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1.
Mol Pharmacol ; 103(4): 211-220, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36720643

RESUMO

The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.


Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Masculino , Humanos , Receptores Androgênicos/genética , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Domínios Proteicos , Peptídeos/uso terapêutico , Neoplasias da Próstata/metabolismo , DNA , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Elk-1 do Domínio ets/uso terapêutico
2.
Protein Expr Purif ; 203: 106216, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36528218

RESUMO

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Elk-1 do Domínio ets , Animais , Humanos , DNA/metabolismo , Escherichia coli/metabolismo , Células HEK293 , Mamíferos , Fosforilação , Receptores Androgênicos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Elk-1 do Domínio ets/biossíntese , Proteínas Elk-1 do Domínio ets/metabolismo , Células Sf9/metabolismo
3.
Biochem J ; 479(14): 1519-1531, 2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-35781489

RESUMO

Prostate cancer (PCa) growth requires tethering of the androgen receptor (AR) to chromatin by the ETS domain transcription factor ELK1 to coactivate critical cell proliferation genes. Disruption of the ELK1-AR complex is a validated potential means of therapeutic intervention in PCa. AR associates with ELK1 by coopting its two ERK docking sites, through the amino-terminal domain (A/B domain) of AR. Using a mammalian two-hybrid assay, we have now functionally mapped amino acids within the peptide segments 358-457 and 514-557 in the A/B domain as required for association with ELK1. The mapping data were validated by GST (glutathione S-transferase)-pulldown and BRET (bioluminescence resonance energy transfer) assays. Comparison of the relative contributions of the interacting motifs/segments in ELK1 and AR to coactivation of ELK1 by AR suggested a parallel mode of binding of AR and ELK1 polypeptides. Growth of PCa cells was partially inhibited by deletion of the upstream segment in AR and nearly fully inhibited by deletion of the downstream segment. Our studies have identified two peptide segments in AR that mediate the functional association of AR with its two docking sites in ELK1. Identification of the ELK1 recognition sites in AR should enable further structural studies of the ELK1-AR interaction and rational design of small molecule drugs to disrupt this interaction.


Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Mamíferos/metabolismo , Peptídeos/genética , Peptídeos/uso terapêutico , Neoplasias da Próstata/genética , Receptores Androgênicos/química , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Elk-1 do Domínio ets/uso terapêutico
4.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066106

RESUMO

Genome expansion, whole genome and gene duplication events during metazoan evolution produced an extensive family of ETS genes whose members express transcription factors with a conserved winged helix-turn-helix DNA-binding domain. Unravelling their biological roles has proved challenging with functional redundancy manifest in overlapping expression patterns, a common consensus DNA-binding motif and responsiveness to mitogen-activated protein kinase signalling. Key determinants of the cellular repertoire of ETS proteins are their stability and turnover, controlled largely by the actions of selective E3 ubiquitin ligases and deubiquitinases. Here we discuss the known relationships between ETS proteins and enzymes that determine their ubiquitin status, their integration with other developmental signal transduction pathways and how suppression of ETS protein ubiquitination contributes to the malignant cell phenotype in multiple cancers.


Assuntos
Neoplasias/patologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Neoplasias/metabolismo , Ubiquitinação
5.
J Biol Chem ; 296: 100214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33428929

RESUMO

The mitogen-responsive, ETS-domain transcription factor ELK-1 stimulates the expression of immediate early genes at the onset of the cell cycle and participates in early developmental programming. ELK-1 is subject to multiple levels of posttranslational control, including phosphorylation, SUMOylation, and ubiquitination. Recently, removal of monoubiquitin from the ELK-1 ETS domain by the Ubiquitin Specific Protease USP17 was shown to augment ELK-1 transcriptional activity and promote cell proliferation. Here we have used coimmunoprecipitation experiments, protein turnover and ubiquitination assays, RNA-interference and gene expression analyses to examine the possibility that USP17 acts antagonistically with the F-box protein FBXO25, an E3 ubiquitin ligase previously shown to promote ELK-1 ubiquitination and degradation. Our data confirm that FBXO25 and ELK-1 interact in HEK293T cells and that FBXO25 is active toward Hand1 and HAX1, two of its other candidate substrates. However, our data indicate that FBXO25 neither promotes ubiquitination of ELK-1 nor impacts on its transcriptional activity and suggest that an E3 ubiquitin ligase other than FBXO25 regulates ELK-1 ubiquitination and function.


Assuntos
Endopeptidases/metabolismo , Proteínas F-Box/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Proliferação de Células , Endopeptidases/genética , Proteínas F-Box/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Fosforilação , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sumoilação , Transfecção , Ubiquitinação , Proteínas Elk-1 do Domínio ets/genética
6.
Int J Biochem Cell Biol ; 130: 105886, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33227393

RESUMO

Eukaryotic cells perform a range of complex processes, some essential for life, others specific to cell type, all of which are governed by post-translational modifications of proteins. Among the repertoire of dynamic protein modifications, ubiquitination is arguably the most arcane and profound due to its complexity. Ubiquitin conjugation consists of three main steps, the last of which involves a multitude of target-specific ubiquitin ligases that conjugate a range of ubiquitination patterns to protein substrates with diverse outcomes. In contrast, ubiquitin removal is catalysed by a relatively small number of de-ubiquitinating enzymes (DUBs), which can also display target specificity and impact decisively on cell function. Here we review the current knowledge of the intriguing ubiquitin-specific protease 17 (USP17) family of DUBs, which are expressed from a highly copy number variable gene that has been implicated in multiple cancers, although available evidence points to conflicting roles in cell proliferation and survival. We show that key USP17 substrates populate two pathways that drive cell cycle progression and that USP17 activity serves to promote one pathway but inhibit the other. We propose that this arrangement enables USP17 to stimulate or inhibit proliferation depending on the mitogenic pathway that predominates in any given cell and may partially explain evidence pointing to both oncogenic and tumour suppressor properties of USP17.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Ciclo Celular/fisiologia , Endopeptidases/metabolismo , Humanos , Neoplasias/patologia
7.
PLoS One ; 15(12): e0244255, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332446

RESUMO

Reactive oxygen species are bona fide intracellular second messengers that influence cell metabolism and aging by mechanisms that are incompletely resolved. Mitochondria generate superoxide that is dis-mutated to hydrogen peroxide, which in turn oxidises cysteine-based enzymes such as phosphatases, peroxiredoxins and redox-sensitive transcription factors to modulate their activity. Signal Transducer and Activator of Transcription 3 (Stat3) has been shown to participate in an oxidative relay with peroxiredoxin II but the impact of Stat3 oxidation on target gene expression and its biological consequences remain to be established. Thus, we created murine embryonic fibroblasts (MEFs) that express either WT-Stat3 or a redox-insensitive mutant of Stat3 (Stat3-C3S). The Stat3-C3S cells differed from WT-Stat3 cells in morphology, proliferation and resistance to oxidative stress; in response to cytokine stimulation, they displayed elevated Stat3 tyrosine phosphorylation and Socs3 expression, implying that Stat3-C3S is insensitive to oxidative inhibition. Comparative analysis of global gene expression in WT-Stat3 and Stat3-C3S cells revealed differential expression (DE) of genes both under basal conditions and during oxidative stress. Using differential gene regulation pattern analysis, we identified 199 genes clustered into 10 distinct patterns that were selectively responsive to Stat3 oxidation. GO term analysis identified down-regulated genes to be enriched for tissue/organ development and morphogenesis and up-regulated genes to be enriched for cell-cell adhesion, immune responses and transport related processes. Although most DE gene promoters contain consensus Stat3 inducible elements (SIEs), our chromatin immunoprecipitation (ChIP) and ChIP-seq analyses did not detect Stat3 binding at these sites in control or oxidant-stimulated cells, suggesting that oxidised Stat3 regulates these genes indirectly. Our further computational analysis revealed enrichment of hypoxia response elements (HREs) within DE gene promoters, implying a role for Hif-1. Experimental validation revealed that efficient stabilisation of Hif-1α in response to oxidative stress or hypoxia required an oxidation-competent Stat3 and that depletion of Hif-1α suppressed the inducible expression of Kcnb1, a representative DE gene. Our data suggest that Stat3 and Hif-1α cooperate to regulate genes involved in immune functions and developmental processes in response to oxidative stress.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , Elementos de Resposta , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/fisiologia , Animais , Fibroblastos/citologia , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Transdução de Sinais , Ativação Transcricional
8.
Nucleic Acids Res ; 47(9): 4495-4508, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30854565

RESUMO

ELK-1 is a transcription factor involved in ERK-induced cellular proliferation. Here, we show that its transcriptional activity is modulated by ubiquitination at lysine 35 (K35). The level of ubiquitinated ELK-1 rises in mitogen-deprived cells and falls upon mitogen stimulation or oncogene expression. Ectopic expression of USP17, a cell cycle-dependent deubiquitinase, decreases ELK-1 ubiquitination and up-regulates ELK-1 target-genes with a concomitant increase in cyclin D1 expression. In contrast, USP17 depletion attenuates ELK-1-dependent gene expression and slows cell proliferation. The reduced rate of proliferation upon USP17 depletion appears to be a direct effect of ELK-1 ubiquitination because it is rescued by an ELK-1(K35R) mutant refractory to ubiquitination. Overall, our results show that ubiquitination of ELK-1 at K35, and its reversal by USP17, are important mechanisms in the regulation of nuclear ERK signalling and cellular proliferation. Our findings will be relevant for tumours that exhibit elevated USP17 expression and suggest a new target for intervention.


Assuntos
Proliferação de Células/genética , Endopeptidases/genética , Mitose/genética , Proteínas Elk-1 do Domínio ets/genética , Ciclo Celular/genética , Núcleo Celular/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação , Transdução de Sinais/genética , Fatores de Transcrição/genética , Ubiquitinação/genética
9.
EMBO Mol Med ; 10(8)2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29930174

RESUMO

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-ß subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1-negative lung cancers.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Neoplasias Pulmonares/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular/genética
10.
Sci Signal ; 10(472)2017 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-28351946

RESUMO

Signal transducer and activator of transcription 3 (STAT3) is associated with various physiological and pathological functions, mainly as a transcription factor that translocates to the nucleus upon tyrosine phosphorylation induced by cytokine stimulation. In addition, a small pool of STAT3 resides in the mitochondria, where it serves as a sensor for various metabolic stressors including reactive oxygen species (ROS). Mitochondrially localized STAT3 largely exerts its effects through direct or indirect regulation of the activity of the electron transport chain (ETC). It has been assumed that the amounts of STAT3 in the mitochondria are static. We showed that various stimuli, including oxidative stress and cytokines, triggered a signaling cascade that resulted in a rapid loss of mitochondrially localized STAT3. Recovery of the mitochondrial pool of STAT3 over time depended on phosphorylation of Ser727 in STAT3 and new protein synthesis. Under these conditions, mitochondrially localized STAT3 also became competent to bind to cyclophilin D (CypD). Binding of STAT3 to CypD was mediated by the amino terminus of STAT3, which was also important for reducing mitochondrial ROS production after oxidative stress. These results outline a role for mitochondrially localized STAT3 in sensing and responding to external stimuli.


Assuntos
Ciclofilinas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Peptidil-Prolil Isomerase F , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Interleucina-6/farmacologia , Masculino , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Oxidantes/farmacologia , Estresse Oxidativo , Fator de Transcrição STAT3/genética
11.
J Biol Chem ; 291(50): 25983-25998, 2016 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-27793987

RESUMO

The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10-8 m A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.


Assuntos
Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Células HeLa , Humanos , Masculino , 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 , Neoplasias da Próstata/genética , Ligação Proteica , Receptores Androgênicos/genética , Proteínas Elk-1 do Domínio ets/genética
12.
FEBS J ; 283(6): 1025-38, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26613204

RESUMO

Metazoans have multiple ETS paralogues with overlapping or indiscriminate biological functions. Elk-1, one of three mammalian ternary complex factors (TCFs), is a well-conserved, ETS domain-containing transcriptional regulator of mitogen-responsive genes that operates in concert with serum response factor (SRF). Nonetheless, its genetic role remains unresolved because the elk-1 gene could be deleted from the mouse genome seemingly without adverse effect. Here we have explored the evolution of Elk-1 to gain insight into its conserved biological role. We identified antecedent Elk-1 proteins in extant early metazoans and used amino acid sequence alignments to chart the appearance of domains characteristic of human Elk-1. We then performed biochemical studies to determine whether putative domains apparent in the Elk-1 protein of a primitive hemichordate were functionally orthologous to those of human Elk-1. Our findings imply the existence of primordial Elk-1 proteins in primitive deuterostomes that could operate as mitogen-responsive ETS transcription factors but not as TCFs. The role of TCF was acquired later, but presumably prior to the whole genome duplications in the basal vertebrate lineage. Thus its evolutionary origins link Elk-1 to the appearance of mesoderm.


Assuntos
Evolução Molecular , Proteínas Elk-1 do Domínio ets/química , Proteínas Elk-1 do Domínio ets/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Elk-1 do Domínio ets/metabolismo
13.
Nucleic Acids Res ; 41(22): 10241-53, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24049075

RESUMO

The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Complexo Mediador/metabolismo , Mitógenos/farmacologia , Regiões Promotoras Genéticas , Ativação Transcricional , Proteínas Elk-1 do Domínio ets/metabolismo , Animais , Genes Precoces , Genes ras , Células HEK293 , Células HeLa , Humanos , Complexo Mediador/genética , Camundongos , Mutação , Células NIH 3T3 , Fosforilação
14.
J Cheminform ; 3(1): 49, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22044511

RESUMO

BACKGROUND: The Elk-1 transcription factor is a member of a group of proteins called ternary complex factors, which serve as a paradigm for gene regulation in response to extracellular signals. Its deregulation has been linked to multiple human diseases including the development of tumours. The work herein aims to inform the design of potential peptidomimetic compounds that can inhibit the formation of the Elk-1 dimer, which is key to Elk-1 stability. We have conducted molecular dynamics simulations of the Elk-1 ETS domain followed by virtual screening. RESULTS: We show the ETS dimerisation site undergoes conformational reorganisation at the α1ß1 loop. Through exhaustive screening of di- and tri-peptide libraries against a collection of ETS domain conformations representing the dynamics of the loop, we identified a series of potential binders for the Elk-1 dimer interface. The di-peptides showed no particular preference toward the binding site; however, the tri-peptides made specific interactions with residues: Glu17, Gln18 and Arg49 that are pivotal to the dimer interface. CONCLUSIONS: We have shown molecular dynamics simulations can be combined with virtual peptide screening to obtain an exhaustive docking protocol that incorporates dynamic fluctuations in a receptor. Based on our findings, we suggest experimental binding studies to be performed on the 12 SILE ranked tri-peptides as possible compounds for the design of inhibitors of Elk-1 dimerisation. It would also be reasonable to consider the score-ranked tri-peptides as a comparative test to establish whether peptide size is a determinant factor of binding to the ETS domain.

15.
Nucleic Acids Res ; 39(15): 6390-402, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21543455

RESUMO

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin-proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Elk-1 do Domínio ets/química , Sequência de Aminoácidos , Linhagem Celular , DNA/metabolismo , Dimerização , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Proteínas Elk-1 do Domínio ets/metabolismo
16.
FEBS Lett ; 585(7): 1089-96, 2011 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-21402070

RESUMO

LIMD1 is a tumour suppressor gene (TSG) down regulated in ∼80% of lung cancers with loss also demonstrated in breast and head and neck squamous cell carcinomas. LIMD1 is also a candidate TSG in childhood acute lymphoblastic leukaemia. Mechanistically, LIMD1 interacts with pRB, repressing E2F-driven transcription as well as being a critical component of microRNA-mediated gene silencing. In this study we show a CpG island within the LIMD1 promoter contains a conserved binding motif for the transcription factor PU.1. Mutation of the PU.1 consensus reduced promoter driven transcription by 90%. ChIP and EMSA analysis demonstrated that PU.1 specifically binds to the LIMD1 promoter. siRNA depletion of PU.1 significantly reduced endogenous LIMD1 expression, demonstrating that PU.1 is a major transcriptional activator of LIMD1.


Assuntos
Genes Supressores de Tumor , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Animais , Linhagem Celular Tumoral , Sequência Consenso/genética , Ilhas de CpG/genética , Técnicas de Silenciamento de Genes , Hematopoese/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM , Dados de Sequência Molecular , Mutagênese , Mutação , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , RNA Interferente Pequeno/genética , Transativadores/química , Transativadores/deficiência , Transativadores/genética
17.
Cell Cycle ; 9(21): 4294-6, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20962592

RESUMO

Maintaining the intracellular environment is important for the survival and proliferation of eukaryotic cells. How a cell regulates its redox potential during fluctuations in the generation of intracellular reactive oxygen species and exposure to extracellular oxidants is unclear. The recent findings that Signal Transducer and Activator of Transcription 3 (STAT3) regulates mitochondrial respiration and can be oxidised directly by peroxide to form multimers may have revealed features of a homeostatic mechanism coupling cell cycle progression to the intracellular redox potential.


Assuntos
Mitocôndrias/metabolismo , Fator de Transcrição STAT3/metabolismo , Ciclo Celular , Respiração Celular , Dimerização , Potencial da Membrana Mitocondrial , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/química
18.
Cancer Res ; 70(20): 8222-32, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20807804

RESUMO

Reactive oxygen species (ROS) promote tumor cell proliferation and survival by directly modulating growth-regulatory molecules and key transcription factors. The signal transducer and activator of transcription 3 (STAT3) is constitutively active in a variety of tumor cell types, where the effect of ROS on the Janus kinase/STAT pathway has been examined. We report here that STAT3 is directly sensitive to intracellular oxidants. Oxidation of conserved cysteines by peroxide decreased STAT3 binding to consensus serum-inducible elements (SIE) in vitro and in vivo and diminished interleukin (IL)-6-mediated reporter expression. Inhibitory effects produced by cysteine oxidation in STAT3 were negated in redox-insensitive STAT3 mutants. In contrast, ROS had no effect on IL-6-induced STAT3 recruitment to the c-myc P2 promoter. Expression of a redox-insensitive STAT3 in breast carcinoma cells accelerated their proliferation while reducing resistance to oxidative stress. Our results implicate STAT3 in coupling intracellular redox homeostasis to cell proliferation and survival.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição STAT3/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Neoplasias da Mama/patologia , Divisão Celular , Sequência Conservada , Cisteína/genética , Cisteína/metabolismo , Feminino , Genes Reporter , Humanos , Dados de Sequência Molecular , Estresse Oxidativo , Fator de Transcrição STAT1/genética , Especificidade da Espécie , Quimeras de Transplante , Vertebrados
19.
Methods Mol Biol ; 647: 279-89, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20694674

RESUMO

ETS transcription factors are implicated in gene regulation during cell proliferation and in the development of the haematopoietic cell lineage. Characteristically, ETS proteins act in concert with other transcription factors and are regulated by post-translational modifications, most frequently phosphorylation. These events have been shown to modulate the DNA binding affinity and interactions of ETS transcription factors with co-activators, events that can ultimately determine the formation of productive transcription complexes on target gene promoters. However, direct implication of a transcription factor or one of its post-translational modifications in the regulation of a given gene requires detection of the modified factor at the target gene promoter. Chromatin immunoprecipitation assays were originally adopted to probe modifications to histone proteins associated with transcriptionally active genes in yeast. They have since been used to confirm the presence of numerous proteins at diverse gene promoters including, for example, recruitment of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 to the promoters of mitogen-responsive genes. Here chromatin immunoprecipitation is used to demonstrate the inducible appearance of phosphorylated Elk-1 at the human c-fos promoter.


Assuntos
Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-ets/metabolismo , Extratos Celulares , Imunoprecipitação da Cromatina , DNA/genética , DNA/isolamento & purificação , Células HeLa , Humanos , Mitógenos/farmacologia , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Sonicação , Proteínas Elk-1 do Domínio ets/metabolismo
20.
Methods Mol Biol ; 647: 291-303, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20694675

RESUMO

Regulation of gene expression is essential for coordinated cell growth and development. The de-regulation of certain genes is also recognised to contribute to both heritable and acquired disease. Transcription factors influence the assembly and activity of transcription complexes, which they achieve in part by recruiting co-activators to gene promoters to participate in the dynamic cycle of polymerase binding, initiation and escape from the promoter. Co-activator recruitment and accompanying post-translational modifications to components of promoter complexes appear to differ between genes and as a consequence of varying signal input. Thus a full understanding of transcriptional initiation and control will ultimately require the elucidation of these processes. The method described here was designed to detect the presence of proteins and post-translational modifications in complexes formed in vitro on gene-specific promoters. It has been used, among other things, to detect the recruitment of the Mitogen-Activated Protein (MAP) kinases ERK1 and ERK2 to the promoters of mitogen-responsive genes.


Assuntos
Proteínas Quinases/metabolismo , Transcrição Gênica , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Immunoblotting , Espectrometria de Massas , Mitógenos/farmacologia , Regiões Promotoras Genéticas/genética , Transcrição Gênica/efeitos dos fármacos
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