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1.
J Biol Chem ; 290(26): 15996-6020, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-25940091

RESUMO

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1.


Assuntos
Autoantígenos/metabolismo , Regulação para Baixo , Glicoproteínas de Membrana/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribonucleoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autoantígenos/genética , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Glicoproteínas de Membrana/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , RNA Longo não Codificante , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteína Regulatória Associada a mTOR , Ribonucleoproteínas/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Antígeno SS-B
2.
Proc Natl Acad Sci U S A ; 107(4): 1576-81, 2010 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-20080710

RESUMO

Oncolytic viruses constitute a promising therapy against malignant gliomas (MGs). However, virus-induced type I IFN greatly limits its clinical application. The kinase mammalian target of rapamycin (mTOR) stimulates type I IFN production via phosphorylation of its effector proteins, 4E-BPs and S6Ks. Here we show that mouse embryonic fibroblasts and mice lacking S6K1 and S6K2 are more susceptible to vesicular stomatitis virus (VSV) infection than their WT counterparts as a result of an impaired type I IFN response. We used this knowledge to employ a pharmacoviral approach to treat MGs. The highly specific inhibitor of mTOR rapamycin, in combination with an IFN-sensitive VSV-mutant strain (VSV(DeltaM51)), dramatically increased the survival of immunocompetent rats bearing MGs. More importantly, VSV(DeltaM51) selectively killed tumor, but not normal cells, in MG-bearing rats treated with rapamycin. These results demonstrate that reducing type I IFNs through inhibition of mTORC1 is an effective strategy to augment the therapeutic activity of VSV(DeltaM51).


Assuntos
Glioma/metabolismo , Glioma/terapia , Interferon Tipo I/biossíntese , Fatores de Transcrição/metabolismo , Estomatite Vesicular/metabolismo , Vesiculovirus/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Glioma/genética , Glioma/virologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Transplante de Neoplasias , Terapia Viral Oncolítica , Proteínas , Ratos , Ratos Endogâmicos F344 , Proteínas Quinases S6 Ribossômicas/deficiência , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiência , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Estomatite Vesicular/genética , Estomatite Vesicular/virologia , Vesiculovirus/genética
3.
Proc Natl Acad Sci U S A ; 107(32): 14134-9, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20679199

RESUMO

Translational regulation plays a critical role in the control of cell growth and proliferation. A key player in translational control is eIF4E, the mRNA 5' cap-binding protein. Aberrant expression of eIF4E promotes tumorigenesis and has been implicated in cancer development and progression. The activity of eIF4E is dysregulated in cancer. Regulation of eIF4E is partly achieved through phosphorylation. However, the physiological significance of eIF4E phosphorylation in mammals is not clear. Here, we show that knock-in mice expressing a nonphosphorylatable form of eIF4E are resistant to tumorigenesis in a prostate cancer model. By using a genome-wide analysis of translated mRNAs, we show that the phosphorylation of eIF4E is required for translational up-regulation of several proteins implicated in tumorigenesis. Accordingly, increased phospho-eIF4E levels correlate with disease progression in patients with prostate cancer. Our findings establish eIF4E phosphorylation as a critical event in tumorigenesis. These findings raise the possibility that chemical compounds that prevent the phosphorylation of eIF4E could act as anticancer drugs.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Neoplasias/etiologia , Neoplasias/patologia , Animais , Progressão da Doença , Fator de Iniciação 4E em Eucariotos/genética , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Proteínas de Neoplasias/genética , Neoplasias/genética , Fosforilação/fisiologia , Regulação para Cima
4.
Nat Cell Biol ; 7(3): 286-94, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15723049

RESUMO

The mammalian target of rapamycin (mTOR) and Akt proteins regulate various steps of muscle development and growth, but the physiological relevance and the downstream effectors are under investigation. Here we show that S6 kinase 1 (S6K1), a protein kinase activated by nutrients and insulin-like growth factors (IGFs), is essential for the control of muscle cytoplasmic volume by Akt and mTOR. Deletion of S6K1 does not affect myoblast cell proliferation but reduces myoblast size to the same extent as that observed with mTOR inhibition by rapamycin. In the differentiated state, S6K1(-/-) myotubes have a normal number of nuclei but are smaller, and their hypertrophic response to IGF1, nutrients and membrane-targeted Akt is blunted. These growth defects reveal that mTOR requires distinct effectors for the control of muscle cell cycle and size, potentially opening new avenues of therapeutic intervention against neoplasia or muscle atrophy.


Assuntos
Músculo Esquelético/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/fisiologia , Animais , Atrofia , Peso Corporal , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Colágeno/farmacologia , Combinação de Medicamentos , Deleção de Genes , Vetores Genéticos , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Homozigoto , Humanos , Immunoblotting , Laminina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Músculos/patologia , Plasmídeos/metabolismo , Ligação Proteica , Proteoglicanas/farmacologia , Retroviridae/genética , Transdução de Sinais , Somatomedinas/metabolismo , Serina-Treonina Quinases TOR , Fatores de Tempo , Transfecção
5.
Proc Natl Acad Sci U S A ; 106(52): 22217-22, 2009 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-20018725

RESUMO

Translational control plays an important role in cell growth and tumorigenesis. Cap-dependent translation initiation of mammalian mRNAs with structured 5'UTRs requires the DExH-box protein, DHX29, in vitro. Here we show that DHX29 is important for translation in vivo. Down-regulation of DHX29 leads to impaired translation, resulting in disassembly of polysomes and accumulation of mRNA-free 80S monomers. DHX29 depletion also impedes cancer cell growth in culture and in xenografts. Thus, DHX29 is a bona fide translation initiation factor that potentially can be exploited as a target to inhibit cancer cell growth.


Assuntos
Proliferação de Células , Neoplasias/etiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , RNA Helicases/metabolismo , Regiões 5' não Traduzidas , Animais , Regulação para Baixo , Células HeLa , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Transplante Heterólogo
6.
EMBO J ; 26(23): 4812-23, 2007 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17962806

RESUMO

Miscoordination of growth and proliferation with the cellular stress response can lead to tumorigenesis. Mammalian target of rapamycin (mTOR), a central cell growth controller, is highly activated in some malignant neoplasms, and its clinical implications are under extensive investigation. We show that constitutive mTOR activity amplifies p53 activation, in vitro and in vivo, by stimulating p53 translation. Thus, loss of TSC1 or TSC2, the negative regulators of mTOR, results in dramatic accumulation of p53 and apoptosis in response to stress conditions. In other words, the inactivation of mTOR prevents cell death by nutrient stress and genomic damage via p53. Consistently, we also show that p53 is elevated in TSC tumors, which rarely become malignant. The coordinated relationship between mTOR and p53 during cellular stress provides a possible explanation for the benign nature of hamartoma syndromes, including TSC. Clinically, this also suggests that the efficacy of mTOR inhibitors in anti-neoplastic therapy may also depend on p53 status, and mTOR inhibitors may antagonize the effects of genotoxic chemotherapeutics.


Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Genes p53 , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/genética , Animais , Dano ao DNA , Privação de Alimentos , Hamartoma/genética , Hamartoma/patologia , Camundongos , Mutagênicos/química , Mutagênicos/metabolismo , Fosforilação , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Esclerose Tuberosa/genética , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa
7.
J Clin Invest ; 117(2): 387-96, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17273556

RESUMO

The most common pathology associated with obesity is insulin resistance, which results in the onset of type 2 diabetes mellitus. Several studies have implicated the mammalian target of rapamycin (mTOR) signaling pathway in obesity. Eukaryotic translation initiation factor 4E-binding (eIF4E-binding) proteins (4E-BPs), which repress translation by binding to eIF4E, are downstream effectors of mTOR. We report that the combined disruption of 4E-BP1 and 4E-BP2 in mice increased their sensitivity to diet-induced obesity. Increased adiposity was explained at least in part by accelerated adipogenesis driven by increased expression of CCAAT/enhancer-binding protein delta (C/EBPdelta), C/EBPalpha, and PPARgamma coupled with reduced energy expenditure, reduced lipolysis, and greater fatty acid reesterification in the adipose tissue of 4E-BP1 and 4E-BP2 double KO mice. Increased insulin resistance in 4E-BP1 and 4E-BP2 double KO mice was associated with increased ribosomal protein S6 kinase (S6K) activity and impairment of Akt signaling in muscle, liver, and adipose tissue. These data clearly demonstrate the role of 4E-BPs as a metabolic brake in the development of obesity and reinforce the idea that deregulated mTOR signaling is associated with the development of the metabolic syndrome.


Assuntos
Fatores de Iniciação em Eucariotos/deficiência , Resistência à Insulina/fisiologia , Obesidade/etiologia , Fosfoproteínas/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Adipogenia , Animais , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Primers do DNA/genética , Dieta/efeitos adversos , Fatores de Iniciação em Eucariotos/genética , Humanos , Resistência à Insulina/genética , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Síndrome Metabólica/etiologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Mutantes , Obesidade/genética , Obesidade/fisiopatologia , Fosfoproteínas/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR
8.
RNA ; 14(7): 1318-27, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18515545

RESUMO

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/análise , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/química , Células Cultivadas , Embrião de Mamíferos/citologia , Fator de Iniciação 4E em Eucariotos/análise , Fatores de Iniciação em Eucariotos , Fibroblastos/química , Fibroblastos/citologia , Camundongos , Fosfoproteínas/análise , Fosforilação , RNA Mensageiro/metabolismo
9.
Oncogene ; 23(18): 3172-9, 2004 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-15094766

RESUMO

Over the years, studies have focused on the transcriptional regulation of oncogenesis. More recently, a growing emphasis has been placed on translational control. The Ras and Akt signal transduction pathways play a critical role in regulating mRNA translation and cellular transformation. The question arises: How might the Ras and Akt signaling pathways affect translation and mediate transformation? These pathways converge on a crucial effector of translation, the initiation factor eIF4E, which binds the 5'cap of mRNAs. This review focuses on the role of eIF4E in oncogenesis. eIF4E controls the translation of various malignancy-associated mRNAs which are involved in polyamine synthesis, cell cycle progression, activation of proto-oncogenes, angiogenesis, autocrine growth stimulation, cell survival, invasion and communication with the extracellular environment. eIF4E-mediated translational modulation of these mRNAs plays a pivotal role in both tumor formation and metastasis. Interestingly, eIF4E activity is implicated in mitosis, embryogenesis and in apoptosis. Finally, the finding that eIF4E is overexpressed in several human cancers makes it a prime target for anticancer therapies.


Assuntos
Transformação Celular Neoplásica , Fator de Iniciação 4E em Eucariotos/fisiologia , Biossíntese de Proteínas , Animais , Apoptose , Ciclo Celular , Divisão Celular , Ciclina D1/genética , Genes myc , Humanos , Sistema de Sinalização das MAP Quinases , Fator A de Crescimento do Endotélio Vascular/genética
10.
FASEB J ; 16(8): 899-901, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12039873

RESUMO

Spaceflight, just like aging, causes profound changes in musculoskeletal parameters, which result in decreased bone density and muscular weakness. As these conditions decrease our ability to conduct long-term manned space missions, and increase bone frailty in the elderly, the identification of genes responsible for the apparition of these physiological changes will be of great benefit. Thus, we developed and implemented a new microarray approach to investigate the changes in normal WI38 human fibroblast gene expression that arise as a consequence of space flight. Using our microarray, we identified changes in the level of expression of 10 genes, belonging to either the tumor necrosis factor- (TNF) or interleukin- (IL) related gene families in fibroblasts when WI38 cells exposed to microgravity during the STS-93 Space Shuttle mission were compared with ground controls. The genes included two ligands from the TNF superfamily, TWEAK and TNFSF15; two TNF receptor-associated proteins, NSMAF and PTPN13; three TNF-inducible genes, ABC50, PTX3, and SCYA13; TNF-alpha converting enzyme, IL-1 receptor antagonist, and IL-15 receptor alpha chain. Most of these are involved in either the regulation of bone density, and as such the development of spaceflight osteopenia, or in the development of proinflammatory status.


Assuntos
Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Interleucinas/genética , Voo Espacial , Fator de Necrose Tumoral alfa/genética , Linhagem Celular , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Mol Cell Biol ; 34(6): 1046-53, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24396066

RESUMO

The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initiation factor 4G (eIF4G) and the mRNA 3' poly(A) tail promotes translation initiation. We previously showed that the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3; via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 with Paip1 is regulated by amino acids through the mTORC1 signaling pathway. The Paip1-eIF3 interaction is impaired by the mTORC1 inhibitors, rapamycin and PP242. We show that ribosomal protein S6 kinases 1 and 2 (S6K1/2) promote the interaction of eIF3 with Paip1. The enhancement of Paip1-eIF3 interaction by amino acids is abrogated by an S6K inhibitor or shRNA against S6K1/2. S6K1 interacts with eIF3f and, in vitro, phosphorylates eIF3. Finally, we show that S6K inhibition leads to a reduction in translation by Paip1. We propose that S6K1/2 phosphorylate eIF3 to stimulate Paip1-eIF3 interaction and consequent translation initiation. Taken together, these data demonstrate that eIF3 is a new translation target of the mTOR/S6K pathway.


Assuntos
Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Fosforilação/genética , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores
12.
Cell Stem Cell ; 14(5): 606-16, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24630793

RESUMO

Translational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process.


Assuntos
Reprogramação Celular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Reprogramação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Biológicos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
13.
Cell Cycle ; 9(20): 4106-9, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20948310

RESUMO

Messenger RNA translation or protein synthesis, is a fundamental biological process affecting cell growth, survival and proliferation. Initiation is the rate limiting and hence the most regulated step of translation. In eukaryotes, translation initiation is facilitated by multiple protein factors collectively called eIFs (for eukaryotic translation initiation factors). The complex consisting of the eIF4 group factors including the mRNA cap-binding eIF4E protein, large scaffolding protein eIF4G and RNA helicase eIF4A is assisted by the eIF4B co-factor to unwind local secondary structures and create a ribosome landing pad on mRNA. Recruitment of the ribosome and augmentation in the mRNA scanning process culminates in the positioning of the ribosome over the start codon. Deregulated translational control is believed to play an important role in oncogenic transformation. Indeed, many eIFs are bona fide proto-oncogenes. In many types of human cancers, eIFs are either overexpressed or ectopically activated by Ras-MAPK and PI3K-mTOR signaling cascades, resulting in increased survival and accelerated proliferation. In this review we will analyze the bulk of data describing eIF4B and its role in cell survival and proliferation. Recent studies have shown that eIF4B is phosphorylated and activated by Ras-MAPK and PI3K-mTOR signaling cascades. In addition, eIF4B regulates translation of proliferative and pro-survival mRNAs. Moreover, eIF4B depletion in cancer cells attenuates proliferation, sensitizes them to genotoxic stress-driven apoptosis. Taken together, these findings identify eIF4B as a potential target for development of anti-cancer therapies.


Assuntos
Proliferação de Células , Sobrevivência Celular/fisiologia , Fatores de Iniciação em Eucariotos/metabolismo , Transdução de Sinais/fisiologia , Animais , Fatores de Iniciação em Eucariotos/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas ras/metabolismo
14.
Mol Cell Biol ; 30(6): 1478-85, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20086100

RESUMO

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5' untranslated region (5'UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5'UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecin-induced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Regiões 5' não Traduzidas , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células HeLa , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
Science ; 328(5982): 1172-6, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20508131

RESUMO

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogen and nutrient signals to control cell proliferation and cell size. Hence, mTORC1 is implicated in a large number of human diseases--including diabetes, obesity, heart disease, and cancer--that are characterized by aberrant cell growth and proliferation. Although eukaryotic translation initiation factor 4E-binding proteins (4E-BPs) are critical mediators of mTORC1 function, their precise contribution to mTORC1 signaling and the mechanisms by which they mediate mTORC1 function have remained unclear. We inhibited the mTORC1 pathway in cells lacking 4E-BPs and analyzed the effects on cell size, cell proliferation, and cell cycle progression. Although the 4E-BPs had no effect on cell size, they inhibited cell proliferation by selectively inhibiting the translation of messenger RNAs that encode proliferation-promoting proteins and proteins involved in cell cycle progression. Thus, control of cell size and cell cycle progression appear to be independent in mammalian cells, whereas in lower eukaryotes, 4E-BPs influence both cell growth and proliferation.


Assuntos
Proteínas de Transporte/metabolismo , Crescimento Celular , Proliferação de Células , Fatores de Iniciação em Eucariotos/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Ciclo Celular , Proteínas de Ciclo Celular , Linhagem Celular , Tamanho Celular , Sobrevivência Celular , Fatores de Iniciação em Eucariotos/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas , Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR
16.
Cancer Cell ; 16(5): 439-46, 2009 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-19878875

RESUMO

eIF4E, the mRNA 5' cap-binding translation initiation factor, is overexpressed in numerous cancers and is implicated in mechanisms underlying oncogenesis and senescence. 4E-BPs (eIF4E-binding proteins) inhibit eIF4E activity, and thereby act as suppressors of eIF4E-dependent pathways. Here, we show that tumorigenesis is increased in p53 knockout mice that lack 4E-BP1 and 4E-BP2. However, primary fibroblasts lacking 4E-BPs, but expressing p53, undergo premature senescence and resist oncogene-driven transformation. Thus, the p53 status governs 4E-BP-dependent senescence and transformation. Intriguingly, the 4E-BPs engage in senescence via translational control of the p53-stabilizing protein, Gas2. Our data demonstrate a role for 4E-BPs in senescence and tumorigenesis and highlight a p53-mediated mechanism of senescence through a 4E-BP-dependent pathway.


Assuntos
Transformação Celular Neoplásica/genética , Fator de Iniciação 4E em Eucariotos/genética , Proteína Supressora de Tumor p53/genética , Animais , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Senescência Celular/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Camundongos , Camundongos Knockout , Proteína Supressora de Tumor p53/metabolismo
17.
PLoS One ; 2(2): e242, 2007 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-17311107

RESUMO

BACKGROUND: Translation deregulation is an important mechanism that causes aberrant cell growth, proliferation and survival. eIF4E, the mRNA 5' cap-binding protein, plays a major role in translational control. To understand how eIF4E affects cell proliferation and survival, we studied mRNA targets that are translationally responsive to eIF4E. METHODOLOGY/PRINCIPAL FINDINGS: Microarray analysis of polysomal mRNA from an eIF4E-inducible NIH 3T3 cell line was performed. Inducible expression of eIF4E resulted in increased translation of defined sets of mRNAs. Many of the mRNAs are novel targets, including those that encode large- and small-subunit ribosomal proteins and cell growth-related factors. In addition, there was augmented translation of mRNAs encoding anti-apoptotic proteins, which conferred resistance to endoplasmic reticulum-mediated apoptosis. CONCLUSIONS/SIGNIFICANCE: Our results shed new light on the mechanisms by which eIF4E prevents apoptosis and transforms cells. Downregulation of eIF4E and its downstream targets is a potential therapeutic option for the development of novel anti-cancer drugs.


Assuntos
Epigênese Genética , Fator de Iniciação 4E em Eucariotos/fisiologia , RNA Mensageiro/biossíntese , Regiões 5' não Traduzidas/genética , Animais , Apoptose , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Divisão Celular/genética , Fator de Iniciação 4E em Eucariotos/genética , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Células NIH 3T3/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/fisiologia , Sequências Reguladoras de Ácido Ribonucleico/genética , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética
18.
J Biol Chem ; 282(3): 1757-68, 2007 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-17114181

RESUMO

The mechanisms regulating initiation of mRNA translation for the generation of protein products that mediate interferon (IFN) responses are largely unknown. We have previously shown that both Type I and II IFNs engage the mammalian target of rapamycin (mTOR), resulting in downstream phosphorylation and deactivation of the translational repressor 4E-BP1 (eIF4E-binding protein 1). In the current study, we provide direct evidence that such regulation of 4E-BP1 by IFNalpha or IFNgamma results in sequential dissociation of 4E-BP1 from eukaryotic initiation factor-4E and subsequent formation of a functional complex between eukaryotic initiation factor-4E and eukaryotic initiation factor-4G, to allow initiation of mRNA translation. We also demonstrate that the induction of key IFNalpha- or IFNgamma-inducible proteins (ISG15 (interferon-stimulated gene 15) and CXCL10) that mediate IFN responses are enhanced in 4E-BP1 (4E-BP1(-/-)) knockout MEFs, as compared with wild-type 4E-BP1(+/+) MEFs. On the other hand, IFN-dependent transcriptional regulation of the Isg15 and Cxcl10 genes is intact in the absence of 4E-BP1, as determined by real time reverse transcriptase-PCR assays and promoter assays for ISRE and GAS, establishing that 4E-BP1 plays a selective negative regulatory role in IFN-induced mRNA translation. Interestingly, the induction of expression of ISG15 and CXCL10 proteins by IFNs was also strongly enhanced in cells lacking expression of the tuberin (TSC2(-/-)) or hamartin (TSC1(-/-)) genes, consistent with the known negative regulatory effect of the TSC1-TSC2 complex on mTOR activation. In other work, we demonstrate that the induction of an IFN-dependent antiviral response is strongly enhanced in cells lacking expression of 4E-BP1 and TSC2, demonstrating that these elements of the IFN-activated mTOR pathway exhibit important regulatory effects in the generation of IFN responses. Taken altogether, our data suggest an important role for mTOR-dependent pathways in IFN signaling and identify 4E-BP1 and TSC1-TSC2 as key components in the generation of IFN-dependent biological responses.


Assuntos
Regulação da Expressão Gênica , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antivirais/farmacologia , Proteínas de Ciclo Celular , Quimiocina CXCL10 , Quimiocinas CXC/metabolismo , Humanos , Camundongos , Fosfoproteínas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo
19.
J Biol Chem ; 277(21): 18718-27, 2002 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-11907030

RESUMO

During postnatal brain development the level of peptide elongation factor-1A (eEF1A-1) expression declines and that of the highly homologous isoform, eEF1A-2, increases in neurons. eEF1A-1 is implicated in cytoskeletal interactions, tumorigenesis, differentiation, and the absence of eEF1A-2 is implicated in neurodegeneration in the mouse mutant, wasted. The translation of eEF1A-1 mRNA is up-regulated via mitogenic stimulation. However, it is not known if eEF1A-1 mRNA translation is regulated by neurotrophins or if its synthesis is differentially regulated than that of the neuronal isoform, eEF1A-2. Regulated translation of these factors by neurotrophins, particularly by the Trk class of neurotrophin receptors, would implicate them in differentiation, survival, and neuronal plasticity. In this study, we investigated the effect of nerve growth factor (NGF) stimulation on the synthesis of eEF1A-1 and eEF1A-2. We found that NGF stimulation causes a preferential synthesis of eEF1A-1 over eEF1A-2 in PC12 cells. We analyzed the co-sedimentation of eEF1A-1 mRNA with polyribosome fractions in sucrose gradients, and found that NGF stimulation enriched the presence of eEF1A-1 mRNA in polyribosomes, indicating that the translation of eEF1A-1 mRNA is regulated by NGF. Inhibitors of phosphatidylinositol 3-kinase (LY 294002), mammalian target of rapamycin (rapamycin), and the NGF receptor, TrkA (K-252a), but not of mitogen-activated protein kinase (PD 98059), prevented the recruitment of eEF1A-1 mRNA to polyribosomes. The mobilization of eEF1A-1 mRNA to polyribosomes was rapamycin-sensitive in both proliferating and differentiated PC12 cells, indicating the importance of this pathway during differentiation. Our data shows that after growth factor withdrawal, an NGF-signaling pathway stimulates eEF1A-1 mRNA translation in proliferating and differentiated PC12 cells. Therefore, eEF1A-1 mRNA is a specific translational target of TrkA signaling.


Assuntos
Fator de Iniciação 1 em Eucariotos/genética , Fator de Crescimento Neural/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Ratos , Receptor trkA/metabolismo , Transdução de Sinais
20.
Immunity ; 21(1): 81-93, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15345222

RESUMO

The differentiation of B cells into immunoglobulin-secreting plasma cells is controlled by two transcription factors, Blimp-1 and XBP1. By gene expression profiling, we defined a set of genes whose induction during mouse plasmacytic differentiation is dependent on Blimp-1 and/or XBP1. Blimp-1-deficient B cells failed to upregulate most plasma cell-specific genes, including xbp1. Differentiating xbp1-deficient B cells induced Blimp-1 normally but failed to upregulate genes encoding many secretory pathway components. Conversely, ectopic expression of XBP1 induced a wide spectrum of secretory pathway genes and physically expanded the endoplasmic reticulum. In addition, XBP1 increased cell size, lysosome content, mitochondrial mass and function, ribosome numbers, and total protein synthesis. Thus, XBP1 coordinates diverse changes in cellular structure and function resulting in the characteristic phenotype of professional secretory cells.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Organelas/fisiologia , Plasmócitos/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Animais , Células Produtoras de Anticorpos , Linfócitos B/fisiologia , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Organelas/ultraestrutura , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fatores de Transcrição de Fator Regulador X , Proteína 1 de Ligação a X-Box
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