RESUMEN
The p53 tumour suppressor is involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 stabilization occurs under hypoxic and DNA damage conditions. However, only in the latter scenario is stabilized p53 capable of inducing the expression of its pro-apoptotic targets. Here we present evidence that under hypoxia-mimicking conditions p53 acetylation is reduced to a greater extent at K320 site targeted by P300/CBP-associated factor (PCAF) than at K382 site targeted by p300/CBP. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the p21(WAF-1/CIP-1) gene, which appears to be unaffected by hypoxia, but are not recruited to the BID promoter and hence p53 is incapable of upregulating pro-apoptotic BID in hypoxic conditions. As the K320 p53 acetylation is the site predominantly affected in hypoxia, the PCAF histone acetyltransferase activity is the key regulator of the cellular fate modulated by p53 under these conditions. In addition, we provide evidence that PCAF acetylates hypoxia-inducible factor-1alpha (HIF-1alpha) in hypoxic conditions and that the acetylated HIF-1alpha is recruited to a particular subset of its targets. In conclusion, PCAF regulates the balance between cell-cycle arrest and apoptosis in hypoxia by modulating the activity and protein stability of both p53 and HIF-1alpha.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Apoptosis/genética , Hipoxia de la Célula/genética , Línea Celular Tumoral , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Humanos , Regiones Promotoras GenéticasRESUMEN
The transcription of p53 target genes involves p300/CBP coactivators, which are multiprotein complexes that interact with the p53 activation domain. We report a cofactor in the p300 coactivator complex, Strap, which has an unusual structure, being composed almost entirely of a tandem series of six tetratricopeptide repeat (TPR) motifs. The TPR motif functions as a protein interaction domain, and it is consistent with this property that Strap harbors distinct and dedicated domains that allow it to bind and augment the interaction between different components of the p300 complex. Strap facilitates p53 activity in response to stress, in part through the stress-responsive accumulation of Strap protein and interfering with the MDM2-dependent downregulation of p53.
Asunto(s)
Acetiltransferasas/metabolismo , Secuencias de Aminoácidos , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulación de la Expresión Génica , Proteína p53 Supresora de Tumor/metabolismo , Acetiltransferasas/genética , Secuencia de Aminoácidos , Apoptosis/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Etopósido/farmacología , Genes Reporteros , Histona Acetiltransferasas , Humanos , Immunoblotting , Sustancias Macromoleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Radiación Ionizante , Serina Endopeptidasas , Estrés Fisiológico , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción , Transfección , Proteína p53 Supresora de Tumor/genética , Técnicas del Sistema de Dos Híbridos , Rayos Ultravioleta , Factores de Transcripción p300-CBPRESUMEN
The retinoblastoma tumour-suppressor protein (pRb) and p300/CBP co-activator proteins are important for control of proliferation and in tumour cells these are sequestered by viral oncoproteins such as E1A. pRb is involved in negatively regulating growth, and p300/CBP proteins have histone acetyltransferase (HAT) activity, which influences gene expression. Although it is known that phosphorylation by G1 cyclin-dependent kinases (CDKs) regulates pRb activity, the nature and role of other post-translational modifications is not understood. Here we identify acetylation as a new type of modification and level of control in pRb function. Adenovirus E1A, which binds p300/CBP through an amino-terminal transformation-sensitive domain, stimulates the acetylation of pRb by recruiting p300 and pRb into a multimeric-protein complex. Furthermore, pRb acetylation is under cell-cycle control, and acetylation hinders the phosphorylation of pRb by cyclin-dependent kinases. pRb binds more strongly when acetylated to the MDM2 oncoprotein, which indicates that acetylation may regulate protein-protein interactions in the pRb pathway. The acetylation of pRb defines a new level of cell-cycle control mediated by HAT. Furthermore, our results establish a relationship between p300, pRb and acetylation in which E1A acts to recruit and target a cellular HAT activity to pRb.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteína de Retinoblastoma/metabolismo , Acetilación/efectos de los fármacos , Proteínas E1A de Adenovirus/farmacología , Ciclo Celular/efectos de los fármacos , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/farmacología , Humanos , Mutación , Proteínas Nucleares/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/farmacología , Transactivadores/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process.
Asunto(s)
Receptores de Glucocorticoides/metabolismo , Saccharomyces cerevisiae , Serina/metabolismo , Treonina/metabolismo , Animales , Carcinoma Hepatocelular , Desoxicorticosterona/farmacología , Dexametasona/farmacología , Expresión Génica , Glucocorticoides/farmacología , Ratones , Mapeo Peptídico , Monoéster Fosfórico Hidrolasas/farmacología , Fosforilación , Mutación Puntual , Pregnatrienos/farmacología , Ratas , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Steroid and thyroid hormones act on nuclear gene transcription by activating protein receptors, which in turn bind to hormone response elements (HREs). Among these cell-specific processes regulated by steroid receptors is energy metabolism through increased synthesis of respiratory enzymes. As some of these enzymes are encoded by both nuclear and mitochondrial genes, coordination of their synthesis is probable, inter alia at the transcriptional level. We have postulated a direct effect of steroid hormones on mitochondrial gene transcription and here present the following evidence in support of this hypothesis. 1) The human and rodent mitochondrial genomes contain nucleotide sequences similar both to type I and type II HREs. 2) Glucocorticoid receptors (GR) rapidly translocate from the cytoplasm into mitochondria after administration of glucocorticoids. This process has been reproduced in vitro and deletion of the N-terminal part of the glucocorticoid receptor stops translocation into mitochondria. 3) Gel shift analysis has demonstrated binding of GR to putative mitochondrial GR elements. 4) In transfection experiments, mitochondrial HREs confer dexamethasone inducibility on hybrid reporter constructs, abolished in the presence of excess RU38486. 5) Similar results were obtained for thyroid hormone receptor (TR alpha) localization, import, and binding to TR elements. These findings, taken with the demonstrated effects of steroid (and thyroid) hormones on mitochondrial transcription and respiratory enzyme biosynthesis, strongly support the hypothesis of a direct effect of steroid (and thyroid) hormones on mitochondrial gene transcription.
Asunto(s)
Mitocondrias/genética , Mitocondrias/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/genética , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Secuencia Conservada , Dexametasona/farmacología , Resistencia a Medicamentos/genética , Genes Bacterianos , Glucocorticoides/farmacología , Humanos , Mifepristona/farmacología , Mitocondrias/química , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/efectos de los fármacos , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/metabolismo , Ratas , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Homología de Secuencia de Ácido Nucleico , Esteroides , Transcripción Genética , TransfecciónRESUMEN
Six mitochondrial genome sequences, showing strong similarity to the glucocorticoid responsive element consensus sequence (GRE), four localized within the cytochrome c oxidase (COX) subunit I and II genes (GREs I-IV) and two within the D-loop region (GREs a and b) have been examined as binding sites of glucocorticoid receptor (GR) from rat liver cytosol. Purified GR from rat liver cytosol binds with high specificity to all potential mitochondrial GREs, as shown by filter retention and gel shift assays. Specific binding of protein(s), present in a mitochondrial extract from dexamethasone-induced mice, to all six putative mitochondrial GREs was also documented by the same methodology. Both purified GR and protein(s) from mitochondrial extract give the same band in the gel retardation assay. Using monospecific anti-glucocorticoid receptor polyclonal antibody (EP), a supershift of the gel retarded protein-DNA band was obtained. These results demonstrate that the mitochondrial genome sequences examined have characteristics of GREs, since they show the capacity to specifically bind the respective receptor protein. These findings support the hypothesis that the mitochondrial genome is a primary site of action of steroid and thyroid hormones (Sekeris C.E.: The mitochondrial genome: a possible primary site of action of steroid hormones, In vivo 4 (1990) 317-320).
Asunto(s)
Secuencia de Consenso , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias Hepáticas/metabolismo , Receptores de Glucocorticoides/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Citosol/química , ADN Mitocondrial/genética , Dexametasona/farmacología , Complejo IV de Transporte de Electrones/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Mitocondrias Hepáticas/efectos de los fármacos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Ratas , Ratas Wistar , Receptores de Glucocorticoides/aislamiento & purificación , Homología de Secuencia de Ácido NucleicoRESUMEN
Administration of inducing doses of dexamethasone (10 microg/100 g) to adrenalectomized rats results, within 2-5 min, in import of the glucocorticoid receptor from liver cytoplasm into mitochondria, as demonstrated by Western blotting and by electron microscopy. Furthermore, glucocorticoid receptor (GR) synthesized in an in vitro reticulocyte system programmed with the respective mRNA, enters within minutes to added rat liver mitochondria in the form of intact GR, as demonstrated by Western blotting using either monoclonal or polyclonal antibodies against different domains of GR. In vitro studies show that the import is dependent on temperature and/or activation of the hormone-GR complex. These results, in connection with the presence in the human and rodent mitochondrial genome of sequences showing partial homology to the nuclear glucocorticoid response elements, support the hypothesis that the well documented effects of glucocorticoids on mitochondrial functions result from a direct interaction of the GR complex with the mitochondrial genome.