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1.
Nat Commun ; 11(1): 1168, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127537

RESUMO

Telomerase deficiency leads to age-related diseases and shorter lifespans. Inhibition of the mechanistic target of rapamycin (mTOR) delays aging and age-related pathologies. Here, we show that telomerase deficient mice with short telomeres (G2-Terc-/-) have an hyper-activated mTOR pathway with increased levels of phosphorylated ribosomal S6 protein in liver, skeletal muscle and heart, a target of mTORC1. Transcriptional profiling confirms mTOR activation in G2-Terc-/- livers. Treatment of G2-Terc-/- mice with rapamycin, an inhibitor of mTORC1, decreases survival, in contrast to lifespan extension in wild-type controls. Deletion of mTORC1 downstream S6 kinase 1 in G3-Terc-/- mice also decreases longevity, in contrast to lifespan extension in single S6K1-/- female mice. These findings demonstrate that mTOR is important for survival in the context of short telomeres, and that its inhibition is deleterious in this setting. These results are of clinical interest in the case of human syndromes characterized by critically short telomeres.


Assuntos
Envelhecimento/genética , RNA/genética , Serina-Treonina Quinases TOR/metabolismo , Telomerase/genética , Telômero/genética , Envelhecimento/efeitos dos fármacos , Animais , Dano ao DNA/efeitos dos fármacos , Feminino , Longevidade/efeitos dos fármacos , Longevidade/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Sirolimo/farmacologia , Taxa de Sobrevida , Serina-Treonina Quinases TOR/genética , Telômero/efeitos dos fármacos , Telômero/metabolismo
2.
Nat Commun ; 11(1): 258, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937753

RESUMO

BET bromodomain inhibitors (BETi), such as JQ1, have been demonstrated to effectively kill multiple types of cancer cells. However, the underlying mechanisms for BETi resistance remain largely unknown. Our evidences show that JQ1 treatment evicts BRD4 from the FOXD3-localized MIR548D1 gene promoter, leading to repression of miR-548d-3p. The loss of miRNA restores JunD expression and subsequent JunD-dependent transcription of RPS6KA2 gene. ERK1/2/5 kinases phosphorylate RSK3 (RPS6KA2), resulting in the enrichment of activated RSK3 and blockade of JQ1 killing effect. Dual inhibition of MEKs/ERKs or single EGFR inhibition are able to mimic the effect of JunD/RSK3-knockdown to reverse BETi resistance. Collectively, our study indicates that loss of BRD4/FOXD3/miR-548d-3p axis enhances JunD/RSK3 signalling and determines BET inhibition resistance, which can be reversed by targeting EGFR-MEK1/2/5-ERK1/2/5 signalling.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição Forkhead/metabolismo , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Azepinas/farmacologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Triazóis/farmacologia
3.
PLoS One ; 15(1): e0227340, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910234

RESUMO

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit ß (shCK2ß cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2ß. However, in HK-2 cells the dependence on CK2ß was confirmed by rescue experiments (CK2ß re-expression in shCK2ß HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2ß downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2ß was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2ß HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3ß S9, was increased. Differently to what observed in response to CK2ß down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3ß pS9 mirrored those induced by CK2ß knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3ß pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2ß knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3ß pS9 was not reduced by CK2 blockade suggests that GSK3ß phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3ß pS9, and concomitantly decreased Snail1 levels (a GSK3ß target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2ß-downregulated cells, suggesting that reducing GSK3ß pS9 could be a strategy to control Snail1 levels in any situation where CK2ß is defective, as possibly occurring in cancer cells.


Assuntos
Caseína Quinase II/genética , Glicogênio Sintase Quinase 3 beta/genética , Proteína Oncogênica v-akt/genética , Fatores de Transcrição da Família Snail/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Cromonas/farmacologia , Transição Epitelial-Mesenquimal/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morfolinas/farmacologia , Naftiridinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais/efeitos dos fármacos
4.
Anim Genet ; 51(1): 122-126, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31691328

RESUMO

A GWAS was performed for inborn X-linked facial dysmorphia with severe growth retardation in Labrador Retrievers. This lethal condition was mapped on the X chromosome at 17-21 Mb and supported by eight SNPs in complete LD. Dams of affected male puppies were heterozygous for the significantly associated SNPs and male affected puppies carried the associated alleles hemizygously. In the near vicinity to the associated region, RPS6KA3 was identified as a candidate gene causing facial dysmorphia in humans and mice known as Coffin-Lowry syndrome. Haplotype analysis showed significant association with the phenotypes of all 18 animals under study. This haplotype was validated through normal male progeny from a dam with the not-associated haplotype on both X chromosomes but male affected full-sibs with the associated haplotype.


Assuntos
Craniossinostoses/veterinária , Doenças do Cão/genética , Cães/genética , Genes Letais , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Craniossinostoses/genética , Feminino , Estudos de Associação Genética/veterinária , Haplótipos , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Cromossomo X/genética
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(8): 798-800, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31400131

RESUMO

OBJECTIVE: To identify potential mutations of the CLS gene in a Chinese pedigree affected with Coffin-Lowry syndrome. METHODS: Whole exome sequencing was applied to detect potential mutation in the proband, and the result was verified by Sanger sequencing. RESULTS: The proband was found to carry a c.966_967delAA (p.Arg323Thr fs*11) deletional mutation in the RPS6KA3 gene. The same mutation was also found in his mother. CONCLUSION: The c.966_967delAA (p.Arg323Thr fs*11) deletional mutation of the RPS6KA3 gene probably underlies the disorder in this pedigree.


Assuntos
Síndrome de Coffin-Lowry/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Grupo com Ancestrais do Continente Asiático , China , Humanos , Mutação , Linhagem , Deleção de Sequência
6.
Biomed Res Int ; 2019: 6302950, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31317034

RESUMO

This study aimed to investigate the effects of isoleucine (Ile) on the synthesis and secretion of digestive enzymes and cellular signalling in the pancreatic tissue of dairy goats. The pancreatic tissues were incubated in buffer containing 0, 0.40, 0.80, and 1.60 mM Ile. High levels of Ile significantly increased the buffer release and total concentration of ɑ-amylase in the tissues (P < 0.001). The total trypsin and chymotrypsin concentrations in each of the Ile groups were significantly higher than those in the control group (P < 0.05); however, lipase was not affected. High levels of Ile significantly increased ɑ-amylase mRNA expression (P < 0.001) but had no effect on the mRNA expression of trypsin, chymotrypsin, or lipase. Ile did not affect S6K1 phosphorylation levels. High levels of Ile significantly increased the expression of the γ isoform of 4EBP1 (P < 0.001), which indicated that the phosphorylation of 4EBP1 was significantly increased. The phosphorylation level of eEF2 gradually decreased with the addition of Ile (P < 0.001). These results suggested that high doses of Ile can regulate the excretion of enzymes, especially ɑ-amylase, in the pancreatic tissues of dairy goats by modulating mTOR signalling, and this regulation is independent of the mTOR-S6K1 pathway.


Assuntos
Cabras/metabolismo , Isoleucina/metabolismo , Pâncreas/enzimologia , alfa-Amilases/biossíntese , Animais , Quimotripsina/biossíntese , Quimotripsina/metabolismo , Quinase do Fator 2 de Elongação/genética , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica/genética , Lipase/biossíntese , Lipase/metabolismo , Pâncreas/metabolismo , Fosforilação , RNA Mensageiro/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Tripsina/biossíntese , Tripsina/metabolismo , alfa-Amilases/metabolismo
7.
Mol Med Rep ; 20(2): 1583-1592, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257496

RESUMO

Propofol is a general anesthetic used in surgical operations. Phosphoprotein enriched in astrocytes 15(PEA15) was initially identified in astrocytes. The present study examined the role of PEA15 in the damage induced by propofol in hippocampal neurons. A model of hippocampal neuron damage was established using 50 µmol/l propofol. Cell viability, proliferation and apoptosis of hippocampal neurons were tested by Cell Counting Kit­8 and flow cytometry. Western blotting and reverse transcription­quantitative polymerase chain reaction analysis were performed to measure the expression levels of PEA15, and additional factors involved in apoptosis or in the signaling pathway downstream of PEA15. The present results suggested that propofol significantly decreased PEA15 expression levels in hippocampal neurons. Furthermore, overexpression of PEA15 significantly increased the cell viability and cell proliferation of cells treated with propofol. Additionally, PEA15 overexpression decreased apoptosis, which was promoted by propofol. Treatment with propofol significantly decreased the protein expression levels of pro­caspase­3, B­cell lymphoma-2, phosphorylated extracellular signal­regulated kinases (ERK)1/2, ribosomal S6 kinase 2 (RSK2) and phosphorylated cAMP responsive element binding protein 1 (CREB1). However, propofol upregulated active caspase­3 and Bax expression levels. Notably, PEA15 overexpression was able to reverse the effects of propofol. Collectively, overexpression of PEA15 was able to attenuate the neurotoxicity of propofol in rat hippocampal neurons by increasing proliferation and repressing apoptosis via upregulation of the ERK­CREB­RSK2 signaling pathway.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Propofol/toxicidade , Animais , Animais Recém-Nascidos , Apoptose/genética , Proteínas Reguladoras de Apoptose/agonistas , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Gravidez , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Transfecção , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
8.
Analyst ; 144(12): 3756-3764, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31070195

RESUMO

Protein phosphorylation is a very important regulatory mechanism in a majority of biological processes, and the determination of protein kinase activity plays a key role in the pathological study and drug development of kinase-related diseases. However, it is very challenging to in situ study endogenous protein kinase activity in a single living cell due to the shortage of in vivo efficient methods. Here, we propose a new strategy for direct determination of protein kinase activity in a single living cell by combining single molecule fluorescence correlation spectroscopy (FCS) with activity-based probes (ABPs). Ribosomal S6 kinase-2 (RSK2) was used as a model, and the ABPs were synthesized on the basis of RSK2 inhibitor FMK to specially label active RSK2 in living cells. Conventional FCS and MEMFCS (maximum entropy method) single molecule techniques were used to in situ determine RSK2 activity in living cells based on the difference in molecular weight between free probes and probe-RSK2 complexes. Furthermore, wild-type and mutated RSK2 were fused with enhanced green fluorescent protein (EGFP) using lentivirus infection, and fluorescence cross-correlation spectroscopy (FCCS) was used to verify the selective binding of ABPs to RSK2-EGFP fusion protein in living cells. Finally, FCS with ABPs was applied for in situ monitoring of the activation of endogenous RSK2 in the stimulation of serum, epidermal growth factor, kinase inhibitors and ultraviolet irradiation; we observed that endogenous RSK2 showed different behaviors in the cytoplasm and the nucleus in some stimulation. Our results document that FCS with ABPs is a very promising method for studying endogenous protein kinases in living cells.


Assuntos
Ensaios Enzimáticos/métodos , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Análise de Célula Única/métodos , Espectrometria de Fluorescência/métodos , Compostos de Boro/síntese química , Compostos de Boro/química , Carbocianinas/síntese química , Carbocianinas/química , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Imagem Individual de Molécula/métodos
9.
Mol Med Rep ; 19(6): 5133-5141, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059023

RESUMO

Sepsis is a type of systemic inflammatory response caused by infection. The present study aimed to identify novel targets for the treatment of sepsis. We conducted bioinformatic analysis of the microarray Gene Expression Omnibus dataset GSE12624, which includes data on 34 patients with sepsis and 36 healthy individuals without sepsis. Differentially expressed genes (DEGs) in sepsis patients were identified using Bayesian methods included in the limma package in R. Correlations among the expression values of DEGs were analyzed using the weighted gene co­expression network analysis (WGCNA) to construct a co­expression network. Subsequently, the generated co­expression network was visualized using Cytoscape 3.3 software. Additionally, a protein­protein interaction (PPI) network was constructed based on all the DEGs using STRING. Finally, the integrated regulatory network was constructed based on DEGs, microRNAs (miRNAs) and transcription factors (TFs). A total of 407 DEGs were identified in the sepsis samples, including 227 upregulated DEGs and 180 downregulated DEGs. WGCNA grouped the DEGs into 13 co­expressed modules. Additionally, MAP3K8 and RPS6KA5 in the MEyellow module were enriched in the MAPK and TNF signaling pathways. In addition, the PPI network comprised 48 nodes and 112 edges, which included the pairs MAP3K8­RPS6KA5, MAP3K8­IL10, RPS6KA5­EXOSC4 and EXOSC4­EXOSC5. Lastly, the TF­miRNA­target DEG regulatory network was constructed based on eight TFs (NF­κB), seven miRNAs (miR152, miR­148A/B), and 52 TF­miRNA­target gene triplets (17 upregulated genes, including MAP3K8, and 10 downregulated genes, including RPS6KA5). Our analysis showed that the members of the miR­148 family (miR­148A/B and miR­152) are candidate biomarkers for sepsis.


Assuntos
Biomarcadores/metabolismo , MicroRNAs/metabolismo , Sepse/diagnóstico , Biologia Computacional/métodos , Bases de Dados Genéticas , Regulação para Baixo , Redes Reguladoras de Genes , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sepse/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
10.
J Genet ; 982019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30945684

RESUMO

Microduplications of the X chromosome are a rare cause of X-linked intellectual disability (XLID), a clinically and genetically heterogeneous spectrum of disorders. In the present study, a 950-kb Xp22.12 microduplication including the RPS6KA3 gene was detected in affected members of a family, including the proband (male), his mother and one maternal uncle. Four female carriers had major depression and one of them also had mild intellectual disability. The present and previous cases with overlapping microduplications suggest that Xp22.12 microduplications can be included in the neuropsychiatric copy number variations.


Assuntos
Cromossomos Humanos X/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Deficiência Intelectual/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Esquizofrenia/genética , Adulto , Duplicação Cromossômica , Variações do Número de Cópias de DNA , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Humanos , Deficiência Intelectual/diagnóstico , Masculino , Pessoa de Meia-Idade , Linhagem , Esquizofrenia/diagnóstico
11.
Oncol Rep ; 41(6): 3355-3366, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942462

RESUMO

Aberrant activation of the extracellular signal­regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway is frequently determined in various human tumor types, including liver cancer, and has been considered as a promising target for cancer chemoprevention and therapy. In the present study, using computer­aided virtual screening and molecular docking, isobavachalcone (IBC), a natural chalcone compound, was identified to be an ATP­competitive inhibitor targeting ERK1/2 and RSK2. Cell Counting Kit­8, EdU incorporation and colony formation assays were used to detect the effects of IBC on cell viability and proliferation, and the results demonstrated that IBC effectively inhibited the proliferation of liver cancer HepG2 and Hep3B cells, whereas it had no notable cytotoxic effect on immortal liver L02 cells. Flow cytometric analysis and western blotting further revealed that IBC caused significant levels of apoptosis on liver cancer cells via the caspase­dependent mitochondria pathway. The computer prediction was confirmed with pull­down and in vitro kinase assays, in which IBC directly bound with ERK1/2 and RSK2, and dose­dependently blocked RSK2 kinase activity in liver cancer cells. Treatment of HepG2 or Hep3B cells with IBC significantly attenuated epidermal growth factor­induced phosphorylation of RSK2 and resulted in the reduced activation of its downstream substrates including cAMP response element­binding protein, activating transcription factor 1, histone H3 and activating protein­1. Enforced RSK2 expression in L02 cells could increase the effect of IBC on suppressing cell growth. Conversely, knockdown of RSK2 reduced the inhibitory effect of IBC on HepG2 cell proliferation. Overall, the present data indicated that ERKs/RSK2 signaling serves a pivotal role in IBC­induced suppression of liver cancer cells and that IBC may be a potential therapeutic candidate for human cancer with elevated ERKs/RSK2 activity.


Assuntos
Neoplasias Hepáticas/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/química , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chalconas/química , Chalconas/farmacologia , Células Hep G2 , Humanos , Ligantes , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Medicina Tradicional Chinesa , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Interface Usuário-Computador
12.
Int J Mol Sci ; 20(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018569

RESUMO

Ribosomal S6 kinase 2 (RSK2), regulated by Ras/Raf/MEKs/ERKs, transmits upstream activation signals to downstream substrates including kinases and transcription and epigenetic factors. We observed that ELK members, including ELK1, 3, and 4, highly interacted with RSK2. We further observed that the RSK2-ELK3 interaction was mediated by N-terminal kinase and linker domains of RSK2, and the D and C domains of ELK3, resulting in the phosphorylation of ELK3. Importantly, RSK2-mediated ELK3 enhanced c-fos promoter activity. Notably, chemical inhibition of RSK2 signaling using kaempferol (a RSK2 inhibitor) or U0126 (a selective MEK inhibitor) suppressed EGF-induced c-fos promoter activity. Moreover, functional deletion of RSK2 by knockdown or knockout showed that RSK2 deficiency suppressed EGF-induced c-fos promoter activity, resulting in inhibition of AP-1 transactivation activity and Ras-mediated foci formation in NIH3T3 cells. Immunocytofluorescence assay demonstrated that RSK2 deficiency reduced ELK3 localization in the nucleus. In MDA-MB-231 breast cancer cells, knockdown of RSK2 or ELK3 suppressed cell proliferation with accumulation at the G1 cell cycle phase, resulting in inhibition of foci formation and anchorage-independent cancer colony growth in soft agar. Taken together, these results indicate that a novel RSK2/ELK3 signaling axis, by enhancing c-Fos-mediated AP-1 transactivation activity, has an essential role in cancer cell proliferation and colony growth.


Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fatores de Transcrição/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Fatores de Transcrição/genética
13.
Gene ; 710: 76-90, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-30898702

RESUMO

Small Tail Han Sheep are an excellent local sheep breed in China, and their outstanding reproductive performance is one of their very important biological characteristics. Clarifying the ovary development process of these ewes should provide a theoretical basis for improving their reproductive efficiency. In this study, we identified the differentially expressed (DE) microRNAs (miRNAs) in 2-, 6-, and 12-month-old small-tail Han sheep ovaries by constructing and analyzing the miRNA expression profiles. These findings clarify the molecular mechanisms regulating the excellent reproductive performance of small-tail Han ewes. We used RNA-Seq technology and bioinformatic to analyze these profiles. Eleven, 13, and 19 DE miRNAs were identified in the 2- vs 6-, 6- vs 12-, and 2- vs 12-month-old ovaries, respectively. In total, 54, 37, and 198 predicted target genes of these DE miRNAs were identified in these three groups, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that in the 2- vs 6-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 102 GO terms and seven signaling pathways; in the 6- vs 12-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 52 GO terms and three signaling pathways; and in the 2- vs 12-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 88 GO terms and six signaling pathways. Three miRNA-target regulatory networks were constructed based on these DE miRNA-target interactions. Nine miRNAs were selected to confirm to the accuracy of the miRNA sequencing data with qRT-PCR. The site at which oar-miR-432 binds RPS6KA1 was determined with a dual-luciferase system. This is the first integrated analysis the expression profiles of miRNAs and their targets during ovarian development in small-tail Han sheep. These data clarify the molecular regulatory mechanisms underlying sheep ovarian development and identify biomarkers that influence the reproductive performance of small-tail Han ewes.


Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , Ovário/crescimento & desenvolvimento , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Carneiro Doméstico/crescimento & desenvolvimento , Animais , Regulação para Baixo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Células HEK293 , Humanos , Ovário/química , Análise de Sequência de RNA/métodos , Ovinos , Carneiro Doméstico/genética
14.
Dev Comp Immunol ; 97: 45-56, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30905684

RESUMO

In mammals, toll-like receptor 3 (TLR3) is capable of recognizing double-stranded RNA and then initiates transcription of IFN-ß. TLR3 can activate the innate immune system by phosphorylating extracellular signal-regulated kinase 1 (ERK1) in the mitogen-activated protein kinase (MAPK) pathway. As a downstream signaling protein of ERK1, ribosomal protein S6 kinase alpha 3 (RSK2) is activated through the "classical" MAPK pathway. So RSK2 plays a critical role in response to innate immune system induced by TRL3. However, the innate immune mechanism of RSK2 remains indistinct in fish. In this study, we cloned and characterized a full length cDNA sequence of RSK2 from Ctenopharyngodon idella (named CiRSK2, MH844551). The full length cDNA of CiRSK2 is 3930 bp with a coding sequence of 2202 bp encoding a polypeptide of 734 amino acids. The expression of CiRSK2 was ubiquitous and significantly up-regulated under the stimulation of poly (I:C) in eight different tissues of C. idella and C. idella kidney cells (CIK). In addition, poly (I:C) stimulation also up-regulated the expression of CiERK1 mRNA in CIK cells and the phosphorylation of CiERK1. We also demonstrated that the activated CiERK1 interacted with CiRSK2 by CO-IP assay and immunofluorescence assay. To further investigate the relationship between CiRSK2 and CiERK1, we performed subcellular localization of CiRSK2 at different periods of CiERK1 stimulation. The result showed that CiERK1 can make CiRSK2 enter the nucleus. Subsequently, we found that CiRSK2 increased the transcriptional level of CiBCL-2 and protein level of CiBCL-2 significantly. Then cell apoptosis was inhibited to a certain extent. Overall, our results suggested that CiRSK2 plays important roles in fish innate immunity and is able to inhibit cell apoptosis by up-regulating CiBCL-2.


Assuntos
Apoptose/genética , Carpas/genética , Proteínas de Peixes/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Apoptose/efeitos dos fármacos , Carpas/metabolismo , Células Cultivadas , Feminino , Proteínas de Peixes/classificação , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Rim/citologia , Ovário/citologia , Filogenia , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/classificação , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo
15.
J Mol Neurosci ; 68(2): 191-203, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30919247

RESUMO

Mitogen- and stress-activated kinase (MSK) 1 is a nuclear serine/threonine kinase. In the central nervous system, it plays an important role in regulating cell proliferation and neuronal survival; it is also involved in astrocyte inflammation and the inhibition of inflammatory cytokine production. However, its specific role in spinal cord injury is not clear. Here, we aimed to elucidate this role using an in vivo animal model. In this study, we found that MSK1 is gradually decreased, starting 1 day after spinal cord injury and to its lowest level 3 days post-injury, after which it gradually increased. To further investigate the possible function of MSK1 in spinal cord injury, we interfered with its expression by utilizing a small interfering RNA (siRNA)-encoding lentivirus, which was injected into the injured spinal cord to inhibit local expression. After MSK1 inhibition, we found that the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß were increased. Moreover, the expression of IL-10 was decreased. In addition, neuronal apoptotic cells were increased significantly and expression of the apoptosis-related protein caspase-3 was also increased. Ultrastructural analysis of nerve cells also revealed typical neuronal apoptosis and severe neuronal damage. Finally, we found that hindlimb motor function decreased significantly with MSK1 knockdown. Therefore, our findings suggest that the inhibition of this protein promotes inflammatory responses and apoptosis and suppresses functional recovery after spinal cord injury. MSK1 might thus play an important role in repair after spinal cord injury by regulating inflammation and apoptosis.


Assuntos
Apoptose , Citocinas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Traumatismos da Medula Espinal/metabolismo , Animais , Citocinas/genética , Inativação Gênica , Masculino , Células PC12 , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Traumatismos da Medula Espinal/genética
16.
J Biol Chem ; 294(15): 6062-6072, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30739083

RESUMO

Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants (Kd ). However, only a limited number of parameters have been measured experimentally in living cells. Here we describe an approach for quantifying the concentration and Kd of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy. First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 (MAPK1) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 (RSK2) gene. We then used fluorescence correlation spectroscopy to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, fluorescence cross-correlation spectroscopy analyses revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sistemas CRISPR-Cas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Espectrometria de Fluorescência/métodos
17.
Transl Psychiatry ; 9(1): 50, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30705255

RESUMO

The molecular signature underlying autism spectrum disorder remains largely unknown. This study identifies differential expression of mTOR and MAPK pathways in patients affected by mild and severe idiopathic autism. A total of 55 subjects were enrolled, of which 22 were typically developing individuals and 33 were patients aged between 3 and 11 years, with autism spectrum disorder. A detailed history, including physical examination, developmental evaluation, mental health history and autism diagnostic observation schedule were performed for each patient. Components of the mTOR and MAPK signalling pathways were analysed from peripheral blood at the protein level. Patients were then stratified according to their clinical phenotypes, and the molecular profiling was analysed in relation to the degree of autism severity. In this cohort of patients, we identified increased activity of mTOR and the MAPK pathways, key regulators of synaptogenesis and protein synthesis. Specifically, rpS6, p-eIF4E, TSC1 and p-MNK1 expression discriminated patients according to their clinical diagnosis, suggesting that components of protein synthesis signalling pathways might constitute a molecular signature of clinical severity in autism spectrum disorder.


Assuntos
Transtorno do Espectro Autista/genética , Sistema de Sinalização das MAP Quinases/genética , Neurogênese/genética , Serina-Treonina Quinases TOR/genética , Criança , Pré-Escolar , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética
18.
Mol Cell ; 73(3): 446-457.e6, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30612880

RESUMO

Multisite phosphorylation of kinases can induce on-off or graded regulation of catalytic activity; however, its influence on substrate specificity remains unclear. Here, we show that multisite phosphorylation of ribosomal protein S6 kinase 1 (S6K1) alters target selection. Agonist-inducible phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) by S6K1 in monocytes and adipocytes requires not only canonical phosphorylation at Thr389 by mTORC1 but also phosphorylation at Ser424 and Ser429 in the C terminus by cyclin-dependent kinase 5 (Cdk5). S6K1 phosphorylation at these additional sites induces a conformational switch and is essential for high-affinity binding and phosphorylation of EPRS, but not canonical S6K1 targets, e.g., ribosomal protein S6. Unbiased proteomic analysis identified additional targets phosphorylated by multisite phosphorylated S6K1 in insulin-stimulated adipocytes-namely, coenzyme A synthase, lipocalin 2, and cortactin. Thus, embedded within S6K1 is a target-selective kinase phospho-code that integrates signals from mTORC1 and Cdk5 to direct an insulin-stimulated, post-translational metabolon determining adipocyte lipid metabolism.


Assuntos
Adipócitos/enzimologia , Metabolismo dos Lipídeos , Células Mieloides/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Aminoacil-tRNA Sintetases/metabolismo , Animais , Quinase 5 Dependente de Ciclina/metabolismo , Ativação Enzimática , Células HEK293 , Células Hep G2 , Humanos , Insulina/farmacologia , Interferon gama/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Células Mieloides/efeitos dos fármacos , Fosforilação , Proteômica/métodos , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais , Especificidade por Substrato , Células U937
19.
Mol Carcinog ; 58(5): 767-776, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30604908

RESUMO

TIMM50 (Translocase of the inner mitochondrial membrane 50), also called TIM50, plays an essential role in mitochondrial membrane transportation. The existing literature suggests that TIMM50 may perform as an oncogenetic protein in breast cancer. However, the molecular mechanism, especially in human non-small cell lung cancer (NSCLC), is uncertain to date. In the present study, using immunohistochemistry, we found that TIMM50 expression significantly correlated with larger tumor size (P = 0.049), advanced TNM stage (P = 0.001), positive regional lymph node metastasis (P = 0.007), and poor overall survival (P = 0.001). Proliferation and invasion assay showed that TIMM50 dramatically promoted the ability of proliferation and invasion of NSCLC cells. Subsequent Western blotting results revealed that TIMM50 enhanced the expression of Cyclin D1 and Snail, and inhibited the expression of E-cadherin. Moreover, TIMM50 facilitated the expression of phosphorylated ERK and P90RSK. Incorporation of ERK inhibitor counteracted the upregulating expression of CyclinD1, and Snail, and downregulating expression of E-cadherin expression induced by TIMM50 overexpression. In conclusion, our data indicated that TIMM50 facilitated tumor proliferation and invasion of NSCLC through enhancing phosphorylation of its downstream ERK/P90RSK signaling pathway. We speculated that TIMM50 might be a useful prognosis marker of NSCLC patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Adulto , Idoso , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundário , Estudos de Casos e Controles , Proliferação de Células , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metástase Linfática , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Fosforilação , Prognóstico , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas
20.
IUBMB Life ; 71(2): 261-276, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30452117

RESUMO

Myofibroblast apoptosis is essential for normal resolution of wound repair, including cardiac infarction repair. Impaired cardiac myofibroblast (CMF) apoptosis is associated with excessive extracellular matrix (ECM) deposition, which could be responsible for pathological cardiac fibrosis. Conventionally, angiotensin II (Ang II), a soluble peptide, is implicated in fibrogenesis because it induces cardiac fibroblast (CFb) proliferation, differentiation, and collagen synthesis. However, the role of Ang II in regulation of CMF survival and apoptosis has not been fully clarified. In this report, we cultured neonatal rat CFbs, which transform into CMFs after passage 3 (6-8 days), and investigated the effects of Ang II on CMFs challenged by TNF-α combined with cycloheximide and the underlying mechanisms. Here, we show that Ang II rapidly activates MAPKs but not AKT in CMFs and confers apoptosis resistance, as evidenced by the inhibition of caspase-3 cleavage, early apoptotic cells and late apoptotic cells. This inhibitory effect of Ang II was reversed by blockade of AT1 or inactivation of ERK1/2 or RSK1 but not AT2, indicating that activation of the prosurvival AT1/ERK1/2/RSK1 signaling pathway mediates apoptosis resistance. TGF-ß, a latent fibrotic factor, was found to have no relation to Ang II-induced apoptosis resistance in our study. Furthermore, Ang II-mediated apoptosis resistance, which was conferred by activation of the AT1/ERK1/2/RSK1 signaling pathway, was also confirmed in human adult ventricular cardiac myofibroblasts. Collectively, our findings suggest a novel profibrotic mechanism of Ang II in which it promotes myofibroblast resistance to apoptosis in addition to classical mechanisms, providing a potential novel therapeutic approach by targeting prosurvival signaling pathways. © 2018 IUBMB Life, 71(1):261-276, 2019.


Assuntos
Angiotensina II/farmacologia , Apoptose/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Miofibroblastos/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Apoptose/genética , Butadienos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Flavonoides/farmacologia , Regulação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Losartan/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Nitrilos/farmacologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
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