RESUMEN
Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of (32)P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)(+)-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs.
Asunto(s)
Anexina A2/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Citoesqueleto/metabolismo , Congelación , Ratones , Polirribosomas/metabolismo , Unión Proteica , Células Tumorales CultivadasRESUMEN
Translation of mRNA is a prerequisite for cell proliferation, differentiation and viability. We have studied the effect of ribosome protein factors (GPRE) on acute myeloid leukemia (AML) blast cells. Ribosomes were isolated from MPC-11 cells using ultra-centrifugation. GPRE were extracted using a high KCl procedure. Blast cells from six AML patients were grown in suspension cultures for 24 and 96 h. GPRE or granulocyte macrophage-colony stimulating factor (GM-CSF) were added at the start of the incubation. GPRE, but not GM-CSF, prevented chromatin condensation and fragmentation of blast cell nuclei in AML-M2, -M4 and -M5 and the loss of nucleoli in AML-M2 and -M5. The fraction of phagocytosing blast cells in AML-M1, -M2, -M4 and -M5 was increased by GPRE. GPRE stimulated opsonin-dependent and -independent attachment and internalisation of N. meningitidis. GPRE increased the fraction of blasts expressing CD11b and CD32 in AML-M2 and -M5. GPRE diminished the fraction of AML-M5 cells bearing CD35 and CD32. GPRE also decreased the fraction of CD11c-bearing AML-M2 and -M5 cells. GM-CSF potentiated effects of GPRE in AML-M1, -M2, -M4 and -M5. GPRE and GM-CSF in combination affected phagocytosis and surface antigen expression in blast cells that were not influenced by either factor alone. Neither GPRE nor GM-CSF induced terminal differentiation or DNA-synthesis. We conclude that GPRE affects AML blast cell morphology, function and surface molecule expression, possibly by inhibiting apoptosis. The effects of GPRE may be mediated by ribosomal proteins that regulate translation and modulate the subcellular distribution of mRNA species.
Asunto(s)
Leucemia Mieloide/patología , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/fisiología , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Membrana Celular/inmunología , Supervivencia Celular , ADN/biosíntesis , Femenino , Histocitoquímica , Humanos , Integrina alfaXbeta2/análisis , Leucemia Mieloide/sangre , Leucemia Mieloide/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Luz , Antígeno de Macrófago-1/análisis , Masculino , Persona de Mediana Edad , Fagocitosis , Fenotipo , Polirribosomas/química , Receptores de Complemento 3b/análisis , Receptores de IgG/análisis , Proteínas Ribosómicas/análisis , Dispersión de Radiación , Factores de TiempoRESUMEN
Individual mRNA species have been shown to differ both with respect to localization in the cell, and in their distribution upon stimulation of cells with different signals. In this study we have examined the distribution of actin mRNA in the free, cytoskeletal-bound, and membrane-bound RNA fractions, both in starved cells, and in response to stimulation by feeding. These results were then compared with mRNAs for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and histone H4. The results we obtained showed that actin mRNA was located in the free RNA fraction in starved cells, while upon stimulation it was located both in the free, and in the cytoskeletal fraction; no redistribution of GAPDH mRNA occurred between the three RNA fractions, while H4 mRNA showed a different localization upon stimulation. Incubation with the drugs actinomycin-D and cycloheximide showed that an altered localization of actin mRNA from free in starved cells to free and cytoskeletal mRNA fractions following stimulation, was dependent on RNA synthesis, and not on protein synthesis.
Asunto(s)
Actinas/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/genética , Animales , División Celular , Fraccionamiento Celular , Membrana Celular , Medios de Cultivo , Cicloheximida/farmacología , Citoesqueleto/metabolismo , Dactinomicina/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Histonas/genética , Histonas/metabolismo , Ratones , Microtúbulos/metabolismo , Mieloma Múltiple , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
A three-step detergent/salt extraction procedure (Vedeler et al., Mol Cell Biochem 100: 183-193, 1991) was used to isolate free polysomes (FP), cytoskeletal-bound polysomes (CBP) and membrane-bound polysomes (MBP) from MPC-11 and Krebs II ascites cells. Polysomes were pelleted, washed with high salt buffer and re-pelleted. Proteins in the dialysed high-salt extracts were subjected to poly(A) Sepharose chromatography and poly(A) binding and non-binding proteins were separated by SDS-PAGE. In MPC-11 cells the FP fraction contains thirteen poly(A) binding proteins and four non-poly(A) binding proteins while the corresponding fraction in Krebs II ascites cells has four poly(A) binding proteins and six proteins which do not bind poly(A). The CBP fraction isolated from MPC-11 cells has a complement of ten poly(A) binding proteins, four which are non-poly(A) binding, and a protein of 105 kDa which has both poly(A) binding and non-poly(A) binding properties. In the CBP fraction prepared from Krebs II ascites cells a protein band at 32 kDa exhibits both poly(A) binding and non-poly(A) binding properties. In this fraction there are six poly(A) binding proteins and an additional eight which do not bind poly(A). Of the total number of proteins eight of these have a molecular weight below 40 kDa. The MBP fraction in MPC-11 cells contains three poly(A) binding proteins and eleven with non-poly(A) binding properties. In contrast this fraction in Krebs II ascites cells has a complement of thirteen poly(A) binding and ten non-poly(A) binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Membrana Celular/química , Citoesqueleto/química , Polirribosomas/química , Proteínas de Unión al ARN/análisis , Células 3T3 , Animales , Ascitis , División Celular , Separación Celular , Sistema Libre de Células , Cromatografía de Afinidad , Ratones , Proteínas de Unión a Poli(A) , Células Tumorales CultivadasRESUMEN
It is known that insulin treatment increases the rate of protein synthesis in many cells and tissues and that it causes changes in the distribution of ribosomes between free (FP), cytoskeletal-bound (CBP) and membrane-bound polysome (MBP) populations. This paper concerns an analysis of the pattern of proteins in high-salt extracts of FP, CBP and MBP isolated from Krebs II ascites and MPC-11 cells. A combined detergent/salt extraction procedure was used to isolate the three fractions of polysomes from control cells and from cells following short-term stimulation with insulin. There were differences in the protein patterns in the individual fractions and changes occurred after insulin stimulation.
Asunto(s)
Membrana Celular/química , Citoesqueleto/química , Insulina/farmacología , Polirribosomas/química , Proteínas/análisis , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Polirribosomas/efectos de los fármacos , Células Tumorales CultivadasAsunto(s)
Citoesqueleto de Actina/fisiología , Insulina/farmacología , Biosíntesis de Proteínas , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Tamaño de la Célula/efectos de los fármacos , Transformación Celular Neoplásica , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Polirribosomas/metabolismoRESUMEN
A three-step sequential detergent/salt extraction procedure was used in order to isolate three distinct subcellular fractions containing free (FP), cytoskeletal-bound (CBP) and membrane-bound polysomes (MBP), respectively, from Krebs II ascites cells (Vedeler et al., Mol Cell Biochem 100: 183-193, 1991). The purpose was to study changes in the distribution of polysomes in these three fractions during long-term incubation with insulin under either stationary conditions or in roller suspension culture. Insulin caused a redistribution of polysomes between FP, CBP and MBP fractions. The hormone appeared to promote an entry of ribosomes into polysomes both in CBP and MBP populations. When cells were grown in stationary culture in the presence of insulin and thus promoted to attach to the substratum and undergo morphological changes, a diversion of ribosomes from CBP into MBP was observed. The level of protein synthesis was apparently very high in this latter fraction since more than 70% of ribosomes were in polysomes. Morphological changes observed following insulin treatment were accompanied by a shift of certain proteins among subcellular fractions (for example actin and p35). The fibronectin content was about 20% higher in attached compared to non-attached cells. The results suggest that morphological changes induced by stimulation with insulin are associated with an increased activity of MBP, presumably reflecting a requirement for an increased synthesis of membrane proteins.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Fibronectinas/análisis , Insulina/farmacología , Polirribosomas/efectos de los fármacos , Ribonucleoproteínas/análisis , Ribosomas/efectos de los fármacos , Animales , Ascitis , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Citoesqueleto/metabolismo , Ratones , Ratones Endogámicos BALB C , Ribonucleoproteínas/químicaRESUMEN
The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton x 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50-205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton x 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.
Asunto(s)
Actinas/análisis , Ascitis/metabolismo , Cicloheximida/farmacología , Insulina/farmacología , Proteínas de Microfilamentos/análisis , Proteínas de Neoplasias/análisis , Faloidina/farmacología , Animales , Ascitis/patología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Ratones , Ratones Endogámicos BALB C , Peso MolecularAsunto(s)
Proteínas de Microfilamentos/metabolismo , Neoplasias Experimentales/metabolismo , Animales , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Microfilamentos/aislamiento & purificación , Mitocondrias/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
An increase in the amount of actin associated with the plasma membrane was visualized by immunocytochemistry 5 min after the addition of insulin to Krebs II ascites tumour cells maintained in serum-free medium. At 1 h of incubation the rim of fluorescence at the plasma membrane as measured by image analysis, was about 30% more intense than in control cells indicating that the initial accumulation of actin at the plasma membrane was not of a transient nature. Since an increase in the total cellular actin content in ascites cells did not occur until after a lag period of about 15 min then the increased amount of actin at the plasma membrane seen at 5 min was attributed to a stimulation of the polymerization of actin. An increase in the association of actin at the plasma membrane was also observed in 3T3 fibroblasts in areas of membrane ruffling, while in some cells there was also increased actin accumulation in the perinuclear area. The putative plasma membrane-microfilament linking protein 5'-nucleotidase was shown to be present in association with actin in the cytoskeletal fraction. Incubation of cells with insulin resulted in a shift of the enzyme toward the bottom of gradients indicating association with actin filaments of a greater length. The results demonstrate that insulin causes a stimulation of actin polymerization and that the hormone can be therefore assigned a role in the regulation of the cytoskeleton.
Asunto(s)
5'-Nucleotidasa/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Insulina/farmacología , Faloidina/farmacología , Células 3T3 , Animales , Anticuerpos Monoclonales/inmunología , División Celular/efectos de los fármacos , Membrana Celular/enzimología , Citocalasina B/farmacología , Citoesqueleto/enzimología , Ensayo de Inmunoadsorción Enzimática , Ratones , Células Tumorales CultivadasRESUMEN
Polysomes from Krebs II ascites and 3T3 cells were separated into three populations by using a sequential extraction method. Free polysomes were released by using a combination of low salt (25 mM KCl) and NP-40 detergent in the lysis buffer. The cytoskeletal bound polysomes were subsequently released by raising the salt concentration to 130 mM and finally, polysomes bound to the membranes of the endoplasmic reticulum were extracted by the combined treatment with Triton X-100 and deoxycholate. The results presented here illustrate that the three polysome-containing fractions differ in many parameters such as polysome profiles, cytoskeletal components and phospholipid content. When polyA-containing mRNA was isolated from the three polysome fractions and translated in an in vitro system, some differences were observed in the patterns of proteins being synthesized.
Asunto(s)
Fraccionamiento Celular/métodos , Membrana Celular , Citoesqueleto , Polirribosomas , Animales , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Centrifugación , Colchicina/farmacología , Citocalasina B/farmacología , Proteínas del Citoesqueleto/análisis , Citoesqueleto/química , Citoesqueleto/metabolismo , Electroforesis en Gel de Poliacrilamida , Ratones , Ratones Endogámicos BALB C , Faloidina/farmacología , Fosfolípidos/análisis , Polirribosomas/química , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Células Tumorales CultivadasRESUMEN
From 30 min to 1h of step-up conditions there was a redistribution of polysomes between free, cytoskeletal-bound and membrane-bound fractions such that more polysomes were recovered bound to the cytoskeleton and less in the free fraction. After 1h incubation with insulin there was a higher proportion of polysomes in the cytoskeletal fraction with a decrease occurring in the membrane-bound fraction. At 2h little change was observed in the presence of insulin while a large increase occurred in the cytoskeletal-bound fraction and a decrease in membrane-bound polysomes was seen in cells incubated in the absence of insulin. The results indicate that the proportions of polysomes in the three different fractions can be modulated by physiological stimuli, such as media replenishment and insulin.
Asunto(s)
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Insulina/farmacología , Polirribosomas/metabolismo , Animales , Ascitis/patología , Fraccionamiento Celular , Línea Celular , Ratones , Ratones Endogámicos BALB C , Polirribosomas/efectos de los fármacos , Biosíntesis de ProteínasRESUMEN
Binding of guanosine nucleotides to purified native and ADP-ribosylated wheat germ EF-2 was measured. Both forms of EF-2 bound [3H]GDP to the same extent. [3H]GDP binding to native but not to ADP-ribosylated EF-2 was reduced in the presence of GTP and ribosomes. Binding of [gamma-32P]GTP to EF-2 was significantly reduced upon ADP-ribosylation. ADP-ribosylation almost abolished both the stimulatory effect of ribosomes on GTP binding to EF-2 and the ability of EF-2 to form a high-affinity complex with GuoPP(CH2)P and ribosomes. Low-affinity complex formation between EF-2 X GDP and ribosomes was not influenced by ADP-ribosylation. The results indicate that the inhibition of the elongation process caused by the toxin is probably due to the inability of modified EF-2 to exchange GDP with GTP.