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1.
J Cell Sci ; 132(10)2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31028180

RESUMO

During mitosis, the cell sequentially constructs two microtubule-based spindles to ensure faithful segregation of chromosomes. A bipolar spindle first pulls apart the sister chromatids, then a central spindle further separates them away. Although the assembly of the first spindle is well described, the assembly of the second remains poorly understood. We report here that the inhibition of Aurora A leads to an absence of the central spindle resulting from a lack of nucleation of microtubules in the midzone. In the absence of Aurora A, the HURP (also known as DLGAP5) and NEDD1 proteins that are involved in nucleation of microtubules fail to concentrate in the midzone. HURP is an effector of RanGTP, whereas NEDD1 serves as an anchor for the γ-tubulin ring complex (γTURC). Interestingly, Aurora A phosphorylates HURP and NEDD1 during assembly of the initial bipolar spindle. We show here that the expression of a NEDD1 isoform mimicking phosphorylation by Aurora A is sufficient to restore microtubule nucleation in the midzone under conditions of Aurora A inhibition. These results reveal a new control mechanism of microtubule nucleation by Aurora A during assembly of the central spindle.


Assuntos
Aurora Quinase A/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Anáfase/fisiologia , Aurora Quinase A/antagonistas & inibidores , Linhagem Celular Tumoral , Citocinese/fisiologia , Células HeLa , Humanos , Proteínas de Neoplasias/metabolismo , Fosforilação , Serina/metabolismo , Tubulina (Proteína)/metabolismo
2.
J Cell Biol ; 201(1): 65-79, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23547029

RESUMO

Knowledge of Aurora A kinase functions is limited to premetaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. The involvement of Aurora A in events after metaphase has only been suggested because appropriate experiments are technically difficult. We report here the design of the first human Aurora A kinase (as-AurA) engineered by chemical genetics techniques. This kinase is fully functional biochemically and in cells, and is rapidly and specifically inhibited by the ATP analogue 1-Naphthyl-PP1 (1-Na-PP1). By treating cells exclusively expressing the as-AurA with 1-Na-PP1, we discovered that Aurora A is required for central spindle assembly in anaphase through phosphorylation of Ser 19 of P150Glued. This paper thus describes a new Aurora A function that takes place after the metaphase-to-anaphase transition and a new powerful tool to search for and study new Aurora A functions.


Assuntos
Anáfase/fisiologia , Metáfase/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Anáfase/efeitos dos fármacos , Aurora Quinases , Linhagem Celular , Complexo Dinactina , Humanos , Metáfase/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Serina/genética , Serina/metabolismo , Fuso Acromático/genética
3.
Dis Markers ; 34(2): 63-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23324574

RESUMO

Aurora A kinase is overexpressed in many cancers but the status of this protein in the breast cancer often varies. We investigate the expression and localization of Aurora A protein in relation with tumor emergence and progression in breast cancer. Aurora A kinase status was evaluated in 107 patients using immunohistochemistry. The experimental findings showed that high expression of the Aurora A protein was correlated with elevated nuclear grade, low expression of progesterone receptor and positive nodal status. The experimental results showed also that the localization of this kinase shifts from cytoplasm in non malignant adjacent tissue to both cytoplasmic and nuclear compartments in tumoral tissue, suggesting an oncogenic role of the nuclear accumulation. We have, furthermore, detected the overexpression of this protein in non malignant adjacent tissue. The expression of the Aurora A kinase in non malignant tissue may represent an earlier diagnosis tool for breast cancer.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/enzimologia , Proteínas Serina-Treonina Quinases/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos , Aurora Quinases , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/enzimologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/diagnóstico , Carcinoma Intraductal não Infiltrante/enzimologia , Carcinoma Intraductal não Infiltrante/patologia , Citoplasma/metabolismo , Diagnóstico Precoce , Feminino , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Progesterona/metabolismo , Adulto Jovem
4.
Dis Markers ; 33(6): 333-40, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23151618

RESUMO

We investigate the expression and localization of the tumor suppressor protein pVHL as well as the oncoprotein Aurora A kinase in kidney cancer. Both Aurora A kinase and pVHL protein status were evaluated using immunohistochemistry. The Aurora A expression is correlated with the Fuhrman grade and the TNM stage, while the pVHL expression is correlated with the capsule rupture and the TNM stage. Aurora A kinase expression increases in malignant tissue comparing to the non-malignant one. And there is a decrease in pVHL expression from the adjacent healthy tissues to the tumor`s ones. The two kinds of opposite tumor profiles display significant distribution difference according to TNM stages. It could be proposed that the absence of Aurora A protein associated with a strong expression of pVHL in clear cells kidney carcinoma are of good prognosis for the disease.


Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aurora Quinases , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/enzimologia , Feminino , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/enzimologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Regulação para Cima
5.
J Cell Biol ; 197(1): 19-26, 2012 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-22451695

RESUMO

Aurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA.


Assuntos
Centrossomo/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Aurora Quinases , Células HeLa , Humanos , Proteínas Nucleares/genética , Nucleofosmina , Fosforilação
6.
PLoS One ; 6(10): e26512, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22046298

RESUMO

Aurora kinases belong to a conserved family of serine/threonine kinases key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved mainly in mitosis while Aurora-C is expressed during spermatogenesis and oogenesis and is involved in meiosis. Aurora-C is hardly detectable in normal somatic cells. However all three kinases are overexpressed in many cancer lines. Aurora-A possesses an oncogenic activity while Aurora-B does not. Here we investigated whether Aurora-C possesses such an oncogenic activity. We report that overexpression of Aurora-C induces abnormal cell division resulting in centrosome amplification and multinucleation in both transiently transfected cells and in stable cell lines. Only stable NIH3T3 cell clones overexpressing active Aurora-C formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3T3 stable cell lines overexpressing Aurora-C induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumor aggressiveness was positively correlated with the quantity of active kinase, making Aurora-C a potential anti-cancer therapeutic target.


Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Aurora Quinase A , Aurora Quinase B , Aurora Quinase C , Aurora Quinases , Divisão Celular , Linhagem Celular , Centrossomo/patologia , Humanos , Meiose , Camundongos , Células NIH 3T3 , Transplante de Neoplasias , Neoplasias/etiologia
7.
Biochem Biophys Res Commun ; 408(4): 647-53, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21531210

RESUMO

Aurora-C, a member of the Aurora kinase family, is implicated in the regulation of mitosis. In contrast to Aurora-A and Aurora-B its cellular localization and functions are poorly characterized. TACC1 protein belongs to the transforming acidic coiled-coil family shown to interact with the Aurora kinases. In the present study we analyzed the interaction between Aurora-C and TACC1 by means of immunofluorescence (IF), co-immunoprecipitation (IP) and in vitro phosphorylation experiments. We demonstrated that Aurora-C and TACC1 proteins co-localize to the midbody of HeLa cells during cytokinesis. Immunoprecipitated TACC1 from HeLa cell extracts was associated with Aurora-C. In addition, the interaction of the two proteins was tested by analyzing the phosphorylation of TACC1 in vitro. The results demonstrated that TACC1 is phosphorylated by Aurora-C on a serine at position 228. In conclusion, the study demonstrated that TACC1 localizes at the midbody during cytokinesis and interacts with and is a substrate of Aurora-C, which warrant further investigation in order to elucidate the functional significance of this interaction.


Assuntos
Citocinese , Proteínas Fetais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinase C , Aurora Quinases , Proteínas Fetais/genética , Células HeLa , Humanos , Imunoprecipitação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Serina/genética , Serina/metabolismo
8.
PLoS One ; 6(1): e14600, 2011 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-21297952

RESUMO

BACKGROUND: CDK11(p58) is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis. METHODOLOGY/PRINCIPAL FINDINGS: In addition to these previously described roles, our study shows that CDK11(p58) inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11(p58), which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression. CONCLUSION/SIGNIFICANCE: We thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11(p58) could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase.


Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Ciclina D3/fisiologia , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Ciclina D3/metabolismo , Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico
9.
Cell Cycle ; 4(12): 1783-7, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16258285

RESUMO

Aurora-C is the third member of the aurora serine/threonine kinase family and was found only in mammals. Because Aurora-C is overexpressed in many different types of cancer cells we decided to analyze the consequences of Aurora-C overexpression in human cells. We first investigated the subcellular localization of overexpressed GFP-Aurora-C in mitosis and interphase in HeLa cells. As expected, during mitosis, we found that Aurora-C mimics Aurora-B. Surprisingly, in few interphase cells, we found that Aurora-C localized to the centrosome, like Aurora-A. We then examined the phenotype generated by Aurora-C overexpression. Basically it looked similar to the phenotypes observed after overexpression of the other Aurora kinases. We observed an augmentation of polyploid cells containing more than two centrosomes. More interestingly this phenotype was aggravated in the absence of a functional p53. Although the physiological function of Aurora-C in somatic cells remains to be clarified, our results, just like for the two other Aurora kinases, raised the question of a role of Aurora-C in the development and progression of cancer especially in the presence of mutated p53.


Assuntos
Centrossomo/patologia , Expressão Gênica/genética , Poliploidia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/deficiência , Aurora Quinase B , Aurora Quinase C , Aurora Quinases , Células Cultivadas , Cromossomos Humanos/genética , Células HeLa , Humanos , Interfase/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteína Supressora de Tumor p53/metabolismo
10.
J Biol Chem ; 280(14): 13415-23, 2005 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-15687499

RESUMO

At the end of oogenesis, Xenopus laevis stage VI oocytes are arrested at the G2/M transition (prophase) waiting for progesterone to release the block and begin maturation. Progesterone triggers a cascade of phosphorylation events such as a decrease of pK(a) and an increase of maturating-promoting factor activity. Progression through meiosis was controlled by the sequential synthesis of several proteins. For instance, the MAPK kinase kinase c-Mos is the very first protein to be produced, whereas cyclin B1 appears only after meiosis I. After the meiotic cycles, the oocyte arrests at metaphase of meiosis II with an elevated c-Mos kinase activity (cytostatic factor). By using a two-hybrid screen, we have identified maskin, a protein involved in the control of mRNA sequential translation, as a binding partner of Aurora-A, a protein kinase necessary for oocyte maturation. Here we showed that, in vitro, Aurora-A directly binds to maskin and that both proteins can be co-immunoprecipitated from oocyte extracts, suggesting that they do associate in vivo. We also demonstrated that Aurora-A phosphorylates maskin on a Ser residue conserved in transforming acidic coiled coil proteins from Drosophila to human. When the phosphorylation of this Ser was inhibited in vivo by microinjection of synthetic peptides that mimic the maskin-phosphorylated sequence, we observed a premature maturation. Under these conditions, proteins such as cyclin B1 and Cdc6, which are normally detected only in meiosis II, were massively produced in meiosis I before the occurrence of the nuclear envelope breakdown. This result strongly suggests that phosphorylation of maskin by Aurora-A prevents meiosis II proteins from being produced during meiosis I.


Assuntos
Meiose/fisiologia , Oócitos/fisiologia , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Aurora Quinases , Proteínas de Ciclo Celular/metabolismo , Feminino , Humanos , Dados de Sequência Molecular , Oócitos/citologia , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Serina/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus/genética , Xenopus laevis
11.
Mol Cell Biochem ; 243(1-2): 123-31, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12619897

RESUMO

We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an ELISA (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.


Assuntos
Anticorpos Monoclonais , Proteínas Serina-Treonina Quinases/química , Animais , Anticorpos Monoclonais/química , Aurora Quinase A , Aurora Quinases , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Precipitina , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Recombinantes/metabolismo
12.
Protein Expr Purif ; 24(3): 489-96, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11922766

RESUMO

The plasma membrane of Spiroplasma apis contains a 28-kDa major protein (P28), like other spiroplasmas which also possess a main 26- to 28-kDa membrane polypeptide, called spiralin. In the work described here, we have developed a simple and efficient method for the purification of P28 of this mollicute, a wall-less eubacteria. Proteins were first selectively extracted from the isolated membrane with the mild detergents (i) sodium N-lauroylsarcosinate (Sarkosyl) and (ii) 3-[(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate (Chaps) and subjected to size-exclusion HPLC in the presence of Chaps. The P28-enriched fraction was thereafter subjected to the second chromatographic step involving cation exchange HPLC in the presence of the same detergent. P28 was purified at the milligram level (yield, 40%). Metabolite labeling with [14C]palmitic acid and chemical analysis of P28 indicated that it is covalently modified by two O-ester-bound fatty acids and one amide-linked chain and contains a S-glycerylcysteine at the N-terminus. By charge-shift electrophoresis, Triton X-114 phase separation, and growth inhibition tests it was shown that P28 is a typical amphiphilic protein exposed, at least partly, at the cell surface. Together, our data provided evidence that P28 is a "classical" lipoprotein (i.e., triacylated) like the members of the spiralin family.


Assuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Lipoproteínas/isolamento & purificação , Spiroplasma/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Imunoquímica , Lipoproteínas/genética , Lipoproteínas/metabolismo , Spiroplasma/genética
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