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2.
Oncogene ; 26(53): 7445-56, 2007 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-17563753

RESUMO

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of mesenchymal characteristics and invasive properties by tumour cells. The loss of cell-cell contacts may be the first step of the epithelium mesenchyme transition (EMT) and involves the functional inactivation of the cell-cell adhesion molecule E-cadherin. Repression of E-cadherin expression by the transcription factor Snail is a central event during the loss of epithelial phenotype. Akt kinase activation is frequent in human carcinomas, and Akt regulates various cellular mechanisms including EMT. Here, we show that Snail activation and consequent repression of E-cadherin may depend on AKT-mediated nuclear factor-kappaB (NF-kappaB) activation, and that NF-kappaB induces Snail expression. Expression of the NF-kappaB subunit p65 is sufficient for EMT induction, validating this signalling module during EMT. NF-kappaB pathway activation is associated with tumour progression and metastasis of several human tumour types; E-cadherin acts as a metastasis suppressor protein. Thus, this signalling and transcriptional network linking AKT, NF-kappaB, Snail and E-cadherin during EMT is a potential target for antimetastatic therapeutics.


Assuntos
Carcinoma de Células Escamosas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/biossíntese , Neoplasias da Bexiga Urinária/patologia , Animais , Caderinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Epitélio/patologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Mesoderma/patologia , Regiões Promotoras Genéticas , Ratos , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fator de Transcrição RelA/biossíntese , Fator de Transcrição RelA/genética , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Regulação para Cima , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco
3.
Traffic ; 4(8): 566-75, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12839499

RESUMO

The nuclear transport of both proteins and RNAs has attracted considerable interest in recent years. However, regulation pathways of the nuclear transport machineries are still not well characterized. Previous studies indicated that ubiquitination is involved in poly(A)+ RNA nuclear export. For this reason, we systematically investigated ubiquitin-protein ligasess from the homologous to E6-AP carboxy terminus (HECT) family for potential individual roles in nuclear transport in Saccharomyces cerevisiae. Here we report that Rsp5, an essential yeast ubiquitin ligase involved in many cellular functions, when deleted or mutated in ligase activity, blocks the nuclear export of mRNAs. Affected messenger RNAs include both total poly(A)+ mRNA and heat-shock mRNAs. Mutation of Rsp5 does not affect nuclear protein import or export. Deletion of RSP5 blocks mRNA export, even under conditions where its essential role in unsaturated fatty acids biosynthesis is bypassed. Using domain mapping, we find that the ligase activity is required for proper mRNA export, indicating that ubiquitination by Rsp5 acts directly or indirectly to affect RNA export. The finding that Rsp5p ligase mutations cause a more pronounced defect at high temperatures suggests that ubiquitination of transport factors by Rsp5p may also be essential during stress conditions.


Assuntos
Núcleo Celular/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Ácidos Graxos Dessaturases/metabolismo , Temperatura Alta , Estrutura Terciária de Proteína , Estearoil-CoA Dessaturase , Fatores de Tempo
4.
Traffic ; 2(10): 684-9, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11576444

RESUMO

The evolution of the nucleus imposed on eukaryotic cells the necessity to strictly control exchange of molecules between the nucleus and the remainder of the cell, not only to protect and correctly transmit genetic information, but also to coordinate nuclear and cytoplasmic functions. Studies over the past 10 years have provided major insights into the molecular mechanisms responsible for transport of molecules between the nucleus and the cytoplasm. In addition, regulation of the nucleocytoplasmic distribution of diverse cellular factors has emerged as one of the most efficient mechanism to adapt gene expression to the cell environment, for example by controlling the access of transcriptional regulators to their target genes. In this review, we focus on the molecular basis of protein nuclear export that relies on interactions between targeting sequences present on the cargoes, specific export receptors or exportins and nuclear pore proteins, with special emphasis on the role of the Ran GTPase and associated proteins in this process.


Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Carioferinas/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Humanos , Transporte Proteico/fisiologia
5.
Science ; 293(5528): 300-3, 2001 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-11452125

RESUMO

Interferons (IFN) alpha/beta and gamma induce the formation of two transcriptional activators: gamma-activating factor (GAF) and interferon-stimulated gamma factor 3 (ISGF3). We report a natural heterozygous germline STAT1 mutation associated with susceptibility to mycobacterial but not viral disease. This mutation causes a loss of GAF and ISGF3 activation but is dominant for one cellular phenotype and recessive for the other. It impairs the nuclear accumulation of GAF but not of ISGF3 in heterozygous cells stimulated by IFNs. Thus, the antimycobacterial, but not the antiviral, effects of human IFNs are principally mediated by GAF.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Mutação em Linhagem Germinativa , Imunidade , Interferon-alfa/imunologia , Interferon gama/imunologia , Infecções por Mycobacterium/imunologia , Transativadores/fisiologia , Viroses/imunologia , Adulto , Animais , Linhagem Celular , Criança , Pré-Escolar , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Humanos , Imunidade/genética , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Janus Quinase 1 , Camundongos , Infecções por Mycobacterium/genética , Complexo Mycobacterium avium/imunologia , Infecção por Mycobacterium avium-intracellulare/genética , Infecção por Mycobacterium avium-intracellulare/imunologia , Mycobacterium bovis , Linhagem , Ligação Proteica , Proteínas Tirosina Quinases/genética , Fator de Transcrição STAT1 , Transdução de Sinais , Vírus 40 dos Símios , Transativadores/genética , Fatores de Transcrição/fisiologia , Viroses/genética
6.
J Biol Chem ; 276(28): 25910-8, 2001 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-11342536

RESUMO

Determining the cis-acting elements controlling nuclear export of RNA is critical, because they specify which RNA will be selected for transport. We have characterized the nuclear export motif of the adenoviral VA1 RNA, a small cytoplasmic RNA transcribed by RNA polymerase III. Using a large panel of VA1 mutants in both transfected COS cells and injected Xenopus oocytes, we showed that the terminal stem of VA1 is necessary and sufficient for its export. Surprisingly, we found that the nucleotide sequence within the terminal stem is not important. Rather, the salient features of this motif are its length and its relative position within the RNA. Such stems thus define a novel and degenerate cytoplasmic localization motif that we termed the minihelix. This motif is found in a variety of polymerase III transcripts, and cross-competition analysis in Xenopus oocytes revealed that export of one such RNA, like hY1 RNA, is specifically competed by VA1 or artificial minihelix. Taken together these results show that the minihelix defines a new cis-acting export element and that this motif could be exported via a novel and specific nuclear export pathway.


Assuntos
RNA Polimerase III/química , RNA/química , Animais , Sequência de Bases , Transporte Biológico , Células COS , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA/genética , RNA/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Especificidade por Substrato
7.
Mol Cell Biol ; 21(10): 3482-90, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11313474

RESUMO

Transcriptional activation of NF-kappaB is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. NF-kappaB activation induces a rapid resynthesis of IkappaBalpha which is responsible for postinduction repression of transcription. Following resynthesis, IkappaBalpha translocates to the nucleus, removes template bound NF-kappaB, and exports NF-kappaB to the cytoplasm in a transcriptionally inactive form. Here we demonstrate that IkappaBalpha interacts directly with another nucleocytoplasmic shuttling protein, hnRNPA1, both in vivo and in vitro. This interaction requires one of the N-terminal RNA binding domains of hnRNPA1 and the C-terminal region of IkappaBalpha. Cells lacking hnRNPA1 are defective in NF-kappaB-dependent transcriptional activation, but the defect in these cells is complemented by ectopic expression of hnRNPA1. hnRNPA1 expression in these cells increased the amount of IkappaBalpha degradation, compared to that of the control cells, in response to activation by Epstein-Barr virus latent membrane protein 1. Thus in addition to regulating mRNA processing and transport, hnRNPA1 also contributes to the control of NF-kappaB-dependent transcription.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas I-kappa B , NF-kappa B/genética , Ribonucleoproteínas/genética , Ativação Transcricional , Animais , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Inibidor de NF-kappaB alfa , Fosforilação , Processamento Pós-Transcricional do RNA , Proteínas da Matriz Viral/genética
8.
J Biol Chem ; 276(21): 18102-7, 2001 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-11278458

RESUMO

The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.


Assuntos
Núcleo Celular/metabolismo , Infecções por HIV , Integrase de HIV/metabolismo , HIV-1 , Transporte Biológico , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Humanos , Replicação Viral
9.
J Biol Chem ; 276(16): 12654-9, 2001 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-11124955

RESUMO

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the PsiKXE motif, where Psi represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IkappaBalpha, containing the PsiKXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Sequência Consenso , Proteínas de Ligação a DNA/química , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Humanos , Inibidor de NF-kappaB alfa , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Proteína Supressora de Tumor p53/química , Ubiquitinas/química , Ubiquitinas/genética
10.
J Cell Biol ; 152(1): 141-55, 2001 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-11149927

RESUMO

Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor-substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616-8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm.


Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Carioferinas , Proteínas de Transporte Nucleocitoplasmático , Receptores Citoplasmáticos e Nucleares , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Transporte/genética , Linhagem Celular , Produtos do Gene rev/genética , Produtos do Gene rev/metabolismo , Genes Reporter , Mutagênese , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Proteína Exportina 1
11.
Hum Mol Genet ; 9(20): 3001-9, 2000 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-11115844

RESUMO

Mutations in MEFV, a gene encoding a protein (marenostrin/pyrin) of unknown function, are associated with familial Mediterranean fever, a genetic condition characterized by febrile episodes of serosal inflammation. Based on its primary structure, this 781 residue protein is thought to function as a nuclear effector molecule. However, recent transient expression studies indicated a perinuclear cytoplasmic localization. Here, we describe the isolation and expression of a novel human MEFV isoform, MEFV-d2, generated by in-frame alternative splicing of exon 2. This transcript, expressed in leukocytes, predicts a 570 residue protein designated marenostrin-d2. To investigate differences in subcellular localization between the full-length protein (marenostrin-fl) and marenostrin-d2, while providing against the overexpression of transiently expressed proteins, we have generated CHO cell lines stably expressing these two isoforms fused to the green fluorescent protein. The localization pattern of marenostrin-d2 differs dramatically from that of marenostrin-fl. Marenostrin-fl is homogeneously distributed over the entire cytoplasm, whereas marenostrin-d2 concentrates into the nucleus. To map the critical domain(s) specifying these differences, deletion mutants have been generated. Deletion of the putative nuclear localization signals (NLS) does not alter the nuclear localization of marenostrin-d2 whereas, despite the lack of discernible NLS in the domain encoded by the exon 1-exon 3 splice junction, deletion of this domain indeed disrupts this localization. These data, which challenge the current domain organization model of marenostrin, strongly suggest that MEFV encodes a nuclear protein and raises the possibility that MEFV alternative splicing may control functions of wild-type and mutant marenostrin proteins by regulating their translocation to the nucleus.


Assuntos
Núcleo Celular/metabolismo , Febre Familiar do Mediterrâneo/genética , Proteínas/genética , Processamento Alternativo , Animais , Transporte Biológico , Northern Blotting , Células CHO , Núcleo Celular/fisiologia , Cricetinae , Proteínas do Citoesqueleto , Éxons , Humanos , Leucócitos Mononucleares/fisiologia , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear/fisiologia , Conformação Proteica , Isoformas de Proteínas , Proteínas/metabolismo , Pirina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares
12.
Exp Cell Res ; 260(2): 387-95, 2000 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-11035935

RESUMO

Infections by human and simian immunodeficiency viruses (HIV and SIV) are independent of host cell division since the preintegration complex (PIC), containing the viral DNA, is able to undergo active nuclear import after viral entry. In order to clarify the mechanisms responsible for nuclear import of the PIC, we have analyzed the subcellular distribution and the karyophilic properties of its viral components, matrix protein (MA), integrase (IN), Vpr, and Vpx. Although MA has been reported to contain a nuclear localization signal, the MA/GFP fusions are excluded from the nucleus and associated with cellular membranes. In contrast, both HIV-1 and SIV IN and Vpr localize in the nucleus of transfected cells. Interestingly, only Vpx from SIVsm virus accumulate in the nucleus while SIVsm Vpr is uniformly distributed throughout nucleus and cytoplasm. Coexpression of MA, Vpr, and IN does not induce any change in their respective intracellular localizations. Finally, we confirm the karyophilic properties of HIV-1 IN and Vpr using an in vitro nuclear import assay. These results indicate that the viral proteins IN and Vpr, which are strongly associated with the viral DNA within PIC, may participate in the nuclear import of the HIV PIC.


Assuntos
Produtos do Gene gag/metabolismo , Produtos do Gene vpr/metabolismo , Antígenos HIV/metabolismo , Integrase de HIV/metabolismo , HIV-1/metabolismo , Integrases/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Proteínas da Matriz Viral/metabolismo , Proteínas Virais , Animais , Transporte Biológico , Núcleo Celular/metabolismo , Expressão Gênica , Produtos do Gene gag/genética , Produtos do Gene vpr/genética , Antígenos HIV/genética , Integrase de HIV/genética , Células HeLa , Humanos , Integrases/genética , Líquido Intracelular/metabolismo , Permeabilidade , Proteínas da Matriz Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência Humana
13.
J Biol Chem ; 275(46): 36441-9, 2000 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-10969074

RESUMO

IkappaBalpha inhibits the transcriptional activity of NF-kappaB both in the cytoplasm by preventing the nuclear translocation of NF-kappaB and in the nucleus where it dissociates NF-kappaB from DNA and transports it back to the cytoplasm. Cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes is controlled by mutual masking of nuclear import sequences of NF-kappaB p65 and IkappaBalpha and active CRM1-mediated nuclear export. Here, we describe an additional mechanism accounting for the cytoplasmic anchoring of IkappaBalpha or NF-kappaB/IkappaBalpha complexes. The N-terminal domain of IkappaBalpha contains a sequence responsible for the cytoplasmic retention of IkappaBalpha that is specifically recognized by G3BP2, a cytoplasmic protein that interacts with both IkappaBalpha and IkappaBalpha/NF-kappaB complexes. G3BP2 is composed of an N-terminal domain homologous to the NTF2 protein, followed by an acidic domain sufficient for the interaction with the IkappaBalpha cytoplasmic retention sequence, a region containing five PXXP motifs and a C-terminal domain containing RNA-binding motifs. Overexpression of G3BP2 directly promotes retention of IkappaBalpha in the cytoplasm, indicating that subcellular distribution of IkappaBalpha and NF-kappaB/IkappaBalpha complexes likely results from a equilibrium between nuclear import, nuclear export, and cytoplasmic retention. The molecular organization of G3BP2 suggests that this putative scaffold protein might connect the NF-kappaB signal transduction cascade with cellular functions such as nuclear transport or RNA metabolism.


Assuntos
Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , NF-kappa B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , DNA Helicases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Imunofluorescência , Genes Reporter , Células HeLa , Humanos , Substâncias Macromoleculares , Mutação , Inibidor de NF-kappaB alfa , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose , Testes de Precipitina , Ligação Proteica , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína , Transporte Proteico , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Proteínas de Ligação a RNA , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Transfecção
14.
Mol Cell Biol ; 20(13): 4562-71, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10848583

RESUMO

To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopus extract-dependent manner. U1 snRNA export requires a 5' monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export.


Assuntos
Proteínas de Transporte/metabolismo , Carioferinas , Proteínas de Transporte Nucleocitoplasmático , RNA Nuclear Pequeno/metabolismo , RNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Proteína ran de Ligação ao GTP/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Feminino , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Metilação , Biologia Molecular/métodos , Mutação , RNA/química , Capuzes de RNA , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/química , RNA de Transferência de Metionina/metabolismo , Proteína Exportina 1
15.
Exp Cell Res ; 252(1): 236-41, 1999 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-10502415

RESUMO

Nuclear export of proteins containing a leucine-rich nuclear export sequence (NES) is mediated by a specific NES receptor known as Crm1. This protein, which is related to the karyopherin beta family, interacts directly with NES in a RanGTP-dependent manner. To characterize the domains of Crm1 involved in formation of the trimeric Crm1-NES-RanGTP complex, N- and C-terminal deletion mutants of Crm1 were generated and their ability to bind NES and RanGTP in vitro was analyzed. Our results indicate that two regions of Crm1 are required for the formation of the trimeric Crm1-NES-RanGTP complex, the N-terminal domain of Crm1 and the central domain of the receptor, starting after residue 160 with an essential region between 566 and 720. The N-terminal domain is homologous to the RanGTP-binding domain of karyopherin beta and therefore is likely involved in the interaction with RanGTP. Consequently, the central domain likely corresponds to the NES-binding site of Crm1.


Assuntos
Proteínas de Transporte/química , Carioferinas , Proteínas Nucleares/química , Receptores Citoplasmáticos e Nucleares , Proteína ran de Ligação ao GTP/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Técnicas In Vitro , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1
16.
Cancer Res ; 59(18): 4625-33, 1999 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-10493517

RESUMO

Treatment of human carcinoma cells with Taxol induces focal unraveling of the nuclear lamina and extensive clustering or ectopic localization of the nuclear pore complexes. These striking aberrations develop when the cells are transferred to drug-free medium and are allowed to complete mitosis. As could be confirmed by terminal deoxynucleotidyl transferase-mediated nick end labeling assays, 4,6-diamidino-2-phenylindole staining, 5-bromo-2-deoxyuridine incorporation, and examination of the nuclear lamins by Western blotting, the malformation of the nuclear envelope is not a consequence of apoptosis or G1 arrest. In fact, Taxol-treated cells possessing a defective nuclear envelope remain alive and replication-competent for at least 24 h, undergoing programmed death 72 h after removal of the drug. While still in the nonapoptotic state, these cells lose the ability to import karyophilic proteins into the nucleus. Diminished nucleocytoplasmic transport through the nuclear pore complex can be readily demonstrated by in vitro assays involving digitonin-permeabilized cells or in vivo monitoring of nuclear factor-kappaB translocation upon stimulation with tumor necrosis factor-alpha. These observations reveal novel cellular targets of antimicrotubule drugs and may pave the way for improved schemes of anticancer treatment.


Assuntos
Núcleo Celular/efeitos dos fármacos , NF-kappa B/metabolismo , Membrana Nuclear/efeitos dos fármacos , Paclitaxel/toxicidade , Apoptose , Bromodesoxiuridina/farmacocinética , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Laminas , Microscopia de Vídeo , Índice Mitótico/efeitos dos fármacos , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/efeitos dos fármacos
17.
Mol Cell Biol ; 19(9): 6345-54, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10454581

RESUMO

The X protein of hepatitis B virus (HBV) is a transcriptional activator which is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. It has been suggested that X acts as a nuclear coactivator or stimulates several signal transduction pathways by acting in the cytoplasm. One of these pathways leads to the nuclear translocation of NF-kappaB. A recent report indicates that X activates NF-kappaB by acting on two cytoplasmic inhibitors of this family of transcription factors: IkappaBalpha and the precursor/inhibitor p105. We demonstrate here that X directly interacts with IkappaBalpha, which is able to transport it to the nucleus by a piggyback mechanism. This transport requires a region of IkappaBalpha (the second ankyrin repeat) which has been demonstrated to be involved in its nuclear import following NF-kappaB activation. Using deletion mutants, we showed that amino acids 249 to 253 of IkappaBalpha (located in the C-terminal part of the sixth ankyrin repeat) play a critical role in the interaction with X. This small region overlaps one of the domains of IkappaBalpha mediating the interaction with the p50 and p65 subunits of NF-kappaB and is also close to the nuclear export sequence of IkappaBalpha, therefore providing a potential explanation for the nuclear accumulation of IkappaBalpha with X. This association can also be observed upon the induction of endogenous IkappaBalpha by tumor necrosis factor alpha (TNF-alpha) treatment of Chang cells expressing X. In accordance with this observation, band shift analysis indicates that X induces a sustained NF-kappaB activation following TNF-alpha treatment, probably by preventing the reassociation of newly synthesized nuclear IkappaBalpha with DNA-bound NF-kappaB complexes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Vírus da Hepatite B/metabolismo , Proteínas I-kappa B , NF-kappa B/antagonistas & inibidores , Transativadores/metabolismo , Proteínas Virais/metabolismo , Animais , Sequência de Bases , Transporte Biológico Ativo , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Sondas de DNA/genética , Proteínas de Ligação a DNA/genética , Vírus da Hepatite B/patogenicidade , Humanos , Mutação , Inibidor de NF-kappaB alfa , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Virais Reguladoras e Acessórias
18.
FEBS Lett ; 452(1-2): 82-6, 1999 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-10376683

RESUMO

Studies over the past 10 years have provided major insights into the molecular mechanisms responsible for active transport of macromolecules in and out of the nucleus. Nucleocytoplasmic transport pathways correspond to active and signal-mediated processes that involve substrates, adaptors and receptors. Regulation of both nuclear import and nuclear export is mainly exerted at the level of transport complex formation and has emerged as one of the most efficient mechanisms to adapt gene expression to the cell environment by restricting the access of transcriptional regulators to their target genes.


Assuntos
Regulação da Expressão Gênica/fisiologia , Membrana Nuclear/genética , Transcrição Gênica/fisiologia , Animais , Transporte Biológico Ativo/genética
19.
Oncogene ; 18(8): 1581-8, 1999 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-10102628

RESUMO

According to current models the inhibitory capacity of I(kappa)B(alpha) would be mediated through the retention of Rel/NF-kappaB proteins in the cytosol. However, I(kappa)B(alpha) has also been detected in the nucleus of cell lines and when overexpressed by transient transfection. To gain better insight into the potential role of nuclear I(kappa)B(alpha) in a physiological context we have analysed its presence in the nucleus of human peripheral blood T lymphocytes (PBL). We demonstrate the nuclear localization of I(kappa)B(alpha) in PBL by different techniques: Western blot, indirect immunofluorescence and electron microscopy. Low levels of nuclear I(kappa)B(alpha) were detected in resting cells whereas a superinduction was obtained after PMA activation. The nuclear pool of I(kappa)B(alpha) showed a higher stability than cytosolic I(kappa)B(alpha) and was partially independent of the resynthesis of the protein. Unexpectedly, the presence of nuclear I(kappa)B(alpha) did not inhibit NF-kappaB binding to DNA and this phenomenon was not due to the presence of IkappaBbeta at the nuclear level. Immunoprecipitation experiments failed to demonstrate an association between nuclear I(kappa)B(alpha) and NF-kappaB proteins. Our results demonstrate that in resting and PMA-activated human PBL, I(kappa)B(alpha) is present in the nucleus in an apparently inactive form unable to disrupt NF-kappaB binding from DNA.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas I-kappa B , Linfócitos T/metabolismo , Células Sanguíneas , Western Blotting , Citosol/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Células Jurkat/metabolismo , Ativação Linfocitária , Microscopia Confocal , Microscopia Eletrônica , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-rel , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica
20.
J Biol Chem ; 274(13): 9108-15, 1999 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-10085161

RESUMO

Transcriptional activation of nuclear factor kappaB (NF-kappaB) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. However, NF-kappaB activation induces rapid resynthesis of IkappaBalpha, which is responsible for post-induction repression of transcription. Newly synthesized IkappaBalpha translocates to the nucleus, where it dissociates NF-kappaB from DNA and transports NF-kappaB from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-kappaB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-kappaB-dependent transcriptional activation mediated by interleukin 1beta or tumor necrosis factor alpha. This is a consequence of the inhibition of signal-induced degradation of IkappaBalpha. Although LMB treatment does not affect the signal transduction pathway leading to IkappaBalpha degradation, it blocks IkappaBalpha nuclear export. IkappaBalpha is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IkappaBalpha is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IkappaBalpha is therefore essential for maintaining a low level of IkappaBalpha in the nucleus and allowing NF-kappaB to be transcriptionally active upon cell stimulation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , NF-kappa B/genética , Transdução de Sinais/genética , Ativação Transcricional/genética , Transporte Biológico/genética , Ácidos Graxos Insaturados/farmacologia , Imunofluorescência , Genes Reporter/genética , Células HeLa , Humanos , Proteínas I-kappa B , Interleucina-1/farmacologia , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Transcrição Gênica/genética , Fator de Necrose Tumoral alfa/farmacologia
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