commun ; 11(1): 1396, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170202

RESUMO

The Aurora B abscission checkpoint delays cytokinesis until resolution of DNA trapped in the cleavage furrow. This process involves PKCε phosphorylation of Aurora B S227. Assessing if this PKCε-Aurora B module provides a more widely exploited genome-protective control for the cell cycle, we show Aurora B phosphorylation at S227 by PKCε also occurs during mitosis. Expression of Aurora B S227A phenocopies inhibition of PKCε in by-passing the delay and resolution at anaphase entry that is associated with non-disjunction and catenation of sister chromatids. Implementation of this anaphase delay is reflected in PKCε activation following cell cycle dependent cleavage by caspase 7; knock-down of caspase 7 phenocopies PKCε loss, in a manner rescued by ectopically expressing/generating a free PKCε catalytic domain. Molecular dynamics indicates that Aurora B S227 phosphorylation induces conformational changes and this manifests in a profound switch in specificity towards S29 TopoIIα phosphorylation, a response necessary for catenation resolution during mitosis.


Assuntos
Anáfase/fisiologia , Aurora Quinase B/metabolismo , Metáfase/fisiologia , Mitose/fisiologia , Anáfase/genética , Aurora Quinase B/química , Aurora Quinase B/genética , Caspase 7/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromatina , Cromossomos/metabolismo , Células HEK293 , Humanos , Metáfase/genética , Mitose/genética , Simulação de Dinâmica Molecular , Fosforilação , Proteína Quinase C-épsilon/metabolismo , Proteínas Recombinantes , Transdução de Sinais/fisiologia
8.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32027339

RESUMO

Aurora B kinase is essential for faithful chromosome segregation during mitosis. During (pro)metaphase, Aurora B is concentrated at the inner centromere by the kinases Haspin and Bub1. However, how Haspin and Bub1 collaborate to control Aurora B activity at centromeres remains unclear. Here, we show that either Haspin or Bub1 activity is sufficient to recruit Aurora B to a distinct chromosomal locus. Moreover, we identified a small, Bub1 kinase-dependent Aurora B pool that supported faithful chromosome segregation in otherwise unchallenged cells. Joined inhibition of Haspin and Bub1 activities fully abolished Aurora B accumulation at centromeres. While this impaired the correction of erroneous KT-MT attachments, it did not compromise the mitotic checkpoint, nor the phosphorylation of the Aurora B kinetochore substrates Hec1, Dsn1, and Knl1. This suggests that Aurora B substrates at the kinetochore are not phosphorylated by centromere-localized pools of Aurora B, and calls for a reevaluation of the current spatial models for how tension affects Aurora B-dependent kinetochore phosphorylation.


Assuntos
Aurora Quinase B/metabolismo , Segregação de Cromossomos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinetocoros/enzimologia , Microtúbulos/enzimologia , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesina/genética , Cinesina/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de Tempo
9.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32028528

RESUMO

Aurora B kinase has a critical role in regulating attachments between kinetochores and spindle microtubules during mitosis. Early in mitosis, kinase activity at kinetochores is high to promote attachment turnover, and in later mitosis, activity decreases to ensure attachment stabilization. Aurora B localizes prominently to inner centromeres, and a population of the kinase is also detected at kinetochores. How Aurora B is recruited to and evicted from these regions to regulate kinetochore-microtubule attachments remains unclear. Here, we identified and investigated discrete populations of Aurora B at the centromere/kinetochore region. An inner centromere pool is recruited by Haspin phosphorylation of histone H3, and a kinetochore-proximal outer centromere pool is recruited by Bub1 phosphorylation of histone H2A. Finally, a third pool resides ~20 nm outside of the inner kinetochore protein CENP-C in early mitosis and does not require either the Bub1/pH2A/Sgo1 or Haspin/pH3 pathway for localization or activity. Our results suggest that distinct molecular pathways are responsible for Aurora B recruitment to centromeres and kinetochores.


Assuntos
Aurora Quinase B/metabolismo , Centrômero/enzimologia , Cinetocoros/enzimologia , Mitose , Aurora Quinase B/genética , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Tempo
10.
Nat Commun ; 11(1): 612, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001712

RESUMO

The kinase Aurora B forms the chromosomal passenger complex (CPC) together with Borealin, INCENP, and Survivin to mediate chromosome condensation, the correction of erroneous spindle-kinetochore attachments, and cytokinesis. Phosphorylation of histone H3 Thr3 by Haspin kinase and of histone H2A Thr120 by Bub1 concentrates the CPC at the centromere. However, how the CPC is recruited to chromosome arms upon mitotic entry is unknown. Here, we show that asymmetric dimethylation at Arg2 on histone H3 (H3R2me2a) by protein arginine methyltransferase 6 (PRMT6) recruits the CPC to chromosome arms and facilitates histone H3S10 phosphorylation by Aurora B for chromosome condensation. Furthermore, in vitro assays show that Aurora B preferentially binds to the H3 peptide containing H3R2me2a and phosphorylates H3S10. Our findings indicate that the long-awaited key histone mark for CPC recruitment onto mitotic chromosomes is H3R2me2a, which is indispensable for maintaining appropriate CPC levels in dynamic translocation throughout mitosis.


Assuntos
Arginina/metabolismo , Aurora Quinase B/metabolismo , Segregação de Cromossomos , Cromossomos Humanos/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Sequência de Aminoácidos , Neoplasias da Mama/patologia , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Citocinese , Desmetilação , Progressão da Doença , Feminino , Células HeLa , Histonas/química , Humanos , Células MCF-7 , Metilação , Mitose , Fosforilação , RNA Interferente Pequeno/metabolismo
11.
Cancer Cell ; 37(2): 200-215.e5, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32049046

RESUMO

Deregulation of MYC plays an essential role in T cell acute lymphoblastic leukemia (T-ALL), yet the mechanisms underlying its deregulation remain elusive. Herein, we identify a molecular mechanism responsible for reciprocal activation between Aurora B kinase (AURKB) and MYC. AURKB directly phosphorylates MYC at serine 67, counteracting GSK3ß-directed threonine 58 phosphorylation and subsequent FBXW7-mediated proteasomal degradation. Stabilized MYC, in concert with T cell acute lymphoblastic leukemia 1 (TAL1), directly activates AURKB transcription, constituting a positive feedforward loop that reinforces MYC-regulated oncogenic programs. Therefore, inhibitors of AURKB induce prominent MYC degradation concomitant with robust leukemia cell death. These findings reveal an AURKB-MYC regulatory circuit that underlies T cell leukemogenesis, and provide a rationale for therapeutic targeting of oncogenic MYC via AURKB inhibition.


Assuntos
Aurora Quinase B/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Linfócitos T/imunologia , Animais , Aurora Quinase A/genética , Aurora Quinase A/imunologia , Aurora Quinase B/imunologia , Linhagem Celular Tumoral , Proteína 7 com Repetições F-Box-WD/imunologia , Humanos , Camundongos , Fosforilação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/imunologia , Peixe-Zebra
12.
Oncogene ; 39(12): 2550-2567, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31996785

RESUMO

Aurora B is a serine/threonine kinase that has been implicated in regulating cell proliferation in distinct cancers, including breast cancer. Here we show that Aurora B expression is elevated in basal-like breast cancer (BLBC) compared with other breast cancer subtypes. This high level of expression seems to correlate with poor metastasis-free survival and relapse-free survival in affected patients. Mechanistically, we show that elevated Aurora B expression in breast cancer cells activates AKT/GSK3ß to stabilize Snail1 protein, a master regulator of epithelial-mesenchymal transition (EMT), leading to EMT induction in a kinase-dependent manner. Conversely, Aurora B knock down by short-hairpin RNAs (shRNAs) suppresses AKT/GSK3ß/Snail1 signaling, reverses EMT and reduces breast cancer metastatic potential in vitro and in vivo. Finally, we identified a specific OCT4 phosphorylation site (T343) responsible for mediating Aurora B-induced AKT/GSK3ß/Snail1 signaling and EMT that could be attenuated by Aurora B kinase inhibitor treatment. These findings support that Aurora B induces EMT to promote breast cancer metastasis via OCT4/AKT/GSK3ß/Snail1 signaling. Pharmacologic Aurora B inhibition might be a potential effective treatment for breast cancer patients with metastatic disease.


Assuntos
Aurora Quinase B/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal/metabolismo , Transição Epitelial-Mesenquimal , Metástase Neoplásica , Fatores de Transcrição da Família Snail/metabolismo , Animais , Aurora Quinase B/antagonistas & inibidores , Neoplasias da Mama/patologia , Carcinoma Ductal/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Fator 3 de Transcrição de Octâmero/metabolismo , Organofosfatos/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica , Quinazolinas/farmacologia , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/metabolismo
13.
Genes Dev ; 34(3-4): 209-225, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31919192

RESUMO

The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.


Assuntos
Aurora Quinase B/metabolismo , Cinetocoros/metabolismo , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Microtúbulos/metabolismo , Proteólise
14.
J Cell Biol ; 219(1)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31712254

RESUMO

Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect from aneuploidy, cells possess mechanisms to delay anaphase onset when Topo II is perturbed, providing additional time for decatenation. Molecular insight into this checkpoint is lacking. Here we present evidence that catalytic inhibition of Topo II, which activates the checkpoint, leads to SUMOylation of the Topo II C-terminal domain (CTD). This modification triggers mobilization of Aurora B kinase from inner centromeres to kinetochore proximal centromeres and the core of chromosome arms. Aurora B recruitment accompanies histone H3 threonine-3 phosphorylation and requires Haspin kinase. Strikingly, activation of the checkpoint depends both on Haspin and Aurora B. Moreover, mutation of the conserved CTD SUMOylation sites perturbs Aurora B recruitment and checkpoint activation. The data indicate that SUMOylated Topo II recruits Aurora B to ectopic sites, constituting the molecular trigger of the metaphase checkpoint when Topo II is catalytically inhibited.


Assuntos
Aurora Quinase B/metabolismo , Cromossomos Humanos/genética , DNA Topoisomerases Tipo II/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metáfase , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Sumoilação , Aurora Quinase B/genética , Segregação de Cromossomos , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinetocoros , Fosforilação , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/genética , Inibidores da Topoisomerase II/farmacologia
15.
Elife ; 82019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804178

RESUMO

Errorless chromosome segregation requires load-bearing attachments of the plus ends of spindle microtubules to chromosome structures named kinetochores. How these end-on kinetochore attachments are established following initial lateral contacts with the microtubule lattice is poorly understood. Two microtubule-binding complexes, the Ndc80 and Ska complexes, are important for efficient end-on coupling and may function as a unit in this process, but precise conditions for their interaction are unknown. Here, we report that the Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, or several previously identified functional determinants including the Ndc80-loop and the Ndc80-tail. Both the Ndc80-tail, which we reveal to be essential for microtubule end-tracking, and Ndc80-bound Ska stabilize microtubule ends in a stalled conformation. Modulation of force-coupling efficiency demonstrates that the duration of stalled microtubule disassembly predicts whether a microtubule is stabilized and rescued by the kinetochore, likely reflecting a structural transition of the microtubule end.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Proteínas do Citoesqueleto/genética , Humanos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de Proteína , Fuso Acromático/metabolismo
16.
AAPS J ; 22(1): 14, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31853739

RESUMO

Aurora kinase B (AKB), a Ser/Thr kinase that plays a crucial role in mitosis, is overexpressed in several cancers. Clinical inhibitors targeting AKB bind to the active DFG "in" conformation of the kinase. It would be beneficial, however, to understand if AKB is susceptible to type II kinase inhibitors that bind to the inactive, DFG "out" conformation, since type II inhibitors achieve higher kinome selectivity and higher potency in vivo. The DFG "out" conformation of AKB is not yet experimentally determined which makes the design of type II inhibitors exceedingly difficult. An alternate approach is to simulate the DFG "out" conformation from the experimentally determined DFG "in" conformation using atomistic molecular dynamics (MD) simulation. In this work, we employed metadynamics (MTD) approach to simulate the DFG "out" conformation of AKB by choosing the appropriate collective variables. We examined structural changes during the DFG-flip and determined the interactions crucial to stabilize the kinase in active and inactive states. Interestingly, the MTD approach also identified a unique transition state (DFG "up"), which can be targeted by small molecule inhibitors. Structural insights about these conformations is essential for structure-guided design of next-generation AKB inhibitors. This work also emphasizes the usefulness of MTD simulations in predicting macromolecular conformational changes at reduced computational costs.


Assuntos
Aurora Quinase B/química , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Animais , Aurora Quinase B/metabolismo , Humanos , Oligopeptídeos/metabolismo , Estrutura Secundária de Proteína , Xenopus laevis
17.
Urol Oncol ; 37(12): 900-906, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597600

RESUMO

OBJECTIVES: Overexpression of aurora kinase A (AURKA) confers a poor prognosis in patients with urothelial carcinoma of the bladder. The prognostic value of high aurora kinase B (AURKB) expression in local bladder cancer is not well defined, and whether the prognostic value of either AURKA or AURKB is affected by the use of chemotherapy is unknown. We sought to characterize the impact of high AURKA and AURKB expression on clinical outcome in patients with muscle-invasive bladder cancer (MIBC) who received neoadjuvant chemotherapy (NAC). MATERIALS AND METHODS: Immunohistochemistry for AURKA and AURKB was performed on pretreatment diagnostic transurethral resection of bladder tumor (TURBT) and matched cystectomy specimens in 50 subjects with MIBC who received NAC. Receiver operator characteristic curves (ROC) were calculated to assess the impact of AURKA and AURKB expression on pathologic response rate. Kaplan-Meier techniques and Cox proportional hazards models were used to assess the association with relapse-free survival (RFS) and overall survival (OS). RESULTS: Twenty-two of 50 [44%] patients had residual muscle-invasive (ypT2-4) urothelial carcinoma after NAC. Neither baseline tumor expression of AURKA (ROC = 0.57, P = 0.46) nor AURKB (ROC = 0.56, P = 0.87) predicted for ypT2-4 status. However, baseline expression of AURKA above the 75th percentile for this cohort was associated with an inferior RFS, (HR = 3.88, P = 0.008) and OS, (HR = 6.10, P < 0.001). Similar trends for worse survival outcomes were also observed for high AURKB levels (RFS, [HR = 2.2, P = 0.13] and OS, (HR = 2.25, P = 0.09). CONCLUSIONS: High baseline tumor AURKA and AURKB expression identified MIBC patients with inferior RFS and OS despite the use of NAC and may identify patients who should be prioritized for clinical trial enrollment rather than standard cisplatin-based chemotherapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Bexiga Urinária/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Cisplatino/uso terapêutico , Cistectomia , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante/métodos , Recidiva Local de Neoplasia , Prognóstico , Bexiga Urinária/patologia , Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia
18.
J Cell Biol ; 218(12): 3912-3925, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31570499

RESUMO

Chromosome association of the chromosomal passenger complex (CPC; consisting of Borealin, Survivin, INCENP, and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres, failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos/metabolismo , Nucleossomos/metabolismo , Animais , Aurora Quinase B/metabolismo , Divisão Celular , Centrômero/ultraestrutura , Segregação de Cromossomos , Células HeLa , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Espectrometria de Massas , Microscopia de Fluorescência , Mitose , Fosforilação , Ligação Proteica , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Survivina/metabolismo , Xenopus laevis
19.
Nat Commun ; 10(1): 4513, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586073

RESUMO

The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1ß-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.


Assuntos
Citocinese/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Mapas de Interação de Proteínas/fisiologia , Proteína Fosfatase 1/metabolismo , Aurora Quinase B/metabolismo , Sítios de Ligação/genética , Células HeLa , Humanos , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismo , Imagem com Lapso de Tempo
20.
Virus Res ; 274: 197777, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31626875

RESUMO

Flaviviruses, such as Dengue (DENV), Zika, Yellow Fever, Japanese Encephalitis and West Nile are important pathogens with high morbidity and mortality. The last estimation indicates that ∼390 millions of people are infected by DENV per year. The DENV replicative cycle occurs mainly in the cytoplasm of the infected cells and different cytoplasmic, nuclear and mitochondrial proteins participate in viral replication. In this paper we analyzed the participation of Aurora kinase B (AurKB) in the DENV replicative cycle using the specific AurKB inhibitor ZM 447439. The kinase inhibition does not alter the viral protein production/secretion or genome replication but impaired the viral yield without altering the percentage of infected cells. Moreover, confocal microscopy analysis of DENV-infected ZM 447439-treated cells show a delocalization of viral components from the replicative complexes. In summary, these observations indicate that AurKB participates in DENV viral morphogenesis or release.


Assuntos
Aurora Quinase B/metabolismo , Vírus da Dengue/fisiologia , Dengue/virologia , Liberação de Vírus , Antivirais/farmacologia , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/genética , Benzamidas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dengue/metabolismo , Vírus da Dengue/efeitos dos fármacos , Inativação Gênica , Humanos , Quinazolinas/farmacologia , Liberação de Vírus/efeitos dos fármacos
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