RESUMEN
FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1) gene has been implicated in X-linked intellectual disability (XLID), but the molecular pathogenesis is unknown. We show that Ftsj1 is responsible for 2'-O-methylation of 11 species of cytosolic transfer RNAs (tRNAs) at the anticodon region, and these modifications are abolished in Ftsj1 knockout (KO) mice and XLID patient-derived cells. Loss of 2'-O-methylation in Ftsj1 KO mouse selectively reduced the steady-state level of tRNAPhe in the brain, resulting in a slow decoding at Phe codons. Ribosome profiling showed that translation efficiency is significantly reduced in a subset of genes that need to be efficiently translated to support synaptic organization and functions. Ftsj1 KO mice display immature synaptic morphology and aberrant synaptic plasticity, which are associated with anxiety-like and memory deficits. The data illuminate a fundamental role of tRNA modification in the brain through regulation of translation efficiency and provide mechanistic insights into FTSJ1-related XLID.
RESUMEN
The brain-specific tyrosine phosphatase, STEP (STriatal-Enriched protein tyrosine Phosphatase) is an important regulator of synaptic function. STEP normally opposes synaptic strengthening by increasing N-methyl D-aspartate glutamate receptor (NMDAR) internalization through dephosphorylation of GluN2B and inactivation of the kinases extracellular signal-regulated kinase 1/2 and Fyn. Here we show that STEP61 is elevated in the cortex in the Nrg1+/- knockout mouse model of schizophrenia (SZ). Genetic reduction or pharmacological inhibition of STEP prevents the loss of NMDARs from synaptic membranes and reverses behavioral deficits in Nrg1+/- mice. STEP61 protein is also increased in cortical lysates from the central nervous system-specific ErbB2/4 mouse model of SZ, as well as in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons and Ngn2-induced excitatory neurons, from two independent SZ patient cohorts. In these selected SZ models, increased STEP61 protein levels likely reflect reduced ubiquitination and degradation. These convergent findings from mouse and hiPSC SZ models provide evidence for STEP61 dysfunction in SZ.
Asunto(s)
Proteínas Tirosina Fosfatasas/fisiología , Esquizofrenia/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurregulina-1/genética , Neuronas/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/genética , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/genética , UbiquitinaciónRESUMEN
The actions of dopamine D1 family receptors (D1R) depend upon a signal transduction cascade that modulates the phosphorylation state of important effector proteins, such as glutamate receptors and ion channels. This is accomplished both through activation of protein kinase A (PKA) and the inhibition of protein phosphatase-1 (PP1). Inhibition of PP1 occurs through PKA-mediated phosphorylation of dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP-32) or the related protein inhibitor-1 (I-1), and the availability of DARPP-32 is essential to the functional outcome of D1R activation in the basal ganglia. While D1R activation is critical for prefrontal cortex (PFC) function, especially working memory, the functional role played by DARPP-32 or I-1 is less clear. In order to examine this more thoroughly, we have utilized immunoelectron microscopy to quantitatively determine the localization of DARPP-32 and I-1 in the neuropil of the rhesus monkey PFC. Both were distributed widely in the different components of the neuropil, but were enriched in dendritic shafts. I-1 label was more frequently identified in axon terminals than was DARPP-32, and DARPP-32 label was more frequently identified in glia than was I-1. We also quantified the extent to which these proteins were found in dendritic spines. DARPP-32 and I-1 were present in small subpopulations of dendritic spines, (4.4% and 7.7% and respectively), which were substantially smaller than observed for D1R in our previous studies (20%). Double-label experiments did not find evidence for colocalization of D1R and DARPP-32 or I-1 in spines or terminals. Thus, at the least, not all prefrontal spines which contain D1R also contain I-1 or DARPP-32, suggesting important differences in D1R signaling in the PFC compared to the striatum.
Asunto(s)
Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Corteza Prefrontal/metabolismo , Proteínas/metabolismo , Animales , Espinas Dendríticas/metabolismo , Macaca mulatta , Microscopía Inmunoelectrónica , Neurópilo/metabolismo , Corteza Prefrontal/ultraestructura , Terminales Presinápticos/metabolismo , Proteína Fosfatasa 1/metabolismo , Receptores de Dopamina D1/metabolismoRESUMEN
The reinforcing effect of cocaine is associated with increases in dopamine in the striatum. The phosphoprotein DARPP-32 (dopamine- and cAMP-regulated phosphoprotein) has been shown to mediate the intracellular events after activation of dopamine receptors. DARPP-32 is phosphorylated at multiple sites by different protein kinases, but little is known about the functional role of these different sites. Cocaine self-administration and striatal levels of dopamine after acute "binge" cocaine administration were measured in separate lines of mice with alanine mutations introduced into DARPP-32 at either Thr34 (protein kinase A site, Thr34A), Thr75, (cyclin-dependent kinase 5 site, Thr75A), Ser97 (kinase CK2 site, Ser97A), or Ser130 (kinase CK1 site, Ser130A). Acquisition of stable cocaine self-administration required significantly more time in Thr34A-/- mice. Both Thr34A- and Ser130A-DARPP-32 mutant mice self-administered more cocaine than their respective wild-type controls. Also, cocaine-induced increases of dopamine in dorsal striatum were attenuated in the Thr34A- and Ser130A-DARPP-32 phosphomutant mice compared with wild-type mice. Notably, levels of P-Thr34- and P-Ser130-DARPP-32 were reduced after self-administration of cocaine in wild-type mice. Thus, phosphorylation states of Thr34- and Ser130-DARPP-32 play important roles in modulating the reinforcing effects of cocaine.
Asunto(s)
Cocaína/administración & dosificación , Inhibidores de Captación de Dopamina/administración & dosificación , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Refuerzo en Psicología , Autoadministración , Serina/metabolismo , Treonina/metabolismo , Alanina/genética , Alanina/metabolismo , Análisis de Varianza , Animales , Conducta Animal , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microdiálisis/métodos , Fosforilación , Esquema de Refuerzo , Factores de TiempoRESUMEN
Cerebral ischaemia is associated with brain damage and inhibition of neuronal protein synthesis. A deficit in neuronal metabolism and altered excitatory amino acid release may both contribute to those phenomena. In the present study, we demonstrate that both NMDA and metabolic impairment by 2-deoxyglucose or inhibitors of mitochondrial respiration inhibit protein synthesis in cortical neurons through the phosphorylation of eukaryotic elongation factor (eEF-2), without any change in phosphorylation of initiation factor eIF-2alpha. eEF-2 kinase may be activated both by Ca(2+)-independent AMP kinase or by an increase in cytosolic Ca2+. Although NMDA decreases ATP levels in neurons, only the effects of 2-deoxyglucose on protein synthesis and phosphorylation of elongation factor eEF-2 were reversed by Na(+) pyruvate. Protein synthesis inhibition by 2-deoxyglucose was not as a result of a secondary release of glutamate from cortical neurons as it was not prevented by the NMDA receptor antagonist 5-methyl-10,11-dihydro-5H-dibenzo-(a,d)-cyclohepten-5,10-imine hydrogen maleate (MK 801), nor to an increase in cytosolic-free Ca2+. Conversely, 2-deoxyglucose likely activates eEF-2 kinase through a process involving phosphorylation by AMP kinase. In conclusion, we provide evidence that protein synthesis can be inhibited by NMDA and metabolic deprivation by two distinct mechanisms involving, respectively, Ca(2+)-dependent and Ca(2+)-independent eEF-2 phosphorylation.
Asunto(s)
Antimetabolitos/farmacología , Desoxiglucosa/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Factor 2 de Elongación Peptídica/metabolismo , Animales , Western Blotting/métodos , Calcio/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Células Cultivadas , Corteza Cerebral/citología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Ionóforos/farmacología , Leucina/metabolismo , Ratones , Modelos Biológicos , Neuronas/fisiología , Oligomicinas/farmacología , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Ácido Pirúvico/farmacocinética , Azida Sódica/farmacología , Serina-Treonina Quinasas TOR , Factores de Tiempo , Tritio/metabolismoRESUMEN
In a variety of species memory consolidation following different learning paradigms has been shown to be dependent on protein synthesis. However, it is not known whether modulation of protein synthesis is a critical component of the consolidation process, nor is the identity of any protein(s) subject to translational regulation, known. We report here that phosphorylation of eukaryotic elongation factor-2 (eEF2), an indicator for translational elongation attenuation, is correlated with input that produces taste memory consolidation in the relevant cortex of rat. The temporal pattern of eEF2 phosphorylation is similar to extra-cellular regulated kinase 2 (ERK2) activation and S6K1 phosphorylation, which are known to stimulate translation initiation. In addition, increased eEF2 phosphorylation and increased alphaCaMKII expression is detected in a synaptoneurosomal fraction made from taste cortex following memory consolidation. These results suggest that increased initiation rate together with decreased elongation rate, during memory consolidation, shift the rate-limiting step of protein synthesis, to produce a local switch-like effect in the expression of neuronal proteins.
Asunto(s)
Memoria/fisiología , Gusto/fisiología , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiología , Condicionamiento Operante/efectos de los fármacos , Citosol/enzimología , Citosol/metabolismo , Activación Enzimática/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiología , Masculino , N-Metilaspartato/farmacología , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/fisiología , Fosforilación , Ratas , Ratas Wistar , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/fisiología , Sinaptosomas/enzimología , Sinaptosomas/fisiologíaRESUMEN
Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1-NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Activación del Canal Iónico/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Humanos , Mutagénesis , Nucleótidos/metabolismoRESUMEN
Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. We identified a CaM-binding protein, designated regulator of calmodulin signaling (RCS). G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acted as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocked GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increased this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Animales , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neostriado/citología , Neostriado/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fosforilación , Receptor Muscarínico M1/metabolismo , Receptores de Dopamina D1/metabolismoRESUMEN
The persistent maintenance of long-term potentiation requires both messenger RNA and protein synthesis. While there is mounting evidence for an active role of protein synthesis in hippocampal long-term potentiation, the nature of mechanisms underlying its regulation has not yet been established. We used a previously described chemical long-term potentiation protocol [J Neurosci 19 (1999) 2500] to address the hypothesis that signaling mechanisms, involved in long-lasting long-term potentiation, directly regulate protein synthesis. Chemical long-term potentiation is an N-methyl-D-aspartate receptor-dependent form of plasticity, which relies on both synaptic activity, in the form of spontaneous bursting induced by high concentrations of K(+) and Ca(2+), and cyclic AMP/adenylyl cyclase signaling. We found that chemical long-term potentiation in CA1 of the mouse hippocampus lasts for at least 3 hours and requires both messenger RNA and protein synthesis. However, surprisingly de novo total protein synthesis was paradoxically decreased at 1 hour after long-term potentiation induction. Consistent with the decrease in total protein synthesis in potentiated CA1, phosphorylation of eukaryotic elongation factor 2 was increased and is likely responsible for inhibition of translation at the elongation step. Increased phosphorylation of eukaryotic elongation factor 2 was dependent on coincident cyclic AMP/adenylyl cyclase activation and synaptic activity and required N-methyl-D-aspartate receptor activation. Despite the inhibition in total protein synthesis, the level of the immediate early gene protein Arc (activity regulated cytoskeleton-associated protein) increased at 1 hour after chemical long-term potentiation induction. Taken together, the results suggest that regulation at the elongation step of protein synthesis contributes to persistent forms of long-term potentiation.
Asunto(s)
Adenilil Ciclasas/fisiología , Potenciación a Largo Plazo/fisiología , Biosíntesis de Proteínas/fisiología , Animales , Hipocampo/enzimología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Transmisión Sináptica/fisiologíaRESUMEN
Transient cerebral ischemia, which is accompanied by a sustained release of glutamate, strongly depresses protein synthesis. We have previously demonstrated in cortical neurons that a glutamate-induced increase in intracellular Ca(2+) is likely responsible for the blockade of the elongation step of protein synthesis. In this study, we provide evidence indicating that NMDA mobilizes a thapsigargin-sensitive pool of intracellular Ca(2+). Exposure of cortical neurons to NMDA, in the absence of external Ca(2+), produced a transient rise in intracellular Ca(2+) that was suppressed by pretreatment with thapsigargin. This rise in intracellular Ca(2+) did not result from an influx of Na(+) via reversal of the mitochondrial Na(+)/Ca(2+) exchanger since it persisted in a Na(+)-free medium or in the presence of CGP 37157, an inhibitor of the exchanger. Moreover, the NMDA-induced increase in intracellular Ca(2+) required the presence of D-serine, was blocked by D(-)-2-amino-5-phosphonopentanoic acid, but was not reduced in the presence of external Mg(2+). This unexpected non-ionotropic effect of NMDA was associated with an inhibition of protein synthesis that was also insensitive to the absence of external Ca(2+) or Na(+), or presence of Mg(2+). NMDA treatment resulted in an increase in the phosphorylation of eEF-2 in the absence or presence of external Ca(2+). The initiation step of protein synthesis was not blocked by NMDA since the phosphorylation of initiation factor eIF-2alpha subunit was not altered by NMDA treatment. In conclusion, we provide evidence indicating that NMDA can inhibit protein synthesis in cortical neurons through a process that involves the mobilization of intracellular Ca(2+) stores via a mechanism that is not linked to the ionic properties of NMDA receptors.
Asunto(s)
Corteza Cerebral/citología , Neuronas/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Tapsigargina/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Factor 2 Eucariótico de Iniciación/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Ratones , N-Metilaspartato/farmacología , Fosforilación , Biosíntesis de ProteínasRESUMEN
Regulation of gene expression by dopamine may play an important role in learning, reward, and addiction. Hyman and colleagues now report the characterization of ania-6, a novel cyclin that associates with RNA polymerase II and is induced by dopamine or cocaine in the neostriatum. Ania-6 may thus provide a link between dopamine and gene expression at the level of mRNA processing.
Asunto(s)
Ciclinas/genética , Dopamina/fisiología , ARN Mensajero/metabolismo , Empalme Alternativo , Animales , Cocaína/farmacología , Ciclinas/metabolismo , Dopamina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , ARN Polimerasa II/metabolismoRESUMEN
Synapsins are major neuronal phosphoproteins involved in regulation of neurotransmitter release. Synapsins are well established targets for multiple protein kinases within the nerve terminal, yet little is known about dephosphorylation processes involved in regulation of synapsin function. Here, we observed a reciprocal relationship in the phosphorylation-dephosphorylation of the established phosphorylation sites on synapsin I. We demonstrate that, in vitro, phosphorylation sites 1, 2, and 3 of synapsin I (P-site 1 phosphorylated by cAMP-dependent protein kinase; P-sites 2 and 3 phosphorylated by Ca(2+)-calmodulin-dependent protein kinase II) were excellent substrates for protein phosphatase 2A, whereas P-sites 4, 5, and 6 (phosphorylated by mitogen-activated protein kinase) were efficiently dephosphorylated only by Ca(2+)-calmodulin-dependent protein phosphatase 2B-calcineurin. In isolated nerve terminals, rapid changes in synapsin I phosphorylation were observed after Ca(2+) entry, namely, a Ca(2+)-dependent phosphorylation of P-sites 1, 2, and 3 and a Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6. Inhibition of calcineurin activity by cyclosporin A resulted in a complete block of Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6 and correlated with a prominent increase in ionomycin-evoked glutamate release. These two opposing, rapid, Ca(2+)-dependent processes may play a crucial role in the modulation of synaptic vesicle trafficking within the presynaptic terminal.
Asunto(s)
Calcio/metabolismo , Ácido Glutámico/metabolismo , Terminales Presinápticos/metabolismo , Sinapsinas/metabolismo , Sinaptosomas/metabolismo , 4-Aminopiridina/farmacología , Animales , Sitios de Unión/fisiología , Corteza Cerebral/química , Inhibidores Enzimáticos/farmacología , Cinética , Masculino , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Especificidad por Sustrato/fisiología , Sinapsinas/química , Sinaptosomas/químicaRESUMEN
Previously, eEF-2 phosphorylation has been identified as a reversible mechanism involved in the inhibition of the elongation phase of translation. In this study, an increased level of phosphorylation of eukaryotic elongation factor-2 (eEF-2) was observed in the brains and livers of hibernating ground squirrels. In brain and liver from hibernators, eEF-2 kinase activity was increased relative to that of active animals. The activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF-2, was also decreased in brain and liver from hibernators. This was associated with an increase in the level of inhibitor 2 of PP2A (I(2)(PP2A)), although there was an increase in the level of the catalytic subunit of PP2A (PP2A/C) in hibernating brains and livers. These results indicate that eEF-2 phosphorylation represents a specific and previously uncharacterized mechanism for inhibition of the elongation phase of protein synthesis during hibernation. Increased levels of eEF-2 phosphorylation in hibernators appear to be a component of the regulated shutdown of cellular functions that permits hibernating animals to tolerate severe reductions in cerebral blood flow and oxygen delivery capacity.
Asunto(s)
Encéfalo/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Hibernación/fisiología , Hígado/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Sciuridae/fisiología , Animales , Catálisis , Citosol/enzimología , Quinasa del Factor 2 de Elongación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Subunidades de ProteínaRESUMEN
Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca(2+) currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca(2+) channels.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Metoxihidroxifenilglicol/análogos & derivados , Neostriado/fisiología , Neuronas/fisiología , Proteínas Quinasas/metabolismo , Receptores de Glutamato Metabotrópico/fisiología , Animales , Canales de Calcio/fisiología , Caseína Quinasas , Quinasa 5 Dependiente de la Ciclina , Fosfoproteína 32 Regulada por Dopamina y AMPc , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Cinética , Masculino , Potenciales de la Membrana/fisiología , Metoxihidroxifenilglicol/farmacología , Ratones , Ratones Endogámicos C57BL , Neostriado/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Fosfoproteínas/metabolismo , Fosforilación , Fosfoserina , Fosfotreonina , Receptores de Glutamato Metabotrópico/efectos de los fármacosAsunto(s)
Calcio/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ciclo Celular/fisiología , División Celular/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cisteína Endopeptidasas/metabolismo , Quinasa del Factor 2 de Elongación , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Neuronas/metabolismo , Extensión de la Cadena Peptídica de Translación , Factor 2 de Elongación Peptídica/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal , Homología de Secuencia de Aminoácido , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
Mice were subjected to 60 min occlusion of the left middle cerebral artery (MCA) followed by 1-6 h of reperfusion. Tissue samples were taken from the MCA territory of both hemispheres to analyse ischaemia-induced changes in the phosphorylation of the initiation factor eIF-2alpha, the elongation factor eEF-2 and p70 S6 kinase by western blot analysis. Tissue sections from additional animals were taken to evaluate ischaemia-induced changes in global protein synthesis by autoradiography and changes in eIF-2alpha phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF-2alpha was slightly increased during ischaemia, it was markedly up-regulated after 1 h of reperfusion and it normalized after 6 h of recirculation despite ongoing suppression of protein synthesis. Similar changes in eIF-2alpha phosphorylation were induced in primary neuronal cell cultures by blocking of endoplasmic reticulum (ER) calcium pump, suggesting that disturbances of ER calcium homeostasis may play a role in ischaemia-induced changes in eIF-2alpha phosphorylation. Dephosphorylation of eIF-2alpha was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF-2alpha from phosphorylation, even in the presence of active eIF-2alpha kinase. Phosphorylation of eEF-2 rose moderately during ischaemia, but returned to control levels after 1 h of reperfusion and declined markedly below control levels after 3 and 6 h of recirculation. In contrast to the only short-lasting phosphorylation of eIF-2a and eEF-2, transient focal ischaemia induced a long-lasting dephosphorylation of p70 S6 kinase. The results suggest that blocking of elongation does not play a major role in suppression of protein synthesis induced by transient focal cerebral ischaemia. Investigating the factors involved in ischaemia-induced suppression of the initiation step of protein synthesis and identifying the underlying mechanisms may help to further elucidate those disturbances directly related to the pathological process triggered by transient cerebral ischaemia and leading to neuronal cell injury.
Asunto(s)
Corteza Cerebral/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Ataque Isquémico Transitorio/metabolismo , Neuronas/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/citología , Circulación Cerebrovascular , Inhibidores Enzimáticos/farmacología , Immunoblotting , Inmunohistoquímica , Flujometría por Láser-Doppler , Ratones , Arteria Cerebral Media/cirugía , Neuronas/efectos de los fármacos , Fosforilación , Biosíntesis de Proteínas , Ratas , Ratas Wistar , Tapsigargina/farmacologíaRESUMEN
Increased protein synthesis is the cardinal feature of cardiac hypertrophy. We have studied angiotensin II (ANG II)-dependent regulation of eukaryotic elongation factor-2 (eEF-2), an essential component of protein translation required for polypeptide elongation, in rat neonatal cardiac myocytes. eEF2 is fully active in its dephosphorylated state and is inhibited following phosphorylation by eEF2 kinase. ANG II treatment (10(-10) - 10(-7) M) for 30 min produced an AT(1) receptor-specific and concentration- and time-dependent reduction in the phosphorylation of eEF-2. Protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin, but not the PP2B inhibitor FK506, attenuated ANG II-dependent dephosphorylation of eEF-2. ANG II activated mitogen-activated protein kinase, (MAPK) within 10 min of treatment, and blockade of MAPK activation with PD-98059 (1--20 nM) inhibited eEF-2 dephosphorylation. The effect of ANG II on eEF-2 dephosphorylation was also blocked by LY-29004 (1-20 nM), suggesting a role for phosphoinositide 3-kinase, but the mammalian target rapamycin inhibitor rapamycin (10--100 nM) had no effect. Together these results suggest that the ANG II-dependent increase in protein synthesis includes activation of eEF-2 via dephosphorylation by PP2A by a process that involves both PI3K and MAPK.
Asunto(s)
Angiotensina II/farmacología , Miocardio/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Angiotensina II/fisiología , Animales , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Morfolinas/farmacología , Miocardio/citología , Factor 2 de Elongación Peptídica/biosíntesis , Fosfoproteínas Fosfatasas/fisiología , Fosforilación/efectos de los fármacos , Biosíntesis de Proteínas , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/fisiología , Transducción de Señal/fisiología , Sirolimus/farmacologíaRESUMEN
Transient receptor potential (TRP) channels modulate calcium levels in eukaryotic cells in response to external signals. A novel transient receptor potential channel has the ability to phosphorylate itself and other proteins on serine and threonine residues. The catalytic domain of this channel kinase has no detectable sequence similarity to classical eukaryotic protein kinases and is essential for channel function. The structure of the kinase domain, reported here, reveals unexpected similarity to eukaryotic protein kinases in the catalytic core as well as to metabolic enzymes with ATP-grasp domains. The inclusion of the channel kinase catalytic domain within the eukaryotic protein kinase superfamily indicates a significantly wider distribution for this group of signaling proteins than suggested previously by sequence comparisons alone.
Asunto(s)
Canales de Calcio/química , Proteínas Quinasas Dependientes de AMP Cíclico/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Evolución Molecular , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Fosfotransferasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Canales Catiónicos TRPC , Zinc/metabolismoRESUMEN
Studies on thymic T cell development have usually concentrated on cell surface molecules. However, intracellular proteins expressed only in thymocytes have never been described. Here we report the discovery of a novel thymocyte-specific protein, named TARPP, which represents a high molecular mass ( approximately 100 kDa) variant of the previously identified protein ARPP-21 ( approximately 21 kDa). TARPP is a cytosolic protein that is expressed at high levels in immature thymocytes. It appears concomitant with the commitment to T cell lineage, and its expression is switched off as a consequence of TCR engagement during positive selection. Such an expression pattern, correlating with the rearrangement of the TCR genes and thymocyte education, suggests a role for TARPP during this important phase of thymocyte development.