RESUMEN
Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1B-deficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.
Asunto(s)
Mediadores de Inflamación/metabolismo , Inflamación/enzimología , Activación de Macrófagos , Macrófagos Peritoneales/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Galactosamina , Perfilación de la Expresión Génica/métodos , Humanos , Inmunidad Innata , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Lipopolisacáridos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Tirosina Fosfatasa no Receptora Tipo 1/deficiencia , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2.4 times higher in CB than in PB cells. Importantly, the TPO-induced ploidy level was notably different: whereas 82.7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.
Asunto(s)
Ciclinas/metabolismo , Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas , Megacariocitos/citología , Células Madre/citología , Trombopoyetina/farmacología , Antígenos CD/inmunología , Antígenos CD34/inmunología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Sangre Fetal/metabolismo , Humanos , Integrina beta3 , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Glicoproteínas de Membrana Plaquetaria/inmunología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Factores de TiempoRESUMEN
Certain cell types escape the strict mechanisms imposed on the majority of somatic cells to ensure the faithful inheritance of parental DNA content. This is the case in many embryonic tissues and certain adult cells such as mammalian hepatocytes and megakaryocytes. Megakaryocytic endomitosis is characterized by repeated S phases followed by abortive mitoses, resulting in mononucleated polyploid cells. Several cell cycle regulators have been proposed to play an active role in megakaryocytic polyploidization; however, little is known about upstream factors that could control endomitosis. Here we show that ectopic expression of the transcriptional repressor escargot interferes with the establishment of megakaryocytic endomitosis. Phorbol ester-induced polyploidization was inhibited in stably transfected megakaryoblastic HEL cells constitutively expressing escargot. Analysis of the expression and activity of different cell cycle factors revealed that Escargot affects the G(1)/S transition by influencing Cdk2 activity and cyclin A transcription. Nuclear proteins that specifically bind the Escargot-binding element were detected in endomitotic and non-endomitotic megakaryoblastic cells, but down-regulation occurred only during differentiation of cells that become polyploid. As Escargot was originally implicated in ploidy maintenance of Drosophila embryonic and larval cells, our results suggest that polyploidization in megakaryocytes might respond to mechanisms conserved from early development to adult cells that need to escape normal control of the diploid state.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Megacariocitos/metabolismo , Mitosis , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Transcripción Genética , Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Clonación Molecular , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , ADN/metabolismo , ADN Complementario/metabolismo , Diploidia , Regulación hacia Abajo , Humanos , Immunoblotting , Inmunohistoquímica , Oligonucleótidos/metabolismo , Fenotipo , Ésteres del Forbol/farmacología , Ploidias , Pruebas de Precipitina , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Tiempo , TransfecciónRESUMEN
In the present study, the molecular cloning and characterization of a 49-kDa form of casein kinase (CK)I from Dictyostelium discoideum is reported. The predicted amino acid sequence shares 70% identity with the catalytic domain of the mammalian delta and epsilon isoforms, Drosophila CKIepsilon and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast CK involved in DNA repair. D. discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as detected by Northern-blot analysis. The level of DdCKI expression did not change during the cell cycle. Antibodies raised against a truncated version of the protein recognized a 49-kDa protein from D. discoideum extracts. Protein expression paralleled the pattern found for the RNA. The expression of DdCKI in Escherichia coli resulted in an active enzyme that autophosphorylated and phosphorylated casein. Immunofluorescence assays showed that DdCKI was localized in the cytoplasm and nuclei of Dictyostelium cells. The lack of disruptants of the CKI gene suggests that this protein is essential for the vegetative growth of D. discoideum. Overexpression of DdCKI resulted in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair.
Asunto(s)
Dictyostelium/enzimología , Expresión Génica , Proteínas Quinasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Caseína Quinasas , Dictyostelium/genética , Escherichia coli , Técnica del Anticuerpo Fluorescente , Datos de Secuencia Molecular , Peso Molecular , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Homología de Secuencia de AminoácidoRESUMEN
Megakaryocytes become polyploid by entering a truncated cell cycle, consisting of alternate S phases and abortive mitoses. We have investigated the regulation of the G1/S transition by comparing two megakaryoblastic cell lines, HEL and K562, which respectively do or do not become polyploid in response to phorbol esters. A pronounced downregulation of cyclin A, and to a lesser extent of cyclin E, occurred in K562 cells during the first 24 h after TPA treatment, in contrast with re-replicating HEL cells, in which both cyclins were present in individual G2/M cells. Transactivation experiments suggested that the absence of cyclin A in differentiated K562 cells could be due to a TPA-mediated inhibition of its transcription. To investigate the potential role of cyclin E in the establishment of re-replication cycles, we isolated K562 clones constitutively expressing cyclin E. The resulting clones, and also K562 cells transiently expressing cyclin E, entered re-replication cycles when treated with TPA. The transcriptional activity of the cyclin A promoter was not inhibited after TPA treatment, and although the levels of cyclin A fluctuated during further re-replication cycles, they never decreased below S phase levels. We conclude that the presence of cyclin E in megakaryoblastic G2/M cells determines cyclin A expression and allows the entrance into an extra S phase.
Asunto(s)
Ciclina E/biosíntesis , Megacariocitos/citología , Mitosis , Proteínas Supresoras de Tumor , Células de la Médula Ósea/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/biosíntesis , División Celular , Ciclina A/genética , Ciclina A/metabolismo , Ciclina B/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Regulación hacia Abajo , Fase G2 , Humanos , Células K562 , Megacariocitos/metabolismo , Proteínas Asociadas a Microtúbulos/biosíntesis , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/biosíntesis , Fase S , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética , Fosfatasas cdc25/biosíntesisRESUMEN
We have studied the regulation and role of c-Myc and Max in the differentiation pathways induced in K562 cells by 12-O-tetradecanoyl phorbol-13 acetate (TPA) and staurosporine, an activator and inhibitor, respectively, of protein kinase C (PKC). We found that staurosporine induced megakaryocytic differentiation, as revealed by the cellular ultrastructure, platelet formation, and DNA endoreduplication. In contrast, TPA induced a differentiated phenotype that more closely resembled that of the monocyte-macrophage lineage. c-myc expression was down-regulated in K562 differentiated by both TPA and staurosporine, whereas max expression did not change in either case. Although PKC enzymatic activity was low in cells terminally differentiated with TPA and staurosporine, inhibition of PKC activity by itself did not induce c-myc down-regulation. We conclude that the c-myc gene is switched off as a consequence of the differentiation process triggered by these drugs in a manner independent from PKC. Ectopic overexpression of c-Myc in K562 cells did not affect the monocytic-macrophagic and megakaryocytic differentiation, indicating that c-Myc suppression is not required for these processes in K562. Similarly, both differentiation pathways were not affected by Max overexpression or by concomitant overexpression of c-Myc and Max. This result is in contrast with the inhibition of erythroid differentiation of K562 exerted by c-Myc, suggesting divergent roles for c-Myc/Max, depending on the differentiation pathway.
Asunto(s)
Proteínas de Unión al ADN/genética , Macrófagos/citología , Monocitos/citología , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Carcinógenos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Macrófagos/metabolismo , Monocitos/metabolismo , Fenotipo , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Estaurosporina/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Endoglin is a transmembrane glycoprotein 633 residues in length expressed at the surface of endothelial cells as a disulphide-linked homodimer; the specific cysteine residues involved in endoglin dimerization are unknown. Mutations in the coding region of the endoglin gene are responsible for hereditary haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Many of these mutations, if translated, would lead to truncated forms of the protein. It is therefore of interest to assess the protein expression of different truncated forms of endoglin. Infections in vitro or in vivo with recombinant vaccinia virus, as well as transient transfections with expression vectors, were used to express normal and truncated forms of endoglin. Truncated mutants could be classified into three different groups: (1) those that did not produce stable transcripts; (2) those that produced stable transcripts but did not secrete the protein; and (3) those that secreted a soluble dimeric protein. This is the first time that a recombinant truncated form of endoglin has been found to be expressed in a soluble form. Because a chimaeric construct encoding the N-terminal sequence of platelet/endothelial cell adhesion molecule (PECAM-1) antigen fused to residues Ile281-Ala658 of endoglin also yielded a dimeric surface protein, these results suggest that cysteine residues contained within the fragment Cys330-Cys412 are involved in disulphide bond formation. Infection with vaccinia recombinants encoding an HHT1 mutation did not affect the expression of the normal endoglin, and did not reveal an association of the recombinant soluble form with the transmembrane endoglin, supporting a haploinsufficiency model for HHT1.
Asunto(s)
Expresión Génica , Fragmentos de Péptidos/metabolismo , Eliminación de Secuencia/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Antígenos CD , Línea Celular , Membrana Celular/metabolismo , Cisteína/genética , Cisteína/metabolismo , Dimerización , Disulfuros/metabolismo , Endoglina , Endotelio Vascular/citología , Humanos , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Unión Proteica , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Superficie Celular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sustancias Reductoras , Solubilidad , Telangiectasia Hemorrágica Hereditaria/genética , Transfección , Factor de Crecimiento Transformador beta/metabolismo , Virus Vaccinia/genética , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/química , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
In most eukaryotic cells, a link between S and M phases of the cell cycle must be assured in order to maintain the ploidy of newly divided cells. However, in some cell l/pes, e.g. the precursors of platelets megakaryocytes, extra S-phases can occur in the absence of concomitant mitoses, resulting in polyploidy. We have used two established cell lines with megakaryoblastic characteristics (HEL and MEG-01) to investigate the molecular events that lead these cells to bypass the regular control checkpoints that govern the interdependency of S and M phases. In the presence of the phorbol ester TPA, both cell lines stopped proliferating and displayed additional megakaryocytic features, including polyploidization. Analysis of key cell cycle regulatory factors implicated in the control of G1/S and G2/M transitions revealed a number of differences compared to normally cycling cells. Differentiating megakaryocytes were found to maintain high levels of cdk2, and cyclins E and A. This was accompanied by the appearance of the retinoblastoma protein in the hyperphosphorylated, functionally inactivated form. In addition, TPA-treated cells showed high levels of cyclin B and cdc2 proteins, however no activation of cdc2 was detected. This lack of cdc2 activation which should occur for entry into M phase appeared to be related to the down regulation of cdc25C phosphatase found in both differentiated HEL and MEG-01 cells. Together, our results suggest that in differentiating megakaryoblastic cells endoreplication is accompanied by sustained levels of cyclins A and E, and a lack of cdc2 activation, which is probably mediated through down regulation of cdc25C protein phosphatase.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclinas/genética , Replicación del ADN/genética , Regulación hacia Abajo , Fosfatasas cdc25 , Proteína Quinasa CDC2/metabolismo , Línea Celular , Activación Enzimática , Fase G1 , Humanos , Fase S , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Endoglin is an homodimeric membrane antigen with capacity to bind transforming growth factor-beta (TGF-beta) and whose expression is up-regulated on myeloid cells upon differentiation to macrophages. We have isolated full-length cDNA clones from a lambda gt 10 library, prepared from phorbol 12-myristate 13-acetate-differentiated HL60 cells by screening with an endoglin-specific cDNA probe from endothelial cells. Sequencing of the largest clone (3073 bp), revealed that the leader sequence contains 25 residues and that the 586 amino acids of the extracellular and transmembrane domains were identical to those described for endothelial endoglin. However, the cytoplasmic tail encoded by this cDNA clone contains only 14 amino acids as opposed to the 47 residues previously reported, suggesting the existence of two alternative endoglin variants. The expression of these isoforms was demonstrated by polymerase chain reaction analyses on endothelial cells, myelomonocytic cell lines HL-60 and U-937, and placenta. Independent cDNA constructs corresponding to both forms were transfected into mouse fibroblasts leading to the expression of two distinct endoglin molecules. Both forms were shown to bind TGF-beta 1 and, when overexpressed in transfected mouse fibroblasts, to form disulfide-linked homodimers, indicating that the cysteine residues present in the extracellular domain are responsible for the dimerization.
Asunto(s)
Proteínas Portadoras/análisis , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/análisis , Factor de Crecimiento Transformador beta/metabolismo , Molécula 1 de Adhesión Celular Vascular , Secuencia de Aminoácidos , Antígenos CD , Secuencia de Bases , Citoplasma/química , ADN/aislamiento & purificación , Endoglina , Humanos , Proteínas de Unión a TGF-beta Latente , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Peso Molecular , Receptores de Superficie CelularRESUMEN
Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with betaglycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta 1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta 1 (KD approximately 50 pM) and TGF-beta 3 with high affinity but fails to bind TGF-beta 2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin.
Asunto(s)
Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/química , Factor de Crecimiento Transformador beta/metabolismo , Molécula 1 de Adhesión Celular Vascular , Antígenos CD , Células Cultivadas , Endoglina , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Glicoproteínas de Membrana/inmunología , Peso Molecular , Pruebas de Precipitina , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta , TransfecciónRESUMEN
Time course experiments of the localization of rat LIMP II expressed in COS cells show that the protein is transported directly from the Golgi complex to lysosomes. Substitution of the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II for the native cytoplasmic tails of the plasma membrane proteins CD36 and CD8 resulted in straight transport of both proteins to lysosomes. The synthetic tyrosine-containing heptapeptide, RGTGVYG, did not replace the natural carboxyl cytoplasmic tail of LIMP II in its ability to transport both CD36 and CD8 to lysosomes, and the two constructs were transported to and expressed at the plasma membrane. Substitution of the cytoplasmic tails of either CD36 or CD8 for the carboxyl cytoplasmic tail of LIMP II resulted in transport of the mutants to the plasma membrane where they underwent endocytosis before accumulating into lysosomes. The results indicate that a motif contained in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient to target proteins directly from the Golgi complex to lysosomes.
Asunto(s)
Lisosomas/metabolismo , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Secuencia de Bases , Antígenos CD36 , Antígenos CD8 , Línea Celular , ADN , Aparato de Golgi/metabolismo , Cinética , Proteínas de Membrana de los Lisosomas , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
LIMP II is a glycoprotein expressed in the membrane of lysosomes and secretory granules with lysosomal properties. Sequence analysis of a CNBr-cleaved peptide allowed the synthesis of a 47-mer oligonucleotide that was used to screen a rat liver cDNA library in lambda gt11. This resulted in isolation of a 2-kilobase cDNA containing 1,434 bases encoding the entire protein. The deduced amino acid sequence indicates that LIMP II consists of 478 amino acid residues. The segment spanning residues 4-6 to 26 constitute an uncleavable signal peptide. LIMP II possesses a hydrophobic amino acid segment near the carboxyl end, that together with the uncleaved signal peptide may anchor the protein to the membrane through two distant segments. The major portion of the protein resides on the luminal side and displays 11 potential N-glycosylation sites and 5 cysteine residues. Two short cytoplasmic tails, 2-4 and 20-21 amino acids long, correspond to the NH2- and COOH-terminal ends of the protein, respectively. Transfection of COS cells with the cDNA of LIMP II resulted in expression of the protein and its transport to lysosomes. Comparison of the entire sequence to various data bases of known proteins revealed extensive homology between LIMP II and the cell surface protein CD36 involved in cell adhesion. No significant homology was detected with the two families of lysosomal membrane proteins A and B, recently described.
Asunto(s)
Antígenos CD/química , Lisosomas/metabolismo , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Antígenos CD36 , Adhesión Celular , Clonación Molecular , ADN , Expresión Génica , Proteínas de Membrana de los Lisosomas , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Ratas , Alineación de SecuenciaRESUMEN
Guanine nucleotide exchange factor (GEF) activity in ribosomal high salt wash and cytosolic fractions from suckling (4-10-day-old) and adult (60-day-old) rats was assayed by two different methods, by measuring: (i) its ability to promote binding of [3H]Met-tRNAi to eukaryotic initiation factor-2 (eIF-2) preparations that are partially or wholly in the form of eIF-2-GDP complexes (at Mg2+ concentrations near the optimum for protein synthesis), and (ii) under similar conditions, its ability to catalyze the displacement of [3H]GDP, previously bound to eIF-2, by unlabelled GDP. A purified eIF-2 (GEF-free) from brain was used as the source of eIF-2 activity. GEF activity in ribosomal fractions is higher in the brain of suckling than adults rats, and a direct correlation therefore exists between ribosomal GEF activity and the previously observed age-related decrease in eIF-2 activity in ribosomal high salt wash protein fractions. On the other hand GEF activity in the postmicrosomal supernatant is lower in the brain of suckling than adult rats. These findings further support the hypothesis that the progressive decrease in protein synthesis during brain development is controlled through regulation of the initiation step, by modulation of eIF-2/GEF activities.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Proteínas/metabolismo , Animales , Encéfalo/fisiología , Factor 2 Eucariótico de Iniciación , Factores de Intercambio de Guanina Nucleótido , Guanosina Difosfato/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Ratas , Ribosomas/metabolismo , Fracciones Subcelulares/análisisRESUMEN
About 30% of human tumours contain a mutation in one of the three ras genes leading to the production of p21ras oncoproteins that are thought to make a major contribution to the transformed phenotype of the tumour. The biochemical mode of action of the ras proteins is unknown but as they bind GTP and GDP and have an intrinsic GTPase activity, they may function like regulatory G proteins and control cell proliferation by regulating signal transduction pathways at the plasma membrane. It is assumed that an external signal is detected by a membrane molecule (or detector) that stimulates the conversion of p21.GDP to p21.GTP which then interacts with a target molecule (or effector) to generate an internal signal. Recently a cytoplasmic protein, GAP, has been identified that interacts with the ras proteins, dramatically increasing the GTPase activity of normal p21 but not of the oncoproteins. We report here that GAP appears to interact with p21ras at a site previously identified as the 'effector' site, strongly implicating GAP as the biological target for regulation by p21.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Sitios de Unión , Fibroblastos/metabolismo , GTP Fosfohidrolasas/genética , Proteínas Activadoras de GTPasa , Guanosina Trifosfato/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Unión Proteica , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Recombinantes/metabolismo , Proteínas Activadoras de ras GTPasaAsunto(s)
Transformación Celular Neoplásica , Genes ras , Proteínas de la Membrana/genética , Fosfatidilinositoles/fisiología , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Animales , Membrana Celular/metabolismo , Células Cultivadas , GTP Fosfohidrolasas/metabolismo , Nucleótidos de Guanina/metabolismo , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , TransfecciónRESUMEN
A plasmid has been constructed which contains the normal human N-ras proto-oncogene under the transcriptional control of the steroid-sensitive promoter of the mouse mammary tumor virus long terminal repeat. This plasmid has been introduced into NIH-3T3 cells producing a clone of cells, T15, which is phenotypically normal in the absence of the transcription inducer, dexamethasone, and transformed when treated with high levels of the inducer. At lower levels of dexamethasone, both morphological transformation and stimulation of DNA synthesis are titratable functions of p21N-ras levels. T15 cells have been used to demonstrate that: (i) a 20- to 50-fold over-expression of normal p21ras is required for complete cellular transformation, (ii) p21N-ras expression induces DNA synthesis and the effect can be amplified by epidermal growth factor, (iii) moderate increases in normal p21ras expression can influence cell behaviour.
Asunto(s)
Transformación Celular Neoplásica , Replicación del ADN , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Dexametasona/farmacología , Genes , Genes Virales , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos , Plásmidos , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas p21(ras) , Proto-Oncogenes/efectos de los fármacos , TransfecciónRESUMEN
Developmental changes at the level of initiation step of translation in the rat brain were studied. The level of deacylated tRNAimet in rat brain was measured at two stages of postnatal development. Although the amount of tRNA was slightly lower in adult than in young (4 day old) rats, the charging capacity of initiator tRNAimet in vitro was similar at both ages. No differences during development were found in methionyl-tRNA synthetase activity, which throws doubt on its possible participation in regulation of the initiation step. When assayed in the ribosomal salt wash protein fractions, initiation factor 2 activity decreased during brain development, and increased activities were detected in the supernatant of the microsomal fractions. The decrease in eIF-2 activity paralleled the observed decrease in the rat of overall protein synthesis or initiation activity in vitro, suggesting that the regulation of the initiation step of translation during brain development may be tightly linked to changes in initiation factor 2 activity in brain tissue.
Asunto(s)
Encéfalo/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Proteínas/metabolismo , Envejecimiento , Animales , Encéfalo/crecimiento & desarrollo , Factor 2 Eucariótico de Iniciación , Femenino , Técnicas In Vitro , Metionina-ARNt Ligasa/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Initiation factor 2 from adult rat brain was isolated from salt-washed microsomes using a three-step purification process consisting of heparin-Sepharose, phosphocellulose and diethylaminoethyl-cellulose (DEAE cellulose) column chromatographies. The initiation factor 2(eIF-2) was phosphorylated in subunits alpha and beta by the endogenous protein kinase activity present in the pruified preparation. This protein kinase activity proved to be mostly a casein kinase, although the possible presence of a very specific alpha kinase activity cannot be dismissed.
Asunto(s)
Encéfalo/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Animales , Química Encefálica , Caseínas/metabolismo , Catálisis , Factor 2 Eucariótico de Iniciación , Histonas/metabolismo , Factores de Iniciación de Péptidos/aislamiento & purificación , Fosforilación , Proteínas/aislamiento & purificación , Ratas , Especificidad por SustratoRESUMEN
Eukaryotic initiation factor 2 (eIF-2) was isolated from salt-washed microsomes of 4-day-old rat brain which show a high rate of protein synthesis. A three-step purification scheme was employed, including heparin-Sepharose, phosphocellulose, and DEAE-cellulose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF-2 forms ternary complexes with [3H]methionyl-tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF-2 and explains the high rate of activity of the crude fraction.