RESUMO
The p90 ribosomal S6 kinases (RSK) family of serine/threonine kinases comprises four isoforms (RSK1-4) that lie downstream of the ERK1/2 mitogen-activated protein kinase pathway. RSKs are implicated in fine tuning of cellular processes such as translation, transcription, proliferation, and motility. Previous work showed that pathogens such as Cardioviruses could hijack any of the four RSK isoforms to inhibit PKR activation or to disrupt cellular nucleocytoplasmic trafficking. In contrast, some reports suggest nonredundant functions for distinct RSK isoforms, whereas Coffin-Lowry syndrome has only been associated with mutations in the gene encoding RSK2. In this work, we used the analog-sensitive kinase strategy to ask whether the cellular substrates of distinct RSK isoforms differ. We compared the substrates of two of the most distant RSK isoforms: RSK1 and RSK4. We identified a series of potential substrates for both RSKs in cells and validated RanBP3, PDCD4, IRS2, and ZC3H11A as substrates of both RSK1 and RSK4, and SORBS2 as an RSK1 substrate. In addition, using mutagenesis and inhibitors, we confirmed analog-sensitive kinase data showing that endogenous RSKs phosphorylate TRIM33 at S1119. Our data thus identify a series of potential RSK substrates and suggest that the substrates of RSK1 and RSK4 largely overlap and that the specificity of the various RSK isoforms likely depends on their cell- or tissue-specific expression pattern.
Assuntos
Proteínas Quinases S6 Ribossômicas 90-kDa , Especificidade por Substrato , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Reprodutibilidade dos Testes , MutagêneseRESUMO
Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate the RAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RAS-MAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPRED interactome and evaluate how members of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the N-terminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RAS-MAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics.
Assuntos
Proteínas Quinases Ativadas por Mitógeno , Proteínas Repressoras , Proteínas Quinases S6 Ribossômicas 90-kDa , Transdução de Sinais , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais/genética , Humanos , Linhagem Celular , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Técnicas de Silenciamento de Genes , Transporte Proteico/genética , Ligação Proteica , Estrutura Terciária de Proteína , Modelos Moleculares , Neurofibromina 1/metabolismoRESUMO
Many glycosylated natural products display biological activity and are deglycosylated by the metabolic processes of the body. Although unnatural CF2-glycosides have been proposed as nonhydrolyzable analogues, CF2-derivatives of natural products are exceedingly challenging to synthesize and few examples exist. These difluorinated molecules may have unique conformational behavior as a consequence of changing the glycosidic linkage. In this study, we performed conformational searches using MacroModel followed by molecular dynamics simulations to investigate the conformational behavior of the glycosidic bonds in flavonoid-O-glycosides and in corresponding CF2-glycosylated derivatives. Compared to their O-glycosylated analogues, flavonoid-3-CF2-glycosides and flavonoid-5-CF2-glycosides showed conformational bias, whereas flavonoid-7-CF2-glycosides showed more flexibility. Flavonoid-5-CF2-glycosides were the least flexible compared to all others. Our results show that the site of the glycosylation and the substitution pattern on the flavonoid determine the conformational properties of these molecules. These two factors influence the steric destabilization and/or stereoelectronic stabilization which govern the conformational behavior of the flavonoid glycosides. Moreover, a docking study of quercitrin and its CF2-analogue into murine ribosomal kinase RSK2 demonstrated the potential for flavonoid-CF2-glycosides to retain a similar binding pose as the parent O-glycoside. These findings will assist in designing stable flavonoid-CF2-glycosides for carbohydrate research.
Assuntos
Produtos Biológicos , Flavonoides , Glicosídeos , Animais , Camundongos , Produtos Biológicos/química , Flavonoides/química , Glicosídeos/química , Glicosídeos/metabolismo , Modelos Moleculares , Conformação Molecular , Proteínas Quinases S6 Ribossômicas 90-kDa/químicaRESUMO
It is well known that phosphorylation of extracellular signal-regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signalling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage- and dose-dependent. The absence of Ca2+ reduced PTTH-stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH-stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH-regulated RSK phosphorylation, indicating its redox regulation. A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, decreased PTTH-stimulated RSK phosphorylation, indicating that ERK is an upstream signalling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH-stimulated RSK phosphorylation, and a PKC activator, phorbol 12-myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI-D1870, a specific RSK inhibitor, partly prevented PTTH-stimulated RSK phosphorylation and significantly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH-stimulated ecdysteroidogenesis.
Assuntos
Bombyx , Ecdisona/metabolismo , Hormônios de Inseto/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Animais , Bombyx/metabolismo , Ecdisteroides/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Larva/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , FosforilaçãoRESUMO
The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases that regulate diverse cellular processes, such as cell growth, cell motility, cell survival and cell proliferation. RSKs are downstream effectors of the Ras-extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling cascade. Significant advances in the field of RSK and ERK/MAPK signalling have occurred in the past few years, including biological insights and the discovery of novel substrates and new RSK regulatory mechanisms. Collectively, these data expand the current models of RSK signalling and highlight potential directions of research in RSK-mediated survival, growth, proliferation and migration.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ciclo Celular/fisiologia , Sobrevivência Celular/fisiologia , Ativação Enzimática , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.
Assuntos
Proteínas de Membrana/metabolismo , Modelos Moleculares , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Algoritmos , Sítios de Ligação , Ciclofilina A/química , Ciclofilina A/genética , Ciclofilina A/metabolismo , Humanos , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Oligopeptídeos/química , Oligopeptídeos/genética , Domínios PDZ , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosforilação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Origem de Replicação , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Serina/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPKâMAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/química , Complexos Multienzimáticos/química , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Domínio Catalítico , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca(2+)-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca(2+)-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca(2+)-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a "fuzzy" complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.
Assuntos
Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Ativação Enzimática , Polarização de Fluorescência , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/química , Soluções , Relação Estrutura-Atividade , Triptofano/metabolismoRESUMO
A global survey of cancer has shown that lung cancer is the most common cause of the new cancer cases and cancer deaths in men worldwide. The mortality from lung cancer is more than the combined mortality from breast, prostate and colorectal cancers. The two major histological types of lung cancer are non-small cell lung cancer (NSCLC) accounting for about 85 % of cases and small cell lung cancer accounting for 15 % of cases. NSCLC, the more prevalent form of lung cancer, is often diagnosed at an advanced stage and has a very poor prognosis. Many factors have been shown to contribute to the development of lung cancer in humans including tobacco smoking, exposure to environmental carcinogens (asbestos, or radon) and genetic factors. Despite the advances in treatment, lung cancer remains one of the leading causes of cancer death worldwide. Interestingly, the overall 5 year survival from lung cancer has not changed appreciably in the past 25 years. For this reason, novel and more effective treatments and strategies for NSCLC are critically needed. p90 ribosomal S6 kinase (RSK), a serine threonine kinase that lies downstream of the Ras-MAPK (mitogen activated protein kinase) cascade, has been demonstrated to be involved in the regulation of cell proliferation in various malignancies through indirect (e.g., modulation of transcription factors) or direct effects on the cell-cycle machinery. Increased expression of RSK has been demonstrated in various cancers, including lung cancer. This review focuses on the role of RSK in lung cancer and its potential therapeutic application.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Terapia de Alvo Molecular , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Apoptose/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases S6 Ribossômicas 90-kDa/químicaRESUMO
Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC ß-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).
Assuntos
Simulação de Acoplamento Molecular , Sondas Moleculares/química , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Inibidores de beta-Lactamases/química , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células COS , Cisteína/química , Cisteína/metabolismo , Descoberta de Drogas , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/crescimento & desenvolvimento , Humanos , Interações Hidrofóbicas e Hidrofílicas , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/química , Janus Quinase 3/genética , Ligantes , Sondas Moleculares/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Serina/química , Serina/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
OBJECTIVE: This study was aim to determine the role and underling mechanism of ribosomal protein S6 kinases 1 (S6k1) phosphorylation in Parkinson's disease (PD). METHODS: The dopaminergic neuron MN9D was employed and 1-methyl-4-phenylpyridium (MPP) iodide (MPP(+)) was used to generate PD model in vitro. The S6k1 phosphorylation and UNC-51-like kinase 1 (ULK1) protein levels were analyzed by western blot. The ULK1 mRNA level was evaluated by Real-time RT-PCR. The S6k1 threonine 389 (T389) site-directed mutagenesis, the phosphodeficit T389A (threonine to alanine) and the phosphomimetic T389D (threonine to aspartate) were generated to examine the phosphorylation site of S6k1. RESULTS: An increase in the ULK1 mRNA and protein levels were detected in the MPP(+)-treated MN9D cells compared to control. ULK1 knockdown increased neuronal cell viability, and enhanced S6k1 phosphorylation. Further investigation demonstrated ULK1 knockdown promoted the S6k1 T389 phosphorylation in particular. T389A enhanced the viability of MPP iodide-treated MN9D, whereas T389D decreased the cell viability. CONCLUSION: ULK1 acts to inhibit S6k1 phosphorylation at T389, leading to MN9D viability reduction under MPP(+) treatment. These results provide evidence for a novel mechanism by which the ULK1 inhibit S6k1 T389 phosphorylation contributes to neurodegeneration in MPP(+) treated-MN9D, and suggests a new therapeutic strategy for PD.
Assuntos
Degeneração Neural/etiologia , Degeneração Neural/metabolismo , Transtornos Parkinsonianos/etiologia , Transtornos Parkinsonianos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , 1-Metil-4-fenilpiridínio/toxicidade , Substituição de Aminoácidos , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Sítios de Ligação/genética , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Técnicas de Silenciamento de Genes , Camundongos , Mutagênese Sítio-Dirigida , Degeneração Neural/patologia , Transtornos Parkinsonianos/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genéticaRESUMO
Myricetin is a flavonoid that is abundant in fruits and vegetables and has protective effects against cancer and diabetes. However, the mechanism of action of myricetin against gastric cancer (GC) is not fully understood. We researched myricetin on the proliferation, apoptosis, and cell cycle in GC HGC-27 and SGC7901 cells, to explore the underlying mechanism of action. Cell Counting Kit (CCK)-8 assay, Western blotting, cell cycle analysis, and apoptosis assay were used to evaluate the effects of myricetin on cell proliferation, apoptosis, and the cell cycle. To analyze the binding properties of ribosomal S6 kinase 2 (RSK2) with myricetin, surface plasmon resonance (SPR) analysis was performed. CCK8 assay showed that myricetin inhibited GC cell proliferation. Flow cytometry analysis showed that myricetin induces apoptosis and cell cycle arrest in GC cells. Western blotting indicated that myricetin influenced apoptosis and cell cycle arrest of GC cells by regulating related proteins. SPR analysis showed strong binding affinity of RSK2 and myricetin. Myricetin bound to RSK2, leading to increased expression of Mad1, and contributed to inhibition of HGC-27 and SGC7901 cell proliferation. Our results suggest the therapeutic potential of myricetin in GC.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Flavonoides/administração & dosagem , Proteínas Nucleares/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Neoplasias Gástricas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
All known protein kinases share a bilobal kinase domain with well conserved structural elements. Because of significant structural similarities of nucleotide binding pocket, the development of highly selective kinase inhibitors is a very challenging task. Flavonols, naturally occurring plant metabolites, have long been known to inhibit kinases by mimicking the adenine moiety. Interestingly, recent data show that some flavonol glycosides are more selective, although underlying mechanisms were unknown. Crystallographic data from our laboratory revealed that the N-terminal kinase domain of p90 ribosomal S6 kinase, isoform 2, binds three different flavonol rhamnosides in a highly unusual manner, distinct from other kinase inhibitor interactions. The kinase domain undergoes a reorganization of several structural elements in response to the binding of the inhibitors. Specifically, the main ß-sheet of the N-lobe undergoes a twisting rotation by ~56° around an axis passing through the N- and C-lobes, leading to the restructuring of the canonical ATP-binding pocket into pockets sterically adapted to the inhibitor shape. The flavonol rhamnosides appear to adopt compact, but strained conformations with the rhamnose moiety swept under the B-ring of flavonol, unlike the structure of the free counterparts in solution. These data suggest that the flavonol glycoside scaffold could be used as a template for new inhibitors selective for the RSK family. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
Assuntos
Flavonóis/farmacologia , Glicosídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Cristalografia por Raios X , Flavonóis/química , Flavonóis/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Humanos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Ramnose/química , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
Myricetin, a common dietary flavonoid, is widely distributed in fruits and vegetables and is used as a health food supplement based on its anti-tumor properties. However, the effect and mechanisms of myricetin in esophageal carcinoma are not fully understood. Here, we demonstrated the effect of myricetin on the proliferation, apoptosis, and invasion of the esophageal carcinoma cell lines EC9706 and KYSE30 and explored the underlying mechanism and target protein(s) of myricetin. CCK-8 assay, transwell invasion assay, wound-healing assay, cell cycle analysis, and apoptosis assay were used to evaluate the effects of myricetin on cell proliferation, invasion, and apoptosis. Nude mouse tumor xenograft model was built to understand the interaction between myricetin and NTD RSK2. Pull-down assay was used to verify molecular mechanism. Myricetin inhibited proliferation and invasion and induced apoptosis of EC9706 and KYSE30 cells. Moreover, myricetin was shown to bind RSK2 through the NH2-terminal kinase domain. Finally, myricetin inhibited EC9706 and KYSE30 cell proliferation through Mad1 and induced cell apoptosis via Bad. Myricetin inhibits the proliferation and invasion and induces apoptosis in EC9706 and KYSE30 cells via RSK2. Myricetin exerts anti-proliferative, anti-invasive, and pro-apoptotic effects on esophageal carcinoma EC9706 and KYSE30 cells via RSK2. Our results provide novel insight into myricetin as a potential agent for the prevention and treatment of esophageal carcinoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Flavonoides/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Antineoplásicos/química , Carcinoma/genética , Carcinoma/patologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Feminino , Flavonoides/química , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Modelos Moleculares , Conformação Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Coffin-Lowry syndrome (CLS) is a rare X-linked dominant disorder characterized by intellectual disability, craniofacial abnormalities, short stature, tapering fingers, hypotonia, and skeletal malformations. CLS is caused by mutations in the Ribosomal Protein S6 Kinase, 90 kDa, Polypeptide 3 (RPS6KA3) gene located at Xp22.12, which encodes Ribosomal S6 Kinase 2 (RSK2). Here we analyzed RPS6KA3 in three unrelated CLS patients including one from the historical Coffin-Lowry syndrome family and found two novel mutations. To date, over 140 mutations in RPS6KA3 have been reported. However, the etiology of the very first familial case, which was described in 1971 by Lowry with detailed phenotype and coined the term CLS, has remained unknown. More than 40 years after the report, we succeeded in identifying deposited fibroblast cells from one patient of this historic family and found a novel heterozygous 216 bp in-frame deletion, encompassing exons 15 and 16 of RPS6KA3. Drop episodes in CLS patients were reported to be associated with truncating mutations deleting the C-terminal kinase domain (KD), and only one missense mutation and one single basepair duplication involving the C-terminal KD of RSK2 in the patients with drop episode have been reported thus far. Here we report the first in-frame deletion in C-terminal KD of RPS6KA3 in a CLS patient with drop episodes.
Assuntos
Síndrome de Coffin-Lowry/genética , Mutação/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Criança , Pré-Escolar , Família , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Proteínas Quinases S6 Ribossômicas 90-kDa/químicaRESUMO
An adolescent female presented with intellectual disability, stimulus-induced drop episodes (SIDEs), facial characteristics that include wide set eyes, short nose with wide columella, full and everted lips with wide mouth and progressive skeletal changes: scoliosis, spondylolisthesis and pectus excavatum. These findings were suggestive of Coffin-Lowry syndrome (CLS), and this was confirmed by the identification of a novel mutation in RPS6KA3, a heterozygous one basepair duplication at nucleotide 1570 (c.1570dupA). This mutation occurs within the C-terminal kinase domain of the protein, and, therefore contradicts the previous report that SIDEs is only associated with premature truncation of the protein in the N-terminal kinase domain or upstream of this domain. As CLS is X-linked, it is unusual for a female to have such a classic phenotype.
Assuntos
Síndrome de Coffin-Lowry/diagnóstico , Síndrome de Coffin-Lowry/genética , Genótipo , Fenótipo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Fácies , Feminino , Humanos , Lactente , Mutação , Domínios e Motivos de Interação entre Proteínas , Radiografia , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Escoliose/diagnóstico por imagem , Escoliose/genéticaRESUMO
Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45-346) of mouse RSK2, or RSK2(NTKD), has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3'',4''-di-O-acetyl-α-L-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-L-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2(NTKD) with a dissociation constant (K(d)) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K(d) for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca(2+).
Assuntos
Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quercetina/análogos & derivados , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Animais , Cristalografia por Raios X , Camundongos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Quercetina/metabolismo , Quercetina/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , TermodinâmicaRESUMO
A series of novel indolin-2-ones inhibitors against p90 ribosomal S6 protein kinase 2 (RSK2) were designed and synthesized and their structure-activity relationship (SAR) was studied. The most potent inhibitor, compound 3s, exhibited potent inhibition against RSK2 with an IC50 value of 0.5 µM and presented a satisfactory selectivity against 23 kinases. The interactions of these inhibitors with RSK2 were investigated based on the proposed binding poses with molecular docking simulation. Four compounds and six compounds exhibited moderate anti-proliferation activities against PC 3 cells and MCF-7 cells, respectively.
Assuntos
Indóis/química , Indóis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Masculino , Simulação de Acoplamento Molecular , Neoplasias da Próstata/tratamento farmacológico , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Relação Estrutura-AtividadeRESUMO
The RSK (90 kDa ribosomal S6 kinase) family comprises a group of highly related serine/threonine kinases that regulate diverse cellular processes, including cell growth, proliferation, survival and motility. This family includes four vertebrate isoforms (RSK1, RSK2, RSK3 and RSK4), and single family member orthologues are also present in Drosophila and Caenorhabditis elegans. The RSK isoforms are downstream effectors of the Ras/ERK (extracellular-signal-regulated kinase) signalling pathway. Significant advances in the field of RSK signalling have occurred in the past few years, including several new functions ascribed to the RSK isoforms, the discovery of novel protein substrates and the implication of different RSK isoforms in cancer. Collectively, these new findings increase the diversity of biological functions regulated by RSK, and highlight potential new directions of research. In the present paper, we review the structure, expression and activation mechanisms of the RSK isoforms, and discuss their physiological roles on the basis of established substrates and recent discoveries.
Assuntos
Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Sequência de Aminoácidos , Animais , Pontos de Checagem do Ciclo Celular/fisiologia , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Alinhamento de SequênciaRESUMO
Ribosomal S6 kinase (RSK) family is a group of serine/threonine kinases, including four isoforms (RSK1/2/3/4). As a downstream effector of the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway, RSK participates in many physiological activities such as cell growth, proliferation, and migration, and is intimately involved in tumor occurrence and development. As a result, it is recognized as a potential target for anti-cancer and anti-resistance therapies. There have been several RSK inhibitors discovered or designed in recent decades, but only two have entered clinical trials. Low specificity, low selectivity, and poor pharmacokinetic properties in vivo limit their clinical translation. Published studies performed structure optimization by increasing interaction with RSK, avoiding hydrolysis of pharmacophores, eliminating chirality, adapting to binding site shape, and becoming prodrugs. Besides enhancing efficacy, the focus of further design will move towards selectivity since there are functional differences among RSK isoforms. This review summarized the types of cancers associated with RSK, along with the structural characteristics and optimization process of the reported RSK inhibitors. Furthermore, we addressed the importance of RSK inhibitors' selectivity and discussed future drug development directions. This review is expected to shed light on the emergence of RSK inhibitors with high potency, specificity, and selectivity.