RESUMEN
Apoptosis triggered by endoplasmic reticulum (ER) stress has been implicated in many diseases but its cellular regulation remains poorly understood. Previously, we identified salubrinal (sal), a small molecule that protects cells from ER stress-induced apoptosis by selectively activating a subset of endogenous ER stress-signaling events. Here, we use sal as a probe in a proteomic approach to discover new information about the endogenous cellular response to ER stress. We show that sal induces phosphorylation of the translation elongation factor eukaryotic translation elongation factor 2 (eEF-2), an event that depends on eEF-2 kinase (eEF-2K). ER stress itself also induces eEF-2K-dependent eEF-2 phosphorylation, and this pathway promotes translational arrest and cell death in this context, identifying eEF-2K as a hitherto unknown regulator of ER stress-induced apoptosis. Finally, we use both sal and ER stress models to show that eEF-2 phosphorylation can be activated by at least two signaling mechanisms. Our work identifies eEF-2K as a new component of the ER stress response and underlines the utility of novel small molecules in discovering new cell biology.
Asunto(s)
Apoptosis , Cinamatos/farmacología , Quinasa del Factor 2 de Elongación/metabolismo , Retículo Endoplásmico/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Tiourea/análogos & derivados , Animales , Células Cultivadas , Factor 2 Eucariótico de Iniciación/metabolismo , Ratones , Células PC12 , Proteómica , Ratas , Transducción de Señal , Tiourea/farmacologíaRESUMEN
A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Factor 2 de Elongación Peptídica/metabolismo , Mapeo Peptídico , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Quinasa del Factor 2 de Elongación , Activación Enzimática/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mapeo Peptídico/métodos , Péptidos/síntesis química , Péptidos/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Especificidad por Sustrato/genéticaRESUMEN
Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Domínguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.
Asunto(s)
Factores de Elongación de Péptidos/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Células 3T3 , Animales , Células COS , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Electroforesis en Gel de Poliacrilamida , Quinasa del Factor 2 de Elongación , Fluoruros/metabolismo , Células HeLa , Humanos , Ratones , Factor 2 de Elongación Peptídica , Fosforilación , Timosina/metabolismo , TransfecciónRESUMEN
Mobilization of Ca2+ sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca2+ ([Ca2+]i) due to plasmalemmal Ca2+ influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca2+ stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca2+]i per se causes slowing of peptide chain elongation is lacking. L-type Ca2+ channel activity of GH3 pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca2+]i on eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca2+, resting [Ca2+]i values were high (154-255 nM) and eEF-2 was phosphorylated. The Ca2+ channel antagonist, nisoldipine, lowered [Ca2+]i and reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca2+ channel agonist, Bay K 8644, produced sustained elevations of [Ca2+]i that were associated with 25-50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca2+ influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50-100% above control) and inhibited leucine incorporation by 20-70% but ATP content was reduced by 25-50% and total cell-associated Ca2+ contents rose by 3- to 13-fold. eIF-2alpha was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca2+]i that resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2alpha, not eEF-2. It is concluded that changes in [Ca2+]i occurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.
Asunto(s)
Calcio/fisiología , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Adenosina Trifosfato/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio Tipo L , Citosol/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Guanosina Trifosfato/metabolismo , Ionomicina/farmacología , Ionóforos/farmacología , Leucina/metabolismo , Extensión de la Cadena Peptídica de Translación/fisiología , Fosforilación , Células Tumorales CultivadasRESUMEN
The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of approximately 200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Caenorhabditis elegans/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Dictyostelium/enzimología , Quinasa del Factor 2 de Elongación , Humanos , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Proteínas Protozoarias , Conejos , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reticulocitos/enzimología , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
eEF-2 kinase is a ubiquitous Ca2+/calmodulin-dependent protein kinase that is specific for protein synthesis elongation factor-2 (eEF-2). This study describes an improved procedure for the purification of eEF-2 kinase from rabbit reticulocyte lysate. The eEF-2 kinase preparation was used to raise polyclonal antibodies, which immunoprecipitated eEF-2 kinase protein and activity from rabbit reticulocyte lysate. The antibodies recognized a single 103 kDa band in extracts from several cell lines including NIH 3T3, PC12, C6 glioma, HeLa, and MCF-7 breast carcinoma. However, there was no immunoreactivity in extracts of rabbit or bovine liver or rabbit kidney despite the presence of abundant eEF-2 kinase activity in these tissues. Exposure of PC12 cells to nerve growth factor (NGF) resulted in rapid down-regulation of eEF-2 kinase activity and a decrease in immunoreactivity. After 24 h of incubation with NGF, the activity of the kinase recovered to 80% of initial values. In contrast, the immunoreactivity of eEF-2 kinase continued to decrease. These data suggest that tissue-specific isoforms of eEF-2 kinase may exist and that these isoforms may be regulated by growth factors.
Asunto(s)
Anticuerpos/inmunología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Isoenzimas/análisis , Células 3T3 , Animales , Especificidad de Anticuerpos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/inmunología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/aislamiento & purificación , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Bovinos , Cromatografía en Agarosa , Electroforesis en Gel de Poliacrilamida , Quinasa del Factor 2 de Elongación , Humanos , Técnicas para Inmunoenzimas , Isoenzimas/inmunología , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Ratones , Factores de Crecimiento Nervioso/farmacología , Especificidad de Órganos , Células PC12 , Conejos , Ratas , Células Tumorales CultivadasRESUMEN
There is evidence that resting cells are able to produce molecules with antiproliferative activity, some of which behave as short-lived repressor proteins. We suggest that transient inhibition of protein synthesis in resting cells would lead to a decrease in the levels of these negative growth regulators and might, therefore, promote mitogenic responses. We report that treatment of resting (serum-deprived) NIH 3T3 cells with cyclocheximide (CH) or puromycin induces expression of c-fos, c-jun and c-myc proto-oncogenes in a manner similar to that of platelet-derived growth factor (PDGF). Actinomycin D (Act D) abrogates the induction of proto-oncogene expression. Transient inhibition of protein synthesis by CH or puromycin also induces the resting NIH 3T3 and C3H 1OT1/2 cells to enter the cell cycle. Inhibition of new RNA or protein synthesis abolishes the proliferative response. These findings show that control mechanisms at both transcriptional and translational levels are operative in the resting cells treated with protein synthesis inhibitors. Cell fusion experiments with resting and serum-stimulated NIH 3T3 cells revealed that brief pre-incubation of resting cells with either PDGF, CH or puromycin abrogates their ability to suppress the onset of DNA synthesis in the nuclei of stimulated cells in heterodikaryons. However, the abrogative effect of PDGF disappeared in the presence of Act D, whereas the effects of protein synthesis inhibitors did not, indicating their independence of the induction of transcription. The data suggest that the observed effects of protein synthesis inhibitors are connected with elimination of some short-lived negative growth regulators, since a brief translational arrest is sufficient for the resumption of DNA synthesis in the nuclei of stimulated cells blocked by resting cells in heterodikaryons.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Cicloheximida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proto-Oncogenes , Puromicina/farmacología , Células 3T3/efectos de los fármacos , Animales , Ciclo Celular/genética , Línea Celular , Replicación del ADN/efectos de los fármacos , Dactinomicina/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Genes fos , Genes jun , Genes myc , Células Híbridas/efectos de los fármacos , Células Híbridas/metabolismo , Ratones , Ratones Endogámicos C3H , Factor de Crecimiento Derivado de Plaquetas/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Transcripción Genética/efectos de los fármacosRESUMEN
There are many reports which demonstrate that the rate of protein biosynthesis at the elongation stage is actively regulated in eukaryotic cells. Possible physiological roles for this type of regulation are: the coordination of translation of mRNA with different initiation rate constants; regulation of transition between different physiological states of a cell, such as transition between stages of the cell cycle; and in general, any situation where the maintenance of a particular physiological state is dependent on continuous protein synthesis. A number of covalent modifications of elongation factors offer potential mechanisms for such regulation. Among the various modifications of elongation factors, phosphorylation of eEF-2 by the specific Ca2+calmodulin-dependent eEF-2 kinase is the best studied and perhaps the most important mechanism of regulation of elongation rate. Since this phosphorylation is strictly Ca(2+)-dependent, and makes eEF-2 inactive in translation, this mechanism could explain how changes in the intracellular free Ca2+ concentration may regulate elongation rate. We also discuss some recent findings concerning elongation factors, such as the discovery of developmental stage-specific elongation factors and the regulated binding of eEF-1 alpha to cytoskeletal elements. Together, these observations underline the importance of the elongation stage of translation in the regulation of the cellular processes essential for normal cell life.
Asunto(s)
Regulación de la Expresión Génica , Extensión de la Cadena Peptídica de Translación , Factores de Elongación de Péptidos/metabolismo , Biosíntesis de Proteínas , Animales , Células Eucariotas/metabolismo , Metilación , Fosforilación , Proteína Quinasa C/metabolismoRESUMEN
Phosphorylation of translation elongation factor 2(eEF-2) by a specific Ca2+/calmodulin-dependent eEF-2 kinase plays an important role in the regulation of protein synthesis in mammalian cells. We show here that an eEF-2 kinase similar to the mammalian enzyme is present in tissues of the amphibian Xenopus laevis. We investigated changes in the activity of eEF-2 kinase in extracts of Xenopus oocytes at different stages of oogenesis. The eEF-2 kinase activity was constant from stage I to stage IV of oogenesis, but dramatically decreased after stage IV. Extracts of fully grown stage-VI oocytes showed no eEF-2 kinase activity. However, when extracts were analyzed by two-dimensional gel electrophoresis, eEF-2 was found to be present mostly, if not exclusively, in the dephosphorylated form throughout oogenesis. It is suggested that eEF-2 kinase disappears late in oogenesis to make protein synthesis insensitive to changes in intracellular Ca2+ concentration. This may be important for the induction of meiotic maturation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina , Oocitos/metabolismo , Oogénesis , Factores de Elongación de Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Animales , Calcio/fisiología , Calmodulina/fisiología , Quinasa del Factor 2 de Elongación , Inducción Enzimática , Femenino , Extensión de la Cadena Peptídica de Translación , Factor 2 de Elongación Peptídica , Fosforilación , Procesamiento Proteico-Postraduccional , Transducción de Señal , Xenopus laevisRESUMEN
Elongation factor 2 (eEF-2) is a 100-kD protein that catalyzes the ribosomal translocation reaction, resulting in the movement of ribosomes along mRNA. eEF-2 is the target for a very specific Ca2+/calmodulin-dependent eEF-2 kinase. Phosphorylation of eEF-2 makes it inactive in translation, which suggests that protein synthesis can be regulated by Ca2+ through eEF-2 phosphorylation. Recent data demonstrate that eEF-2 phosphorylation can be involved in cell-cycle regulation and other processes where changes of intracellular Ca2+ concentration induce a new physiological state of a cell. The main role of eEF-2 phosphorylation in these processes is temporary inhibition of overall translation in response to transient elevation of the Ca2+ concentrations in the cytoplasm. Temporary inhibition of translation may trigger the transition of a cell from one physiologic state into another because of the disappearance of short-lived repressors and thus the activation of expression of new genes.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina , Calcio/fisiología , Factores de Elongación de Péptidos/metabolismo , Proteínas Quinasas/fisiología , Procesamiento Proteico-Postraduccional , Vertebrados/genética , Neoplasias de las Glándulas Suprarrenales/patología , Secuencia de Aminoácidos , Animales , Calmodulina/fisiología , Quinasa del Factor 2 de Elongación , Globinas/biosíntesis , Guanosina Trifosfato/metabolismo , Interfase , Mitosis , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/farmacología , Oogénesis , Extensión de la Cadena Peptídica de Translación , Factor 2 de Elongación Peptídica , Feocromocitoma/patología , Fosfoproteínas Fosfatasas/fisiología , Fosforilación , Conejos , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Transcripción Genética , Células Tumorales Cultivadas/efectos de los fármacos , Xenopus laevisRESUMEN
Elongation factor 2 was identified in the two-dimensional gel patterns of asynchronous human amnion cells (AMA) by comigration with purified rabbit reticulocyte elongation factor 2 and by two-dimensional gel immunoblot analysis using a specific rabbit polyclonal antibody. In all, four polypeptides were identified corresponding to isoelectric focusing polypeptides 2713 (95.0 kDa), 2714 (94.8 kDa), 3727 (94.8 kDa), and 3702 (93.6 kDa) (listed in order of decreasing pI values) in the computerized comprehensive two-dimensional gel data base of human AMA proteins. The relative proportion of two of these variants (isoelectric focusing polypeptides 3727 and 3702), which are phosphorylated, increased dramatically during mitosis. Since phosphorylation is known to render elongation factor 2 inactive in translation, this observation may partly explain the decline in the rate of protein synthesis observed during cell division.
Asunto(s)
Mitosis , Factores de Elongación de Péptidos/metabolismo , Biosíntesis de Proteínas , Amnios , Electroforesis en Gel Bidimensional , Humanos , Técnicas In Vitro , Interfase , Punto Isoeléctrico , Factor 2 de Elongación Peptídica , Fosfoproteínas/metabolismo , FosforilaciónRESUMEN
Monoclonal antibodies referred to as Am1, Am2 and Am3 against highly purified bovine tryptophanyl-tRNA synthetase were prepared. Am2 antibodies inhibit the Trp-tRNA synthetase activity and interact with the active truncated enzyme forms (dimers of either 40-kDa or 51-kDa fragments) produced by limited proteolysis. Am1 and Am3 antibodies exert no effect on the Trp-tRNA synthetase activity; epitopes recognized by them are mapped close to one another and reside at the dispensable part of the Trp-tRNA synthetase molecule. Am1 cross-reacts with Trp-tRNA synthetases of eukaryotic, prokaryotic and archaebacterial species, as revealed by immunoblot analysis. A rapid two-step technique was developed for isolating electrophoretically homogeneous Trp-tRNA synthetase from Escherichia coli. The purified enzyme interacted with Am1, but not with Am2 and Am3 antibodies taken at the same concentrations. As in the case of eukaryotic Trp-tRNA synthetase, Am1 did not influence the activity of Trp-tRNA synthetase from E. coli. From the aforementioned results it follows that: (a) the conservation of part of the Trp-tRNA synthetase structure which is not directly involved in the formation of the catalytic centre of prokaryotic and eukaryotic Trp-tRNA synthetases suggests that the dispensable part of the molecule might be involved in some additional biological function(s) of Trp-tRNA synthetase besides tRNA(Trp) charging; (b) the common antigenic determinant in Trp-tRNA synthetase of eukaryotes, prokaryotes and archaebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.
Asunto(s)
Aminoacil-ARNt Sintetasas/inmunología , Anticuerpos Monoclonales , Archaea/enzimología , Bacterias/enzimología , Epítopos/análisis , Triptófano-ARNt Ligasa/inmunología , Animales , Evolución Biológica , Bovinos , Escherichia coli/enzimología , Ratones , Ratones Endogámicos BALB C , Mapeo Peptídico , Triptófano-ARNt Ligasa/análisis , Triptófano-ARNt Ligasa/aislamiento & purificaciónRESUMEN
Previously we have found that elongation factor 2 (EF-2) from mammalian cells can be phosphorylated by a special Ca2+/calmodulin-dependent protein kinase (EF-2 kinase). Phosphorylation results in complete inactivation of EF-2 in the poly(U)-directed cell-free translation system. However, the partial function of EF-2 affected by phosphorylation remained unknown. Here we show that phosphorylated EF-2, unlike non-phosphorylated EF-2, is unable to switch ribosomes carrying poly(U) and Phe-tRNA in the A site to a puromycin-reactive state. Thus, phosphorylation of EF-2 seems to block its ability to promote a shift of the aminoacyl(peptidyl)-tRNA from the A site to the P site, i.e. translocation itself.
Asunto(s)
Calcio/farmacología , Calmodulina/farmacología , Factores de Elongación de Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Animales , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacología , Guanilil Imidodifosfato/farmacología , Factor 2 de Elongación Peptídica , Fosforilación , Poli U/metabolismo , Puromicina/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Conejos , Ribosomas/metabolismo , Relación Estructura-ActividadRESUMEN
A model is proposed uniting two groups of facts: the adsorption of enzymes on subcellular structures and the direct ('from hand to hand') transfer of metabolites between enzymes. The basic idea is that the binding of metabolites (substrates and/or products) results in desorption of the enzymes from subcellular structures during each catalytic act. This makes the enzymes mobile and capable of directly (from hand to hand) transferring metabolites to other enzymes adsorbed on subcellular structures. The model leads to a mechanism by means of which soluble enzymes can be compartmentalized in defined regions of the cytoplasm.
Asunto(s)
Células/enzimología , Enzimas/metabolismo , Adsorción , Citosol/enzimología , Modelos TeóricosRESUMEN
A new Ca2+/calmodulin-dependent protein kinase has been recently discovered in mammalian cells. The major substrate of this kinase, a protein of relative molecular mass (Mr) approximately equal to 100,000 (100K), has been identified as elongation factor 2 (EF-2), which participates in protein synthesis. The in vivo activity of the EF-2 kinase depends upon growth factors and other agents affecting the level of Ca2+ and cAMP. Its effect on EF-2 activity, however, remained obscure. This work shows that the phosphorylation of EF-2 by the EF-2 kinase results in a drastic inhibition of polyphenylalanine synthesis in poly(U)-directed translation. Phosphorylated EF-2 is completely inactive in translation and, moreover, inhibits the activity of non-phosphorylated EF-2. Dephosphorylation of EF-2 by phosphatase restores its activity. Hence, the phosphorylation of EF-2 directly affects the elongation stage of translation and thus represents a novel mechanism of translational control.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina , Factores de Elongación de Péptidos/fisiología , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , Animales , Sistema Libre de Células , Quinasa del Factor 2 de Elongación , Regulación de la Expresión Génica , Técnicas In Vitro , Factor 2 de Elongación Peptídica , Fosfoproteínas/fisiología , Fosforilación , ConejosRESUMEN
Elongation factor 2 (EF-2) has been recently shown to be extensively phosphorylated in a Ca2+/calmodulin-dependent manner in extracts of mammalian cells (A. G. Ryazanov (1987) FEBS Lett. 214, 331-334). In the present study, we partially purified the protein kinase which phosphorylates EF-2 from rabbit reticulocytes. The molecular weight of the enzyme determined by gel filtration was about 140,000. Unlike the substrate, the EF-2 kinase had no affinity for RNA and therefore could be separated from EF-2 by chromatography on RNA-Sepharose. After chromatography on hydroxyapatite, the kinase activity became calmodulin-dependent. Two-dimensional separation of the phosphorylated EF-2 according to O'Farrell's technique revealed that there were two phosphorylation sites within the EF-2 molecule; in both cases, the phosphorylated amino acid was threonine. The EF-2 kinase differed from the four known types of Ca2+/calmodulin-dependent protein kinases. Thus, the system of EF-2 phosphorylation represents the novel (fifth) Ca2+/calmodulin-dependent system of protein phosphorylation. This system is supposed to be responsible for the regulation of the elongation rate of protein biosynthesis in eukaryotic cells.
Asunto(s)
Calcio/farmacología , Calmodulina/farmacología , Factores de Elongación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Cromatografía , Durapatita , Hidrólisis , Hidroxiapatitas , Peso Molecular , Factor 2 de Elongación Peptídica , Fosforilación , Fosfotreonina/metabolismo , Proteínas Quinasas/aislamiento & purificación , ARN , Conejos , Reticulocitos/análisis , SefarosaRESUMEN
The addition of 5 mM cAMP to a cell-free translation system from rabbit reticulocytes increases the rate of protein synthesis 3 5-fold. Lower concentrations of cAMP (0.005, 0.05 and 0.5 mM) have no effect on translation in this system. cAMP at all the concentrations tested stimulates the phosphorylation of the same pattern of polypeptides, while 5 mM cAMP additionally stimulates dephosphorylation of the 95 kDa polypeptide identified as elongation factor 2 (EF-2). Testing of the preparations of EF-2 with a different content of the phosphorylated form in poly(U)-directed poly(Phe) synthesis reveals that the EF-2 activity correlates with the fraction of non-phosphorylated EF-2. Thus cAMP-dependent activation of protein synthesis seems to be due to dephosphorylation of EF-2.
Asunto(s)
AMP Cíclico/farmacología , Factores de Elongación de Péptidos/metabolismo , Péptidos , Biosíntesis de Proteínas , Adenosina Trifosfato/metabolismo , Animales , Sistema Libre de Células , Punto Isoeléctrico , Cinética , Biosíntesis de Péptidos , Factor 2 de Elongación Peptídica , Fosforilación , Poli U/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Conejos , Reticulocitos/metabolismoRESUMEN
It has been shown recently that glyceraldehyde-3-phosphate dehydrogenase (GAPD) is one of the three major RNA-binding proteins of rabbit reticulocytes [Ryazanov, A. G. (1985) FEBS Lett. 192, 131-134]. It was suggested that, due to its RNA-binding capacity, GAPD can form loose dynamic complexes with polyribosomes. This communication reports that a considerable amount of GAPD activity can be found in the mono- and polyribosome fraction after sucrose gradient centrifugation of rabbit reticulocyte lysate. An increase of ionic strength, as well as the addition of exogenous RNA to the extract, result in the removal of GAPD from the complex with mono- and polyribosomes. It appears that GAPD forms the complex with polyribosomes due to the interaction with some exposed RNA regions of these structures. Although the interaction of GAPD with ribosomes is weak, it can be detected under physiological ionic conditions by the difference boundary sedimentation velocity technique. Association of GAPD with mono- and polyribosomes can be prevented by a low concentration (10 microM) of NADH, but not NAD+. A nitrocellulose filter binding assay also shows that NADH has a stronger inhibitory effect on the enzyme-RNA complex formation, as compared with NAD+. We propose that the RNA-mediated association of GAPD with mono- and polyribosomes can provide compartmentation of the energy-supplying system on these structures within the cell. This can maintain a high local concentration of ATP and GTP near the sites of protein synthesis.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Polirribosomas/metabolismo , Ribosomas/metabolismo , Animales , Cromatografía en Gel , Escherichia coli , Técnicas In Vitro , NAD/metabolismo , Concentración Osmolar , Cloruro de Potasio/farmacología , Unión Proteica , ARN Ribosómico 23S/metabolismo , ConejosRESUMEN
Incubation of a ribosome-free extract of rabbit reticulocytes or rat liver with [gamma-32P]ATP and Ca2+ results in incorporation of 32P predominantly into a single polypeptide of Mr approximately 100,000. This polypeptide is identified as elongation factor 2 (EF-2). Phosphorylation of EF-2 is strictly Ca2+-dependent and can be inhibited by the calmodulin antagonist trifluoperazine. It is suggested that the Ca2+/calmodulin-dependent phosphorylation of EF-2 is involved in regulation of protein biosynthesis.