RESUMEN
Estrogen is an important vasoprotective molecule that causes the rapid dilation of blood vessels by activating endothelial nitric oxide synthase (eNOS) through an unknown mechanism. In studies of intact ovine endothelial cells, 17beta-estradiol (E2) caused acute (five-minute) activation of eNOS that was unaffected by actinomycin D but was fully inhibited by concomitant acute treatment with specific estrogen receptor (ER) antagonists. Overexpression of the known transcription factor ERalpha led to marked enhancement of the acute response to E2, and this was blocked by ER antagonists, was specific to E2, and required the ERalpha hormone-binding domain. In addition, the acute response of eNOS to E2 was reconstituted in COS-7 cells cotransfected with wild-type ERalpha and eNOS, but not by transfection with eNOS alone. Furthermore, the inhibition of tyrosine kinases or mitogen-activated protein (MAP) kinase kinase prevented the activation of eNOS by E2, and E2 caused rapid ER-dependent activation of MAP kinase. These findings demonstrate that the short-term effects of estrogen central to cardiovascular physiology are mediated by ERalpha functioning in a novel, nongenomic manner to activate eNOS via MAP kinase-dependent mechanisms.
Asunto(s)
Endotelio Vascular/metabolismo , Estrógenos/farmacología , Óxido Nítrico Sintasa/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Activación Enzimática , Receptor alfa de Estrógeno , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III , Receptores de Estrógenos/agonistas , Ovinos , Transducción de Señal/efectos de los fármacosRESUMEN
The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ciclo Celular/fisiología , Factores de Elongación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Proteínas Portadoras/química , División Celular , Línea Celular , Secuencia de Consenso , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fase G2 , Eliminación de Gen , Genes Fúngicos , Genotipo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mamíferos , Proteínas de Transporte de Membrana , Ratones , Mitosis , Datos de Secuencia Molecular , Factor 1 de Elongación Peptídica , Factores de Elongación de Péptidos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/citología , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Dedos de ZincRESUMEN
The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Nucléolo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , División Celular/efectos de los fármacos , Nucléolo Celular/ultraestructura , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Clonación Molecular , Secuencia de Consenso , Secuencia Conservada , Factor de Crecimiento Epidérmico/farmacología , Biblioteca de Genes , Prueba de Complementación Genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Transporte de Membrana , Ratones , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
ZPR1 is a zinc finger protein that binds to the cytoplasmic tyrosine kinase domain of the epidermal growth factor receptor (EGFR). Deletion analysis demonstrated that this binding interaction is mediated by the zinc fingers of ZPR1 and subdomains X and XI of the EGFR tyrosine kinase. Treatment of mammalian cells with EGF caused decreased binding of ZPR1 to the EGFR and the accumulation of ZPR1 in the nucleus. The effect of EGF to regulate ZPR1 binding is dependent on tyrosine phosphorylation of the EGFR. ZPR1 therefore represents a prototype for a class of molecule that binds to the EGFR and is released from the receptor after activation.
Asunto(s)
Proteínas Portadoras/metabolismo , Receptores ErbB/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Línea Celular , Núcleo Celular/metabolismo , Clonación Molecular , Citoplasma/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/química , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas de Transporte de Membrana , Ratones , Datos de Secuencia Molecular , Fosforilación , Fosfotirosina/metabolismo , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Testículo/metabolismo , Fosfolipasas de Tipo C/metabolismo , Vanadatos/farmacología , Dominios Homologos srcRESUMEN
Incubation of cultured human fibroblasts with epidermal growth factor (EGF) causes a proliferative response that is mediated by the binding of the growth factor to specific cell surface receptors. One event that occurs rapidly following EGF binding is the covalent modification of the EGF receptor (EGF-R) by phosphorylation on Ser, Thr, and Tyr residues. Here we report the identification of ubiquitination as a second form of EGF-stimulated covalent modification of the receptor. The LGF receptor was not ubiquitinated in serum-starved cells. However, treatment with EGF caused a rapid increase in EGF-R ubiquitination. In contrast, no EGF-stimulated ubiquitination was found in experiments using cells that express a mutant tyrosine kinase-negative EGF-R. Similarly, ubiquitination of the EGF-R was not observed at 4 degrees C or if the cells are depleted of intracellular K+. Together, these data establish ubiquitination as a form of EGF-stimulated covalent modification of the EGF-R.
Asunto(s)
Receptores ErbB/metabolismo , Ubiquitinas/metabolismo , Animales , Células CHO , Frío , Cricetinae , Endocitosis , Factor de Crecimiento Epidérmico/farmacología , Humanos , Fosforilación , Ovinos , Tirosina/metabolismoRESUMEN
The osmotic balance between the cytoplasmic and extracellular compartments of cells is critical for the control of cell volume. A mammalian protein kinase, Jnk, which is a distant relative of the mitogen-activated protein kinase group, was activated by phosphorylation on threonine and tyrosine in osmotically shocked cells. The activation of Jnk may be relevant to the biological response to osmotic shock because the expression of human Jnk in the yeast Saccharomyces cerevisiae rescued a defect in growth on hyper-osmolar media. These data indicate that related protein kinases may mediate osmosensing signal transduction in yeast and mammalian cells.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae , Transducción de Señal/fisiología , Equilibrio Hidroelectrolítico/fisiología , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Cricetinae , Cricetulus , Activación Enzimática , Prueba de Complementación Genética , Proteínas Quinasas JNK Activadas por Mitógenos , Datos de Secuencia Molecular , Presión Osmótica , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
Rat and mouse spermatogenic cells contain a family of 1700-nucleotide (nt) proenkephalin mRNAs that are generated from an alternate, germ cell-specific promoter. This promoter is located approximately 350 base pairs (bp) downstream of the promoter used in somatic cells, within the first intron for the somatic transcript. In a previous study, rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing both promoters were shown to be transcribed selectively from the germ cell promoter and in the correct developmental pattern in spermatogenic cells of transgenic mice. In the present study it was found that spermatogenic cell-specific transgene expression was maintained after deletion of the upstream somatic promoter. This result establishes that the rat proenkephalin germ-line promoter is capable of functioning independently of transcriptional elements associated with the somatic promoter and localizes the requisite spermatogenic cell cis-elements to a 500-bp region encompassing the germ cell initiation sequences. A comprehensive analysis of binding sites for rat spermatogenic cell nuclear factors within this 500-bp region was performed using gel-shift and DNAse I footprinting techniques. Eight distinct binding regions were identified, each of which formed one or more cell-specific complexes with nuclear proteins from rat spermatogenic cells. These results suggest that multiple cis-acting elements may cooperate in the cell-specific and developmental regulation of rat proenkephalin gene transcription during spermatogenesis.
Asunto(s)
Encefalinas/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Precursores de Proteínas/genética , Espermatogénesis , Testículo/metabolismo , Animales , Secuencia de Bases , Secuencia de Consenso , Genes , Técnicas In Vitro , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Especificidad de Órganos , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Testículo/citologíaRESUMEN
To study the DNA sequences contacting the nuclear lamina (NL) in vivo, Ehrlich ascites tumor cells were UV-irradiated. The NL was purified, and the DNA fragments covalently linked to the lamina proteins in vivo were cloned and sequenced. Although heterogeneous in length and composition, the sequences displayed homology to the introns and/or flanking regions of different genes, suggesting that functionally distinct regions are organized in a topologically defined manner at the nuclear periphery.
Asunto(s)
ADN de Neoplasias/genética , Desoxirribonucleoproteínas/genética , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Homología de Secuencia de Ácido Nucleico , Animales , Carcinoma de Ehrlich/genética , Cromatina/metabolismo , Clonación Molecular , Reactivos de Enlaces Cruzados , ADN de Neoplasias/efectos de la radiación , Intrones , Laminas , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/efectos de la radiación , Proteínas Nucleares/efectos de la radiación , Secuencias Repetitivas de Ácidos Nucleicos , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
IgG antibodies to nuclear lamin proteins have been found in serum samples from 31 patients using immunofluorescence on HEp-2 cells, Western blotting, and enzyme-linked immunosorbent assay, performed against a nuclear lamina preparation from Ehrlich ascites tumor cells. Antilamin antibodies were most prevalent among patients with nonerosive, seronegative polyarthritis, or patients showing serum antiphospholipid reactivity as well. It is possible that anti-lamin antibodies may thus be a marker for a subgroup of polyarthritis patients who have a different prognosis from that of those with seropositive rheumatoid arthritis. The mechanism for the combined occurrence of anti-lamin and antiphospholipid autoantibodies is obscure. Future studies will answer whether these two antibodies represent a distinct antibody profile in patients with antiphospholipid antibody syndrome.
Asunto(s)
Anticuerpos Antinucleares/análisis , Proteínas Nucleares/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Síndrome Antifosfolípido/inmunología , Niño , Femenino , Humanos , Laminas , Hepatopatías/inmunología , Masculino , Persona de Mediana EdadRESUMEN
HNF1 is a transcription factor that is required for hepatocyte-specific expression of several genes, including albumin and fibrinogen. Rat HNF1-encoding cDNAs have recently been cloned, revealing that this factor is a distant member of the homeoprotein family. We have now isolated HNF1 clones from a human liver cDNA library by using a rat HNF1 cDNA-derived probe. The longest clone, HCL20, contains a sequence corresponding to the intact rat HNF1-coding region followed by a 3' nontranslated region and a poly(A) tail, hence representing an almost full-length HNF1 cDNA. Alignment of the human and rat sequences shows that HNF1 is highly conserved between the two species. The HNF1 gene was mapped by in situ hybridization and by RFLP analysis of interspecific mouse backcrosses to chromosomes 12q24.3 and 5F in human and mouse, respectively, establishing a new segmental homology between these two chromosomes.
Asunto(s)
Cromosomas Humanos Par 12 , Proteínas de Unión al ADN , Proteínas Nucleares , Factores de Transcripción/genética , Albúminas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cruzamientos Genéticos , Biblioteca de Genes , Ligamiento Genético , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Hibridación de Ácido Nucleico , Polimorfismo de Longitud del Fragmento de Restricción , Homología de Secuencia de Ácido Nucleico , alfa-Fetoproteínas/genéticaRESUMEN
Lamins A, B, and C are the major proteins of a polymeric structure called nuclear lamina, which is intercalated between chromatin and the inner membrane of the nuclear envelope. Using immunofluorescence on HEp-2 cells, specific enzyme-linked immunosorbent assay, and Western blotting performed against nuclear lamina preparation from Ehrlich ascites tumor cells, we characterized three patients, whose sera contained antibodies to nuclear lamins. The reaction pattern observed in two of the patients may result from single or combined occurrence of anti-lamin A and C antibodies. The third patient had antibodies that probably recognized an epitope in the carboxy-terminal region of lamin C. The sera were donated by a heterogeneous group of patients, and no common clinical or laboratory signs seemed to link them together.
Asunto(s)
Autoanticuerpos/análisis , Lamina Tipo A , Proteínas Nucleares/inmunología , Adulto , Anticuerpos Antinucleares , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Immunoblotting , Laminas , Persona de Mediana EdadRESUMEN
We describe a simple method for the purification of DNA fragments associated with the nuclear lamina in vivo. Ehrlich ascite tumor cells are first u.v.-irradiated to crosslink DNA to proteins. The nuclear lamina is then isolated and purified by low-speed centrifugation through a cushion of 40% sucrose. The material sedimenting through the created density barrier represents nuclear lamina of a very high purity, free from any DNA fragments except those which were in a crosslinking distance to it in vivo.
Asunto(s)
ADN/aislamiento & purificación , Matriz Nuclear/análisis , Animales , Carcinoma de Ehrlich/genética , Centrifugación Zonal , ADN de Neoplasias/aislamiento & purificación , Electroforesis en Gel de Agar , Ratones , Matriz Nuclear/ultraestructura , Proteínas Nucleares/aislamiento & purificación , Rayos UltravioletaRESUMEN
The three members of the jun proto-oncogene family c-jun, jun b and jun D were mapped on the mouse chromosome by in situ hybridization. The c-jun locus is on chromosome 4 subregion C5----C7, whereas jun B and jun D are co-localized on chromosome 8 subregion C. RFLP analysis of interspecific hybrids confirmed the mapping of jun B and D and showed that they are situated about 7.3 +/- 3.5 cM apart. Thus despite their possible origin from a single ancestral gene they are not closely linked on the chromosome. Using the same probes, we showed that the human genome also contains sequences homologous to the mouse jun B and jun D. They are located on human chromosome 19 p13.2, a region that may be involved in chromosomal translocation in acute lymphocytic leukemia (ALL), acute nonlymphocytic leukemia (ANLL) and malignant melanoma (MEL). Finally, the present data identify a new segmental homology between mouse and human chromosomes.
Asunto(s)
Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Proto-Oncogenes , Factores de Transcripción/genética , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Hibridación de Ácido Nucleico , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-jun , Homología de Secuencia de Ácido Nucleico , Translocación GenéticaRESUMEN
Ehrlich Ascites Tumour cells were irradiated with UV-light to crosslink DNA to proteins in vivo. The DNA fragments associated with the nuclear lamina were purified and characterized. The results of the Cot analysis and the hybridization experiments suggest that the DNA fragments attached to the nuclear lamina although containing the entire complexity of genomic DNA are enriched in some highly repeated sequences.
Asunto(s)
Núcleo Celular/metabolismo , ADN/metabolismo , Animales , Carcinoma de Ehrlich/metabolismo , Reactivos de Enlaces Cruzados , ADN de Neoplasias/metabolismo , ADN de Neoplasias/efectos de la radiación , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/efectos de la radiación , Proteínas Nucleares/metabolismo , Proteínas Nucleares/efectos de la radiación , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/efectos de la radiación , Rayos UltravioletaRESUMEN
Proteins from Saccharomyces cerevisiae were tested for their crossreactivity with antibodies raised against nuclear lamina proteins of Ehrlich Ascites Tumour cells. The results of the immunoblotting experiments and ELISA suggest the existence of at least two proteins (65 and 59 kDa) which are immunologically related to the nuclear lamina proteins of higher eukaryotes.
Asunto(s)
Proteínas de la Membrana/inmunología , Saccharomyces cerevisiae/análisis , Animales , Carcinoma de Ehrlich/análisis , Carcinoma de Ehrlich/patología , Carcinoma de Ehrlich/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Proteínas Fúngicas/análisis , Inmunoquímica , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/análisis , Membrana Nuclear/análisis , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/ultraestructuraRESUMEN
We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.
Asunto(s)
ADN de Neoplasias/efectos de la radiación , Nucleoproteínas/efectos de la radiación , Animales , Carcinoma de Ehrlich/metabolismo , ADN de Neoplasias/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas de Filamentos Intermediarios/efectos de la radiación , Laminas , Ratones , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/efectos de la radiación , Nucleoproteínas/metabolismo , Rayos UltravioletaRESUMEN
In the course of digestion of DNA with DNAase II or micrococcal nuclease, considerable amounts of single-stranded (ss) regions are formed, as determined by a second digestion with ss-specific nucleases, hyperchromicity measurements, and electron microscopy. Most of the ss stretches are located internally in the DNA molecules. The effect appears to be related to regions of decreased stability arising around single-stranded cuts in the double helix.
Asunto(s)
ADN de Cadena Simple/aislamiento & purificación , ADN , Endodesoxirribonucleasas , Nucleasa Microcócica , Animales , Cromatina/metabolismo , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Fabaceae , Concentración de Iones de Hidrógeno , Hidrólisis , Ratones , Desnaturalización de Ácido Nucleico , Plantas Medicinales , SolubilidadRESUMEN
We have studied the effect of chromatin condensation on the morphology of the residual structures isolated from rat liver nuclei. DNAse I digestion followed by high salt extraction of nuclei in the presence of Mg++ yields residual structures consisting of a dense peripheral layer surrounding an internal network, similar to those described by Berezney and Coffey [6]. These structures are stable at low ionic strength in the presence of EDTA. When nuclei swollen in EDTA are digested with DNAse II in the presence of EDTA, structures devoid of internal network are obtained even without subsequent treatment with high salt. When swollen nuclei are exposed to Mg++ a specific recondensation of chromatin takes place. The residual structures from recondensed nuclei are similar to those isolated from control nuclei in the presence of Mg++. The results suggest that the integrity and stability of the intranuclear matrix are acquired in the course of the isolation procedure and this is favoured by chromatin condensation.