RESUMEN
Non-homologous end joining (NHEJ) is a highly conserved mechanism of DNA double-stranded break (DSB) repair. Here we utilize a computational protein-protein interaction method to identify human PRKACB as a potential candidate interacting with NHEJ proteins. We show that the deletion of its yeast homolog, TPK1 that codes for the protein kinase A catalytic subunit reduces the efficiency of NHEJ repair of breaks with overhangs and blunt ends in plasmid-based repair assays. Additionally, tpk1Δ mutants showed defects in the repair of chromosomal breaks induced by HO-site specific endonuclease. Our double deletion mutant analyses suggest that TPK1 and YKU80, a key player in NHEJ could function in parallel pathways. Altogether, here we report a novel involvement for TPK1 in NHEJ.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN por Unión de Extremidades/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Roturas del ADN de Doble Cadena , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Genes Fúngicos , Genes Sintéticos , Estudios de Asociación Genética , Humanos , Mapas de Interacción de ProteínasRESUMEN
The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, proper chromosome segregation, and stress responses in Schizosaccharomyces pombe. We demonstrated that both the cAMP/PKA pathway and glucose limitation play roles in appropriate spindle formation. Overexpression of Mal3 (1-308), an EB1 family protein, caused growth defects, increased 4C DNA content, and induced monopolar spindle formation. Overproduction of a high-affinity microtubule binding mutant (Q89R) and a recombinant protein possessing the CH and EB1 domains (1-241) both resulted in more severe phenotypes than Mal3 (1-308). Loss of functional Pka1 and glucose limitation rescued the phenotypes of Mal3-overexpressing cells, whereas deletion of Tor1 or Ssp2 did not. Growth defects and monopolar spindle formation in a kinesin-5 mutant, cut7-446, was partially rescued by pka1 deletion or glucose limitation. These findings suggest that Pka1 and glucose limitation regulate proper spindle formation in Mal3-overexpressing cells and the cut7-446 mutant.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Regulación Fúngica de la Expresión Génica , Glucosa/deficiencia , Cinesinas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Huso Acromático/metabolismo , Sustitución de Aminoácidos , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , ADN de Hongos/genética , ADN de Hongos/metabolismo , Eliminación de Gen , Glucosa/farmacología , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Mutación , Fenotipo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/ultraestructuraRESUMEN
The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, meiosis, and stress responses in Schizosaccharomyces pombe. We demonstrated that Pka1 is responsible for calcium tolerance. Loss of functional components of the PKA pathway such as Git3, Gpa2, Cyr1, and Pka1 yields a CaCl2-sensitive phenotype, while loss of Cgs1, a regulatory subunit of PKA, results in CaCl2 tolerance. Cytoplasmic distribution of Cgs1 and Pka1 is increased by the addition of CaCl2, suggesting that CaCl2 induces dissociation of Cgs1 and Pka1. The expression of Prz1, a transcriptional regulator in calcium homeostasis, is elevated in a pka1∆ strain and in a wild type strain under glucose-limited conditions. Accordingly, higher expression of Prz1 in the wild type strain results in a CaCl2-sensitive phenotype. These findings suggest that Pka1 is essential for tolerance to exogenous CaCl2, probably because the expression level of Prz1 needs to be properly regulated by Pka1.
Asunto(s)
Cloruro de Calcio/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/metabolismo , Factores de Transcripción/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Activación Enzimática/efectos de los fármacos , Eliminación de Gen , Homeostasis/efectos de los fármacos , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Factores de Transcripción/genéticaRESUMEN
The Hedgehog (Hh) signaling pathway has numerous roles in the control of cell proliferation, tissue patterning and stem cell maintenance. In spite of intensive study, the mechanisms of Hh signal transduction are not completely understood. Here I review published data and present a novel model of vertebrate Hh signaling suggesting that Smoothened (Smo) functions as a G-protein-coupled receptor in cilia. This is the first model to propose molecular mechanisms for the major steps of Hh signaling, including inhibition of Smo by Patched, Smo activation, and signal transduction from active Smo to Gli transcription factors. It also suggests a novel role for the negative pathway regulators Sufu and PKA in these processes.
Asunto(s)
Proteínas Hedgehog/metabolismo , Transducción de Señal , Cilios/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Mutación , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Factores de Transcripción/metabolismo , Proteína con Dedos de Zinc GLI1RESUMEN
A reduced sinoatrial node (SAN) functional reserve underlies the age-associated decline in heart rate acceleration in response to stress. SAN cell function involves an oscillatory coupled-clock system: the sarcoplasmic reticulum (SR), a Ca(2+) clock, and the electrogenic-sarcolemmal membrane clock. Ca(2+)-activated-calmodulin-adenylyl cyclase/CaMKII-cAMP/PKA-Ca(2+) signaling regulated by phosphodiesterase activity drives SAN cells automaticity. SR-generated local calcium releases (LCRs) activate Na(+)/Ca(2+) exchanger in the membrane clock, which initiates the action potential (AP). We hypothesize that SAN cell dysfunctions accumulate with age. We found a reduction in single SAN cell AP firing in aged (20-24 mo) vs. adult (3-4 mo) mice. The sensitivity of the SAN beating rate responses to both muscarinic and adrenergic receptor activation becomes decreased in advanced age. Additionally, age-associated coincident dysfunctions occur stemming from compromised clock functions, including a reduced SR Ca(2+) load and a reduced size, number, and duration of spontaneous LCRs. Moreover, the sensitivity of SAN beating rate to a cAMP stress induced by phosphodiesterase inhibitor is reduced, as are the LCR size, amplitude, and number in SAN cells from aged vs. adult mice. These functional changes coincide with decreased expression of crucial SR Ca(2+)-cycling proteins, including SR Ca(2+)-ATPase pump, ryanodine receptors, and Na(+)/Ca(2+) exchanger. Thus a deterioration in intrinsic Ca(2+) clock kinetics in aged SAN cells, due to deficits in intrinsic SR Ca(2+) cycling and its response to a cAMP-dependent pathway activation, is involved in the age-associated reduction in intrinsic resting AP firing rate, and in the reduction in the acceleration of heart rate during exercise.
Asunto(s)
Envejecimiento/fisiología , Calcio/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , AMP Cíclico/deficiencia , Transducción de Señal/fisiología , Nodo Sinoatrial/fisiopatología , Potenciales de Acción/fisiología , Animales , Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/deficiencia , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Frecuencia Cardíaca/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Retículo Sarcoplasmático/fisiología , Estrés Fisiológico/fisiologíaRESUMEN
BACKGROUND: The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4. RESULTS: We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA. CONCLUSIONS: In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in combination, in which PKA is often involved. Redundant signalling pathways confer a greater robustness to the response to glucose, while cooperative pathways provide a greater flexibility.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Glucosa/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Silenciador del Gen/efectos de los fármacos , Glucosa/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Activación Transcripcional/efectos de los fármacosRESUMEN
Cyclic-AMP stimulation of GTP cyclohydrolase I (GCH1) gene transcription was investigated in PC12 cells, the protein kinase A-deficient PC12 cell line 126-1B2 and C6 cells using transient transfection assays of proximal promoter reporter constructs and wild type or dominant negative proteins, chromatin immunoprecipitation and real-time quantitative PCR. These studies show that protein kinase A is necessary and sufficient for cAMP-dependent transcription conferred by both the cAMP regulatory element and the adjacent CCAAT-box. In intact cells these cis-elements were shown to bind cAMP response element binding protein, CCAAT-enhancer binding protein beta and nuclear factor-Y, with each protein controlling a different aspect of the cAMP response. Cyclic-AMP acting through protein kinase A stimulated promoter recruitment of CCAAT-enhancer binding protein beta, nuclear factor-Y and RNA polymerase II while depleting the promoter of cyclic-AMP response element binding protein. Stimulation of transcription by cAMP was not associated with increased acetylation of histones H3 and H4 at proximal promoter nucleosomes, indicating that histone acetyltransferases are not involved in this response. Nonetheless, pharmacological inhibition of histone deacetylase activity did increase histone H4 acetylation and the recruitment of RNA polymerase II, indicating that histone acetyltransferases are normally associated with the proximal promoter. Only in C6 cells, however, did inhibition of histone deacetylases stimulate transcription and synergize with cAMP. These experiments provide the first glimpse of the GCH1 gene promoter functioning within intact cells and supply evidence for the involvement of histone acetyltransferase-containing complexes in GCH1 gene transcription.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , GTP Ciclohidrolasa/genética , Histonas/metabolismo , Regiones Promotoras Genéticas/fisiología , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Acetilación/efectos de los fármacos , Animales , Inmunoprecipitación de Cromatina/métodos , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Histona Desacetilasas/metabolismo , Células PC12 , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transfección/métodosRESUMEN
The human PRKAR1A gene encodes the regulatory subunit 1-alpha (RIalpha) of the cAMP-dependent protein kinase A (PKA) holoenzyme. Regulation of the catalytic activity of PKA is the only well-studied function of RIalpha. Inactivating PRKAR1A mutations cause primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC), an inherited syndrome associated with abnormal skin pigmentation and multiple neoplasias, including PPNAD. Histochemistry of tissues from CNC patients is indicative of autophagic deficiency and this led us to investigate the relationship between RIalpha and mammalian autophagy. We found that fluorescently tagged RIalpha associates with late endosomes and autophagosomes in cultured cells. The number of autophagosomes in prkar1a-/- mouse embryonic fibroblasts (MEFs) was reduced compared with wild-type MEFs. RIalpha co-immunoprecipitated with mTOR kinase, a major regulator of autophagy. Phosphorylated-mTOR levels and mTOR activity were dramatically increased in prkar1a-/- mouse cells, and in HEK 293 cells with RIalpha levels reduced by siRNA. Finally, phosphorylated-mTOR levels and mTOR activity were increased in CNC cells and in PPNAD tissues. These data suggest that RIalpha deficiency decreases autophagy by the activation of mTOR, providing a molecular basis to autophagic deficiency in PPNAD.
Asunto(s)
Autofagia/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Enfermedades de la Corteza Suprarrenal/genética , Enfermedades de la Corteza Suprarrenal/metabolismo , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/genética , ADN Complementario/genética , Fibroblastos/metabolismo , Células HeLa , Humanos , Ratones , Ratones Noqueados , Mutación , Fagosomas/metabolismo , Fosforilación , Proteínas/genética , ARN Interferente Pequeño/genética , Serina-Treonina Quinasas TORRESUMEN
The mammalian Golgi complex consists of stacks of cisternae linked laterally into a continuous perinuclear ribbon structure. Protein kinase A is stably associated with the Golgi complex during interphase. To analyze its role in Golgi structural maintenance cells were depleted of protein kinase A regulatory subunits using small interfering RNAs. Under these conditions, the catalytic subunits redistributed to the cytosol and the entire Golgi complex underwent disassembly into multiple juxtanuclear fragments. A similar effect took place following pharmacological inhibition or redistribution of the complete holoenzyme to the cytosol. Golgi fragments maintained their polarization and competence for anterograde protein trafficking. By electron microscopy, they were identified as whorl-like structures composed of concentrically arrayed cisternae. To test a possible role of protein kinase A in Golgi biogenesis we analyzed its involvement during Golgi reassembly from the endoplasmic reticulum. In cells incubated with protein kinase A inhibitors, Golgi reconstruction was arrested at a late step of the reassembly process. This is consistent with the stage of enzyme recruitment from cytosol to emerging Golgi membranes during the reassembly process. We conclude that protein kinase A activity plays a relevant role in the assembly and maintenance of a continuous Golgi ribbon from separated membrane stacks.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Aparato de Golgi/enzimología , Aparato de Golgi/fisiología , Animales , Brefeldino A/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Galactosiltransferasas/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Cinética , Ratones , Unión Proteica/efectos de los fármacosRESUMEN
This Teaching Resource provides an introduction to the use of cultured cells as an experimental approach in undergraduate laboratory research and the study of neuronal differentiation in PC12 cells. In addition, a thought experiment with answers is provided that can be used to assess student understanding of (i) the scientific method, (ii) signaling processes involved in cellular differentiation, and (iii) the use of pharmacological agents to manipulate a cell culture system.
Asunto(s)
Biología Celular/educación , Células Cultivadas , Neuronas/citología , Células PC12/citología , Transducción de Señal , Materiales de Enseñanza , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Genes ras , Humanos , Células PC12/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/deficiencia , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Ratas , Proyectos de Investigación , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Accumulating evidence indicates that adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is involved in the neurobiological responses to ethanol. Previous reports indicate that mice lacking the RIIbeta subunit of PKA (RIIbeta(-/-)) voluntarily consume more ethanol than wild-type controls (RIIbeta(+/+)) using 2-bottle testing procedures. Although such procedures primarily measure consummatory behavior, operant self-administration procedures allow analysis of consummatory as well as appetitive or "ethanol-seeking" behavior (i.e., lever pressing is required to gain access to the ethanol solution). Therefore, we determined whether the high ethanol consumption characteristic of RIIbeta(-/-) mice would be complemented by increased appetitive ethanol-seeking behavior in an operant paradigm. METHODS: RIIbeta(-/-) (n=8) and RIIbeta(+/+) (n=8) mice were initially sucrose-faded until they were lever responding for nonsweetened ethanol (10, 14, and 18%). Following the self-administration testing, RIIbeta(+/+) and RIIbeta(-/-) mice were given access to 2 bottles, one containing water and the other ethanol to replicate the voluntary ethanol drinking data previously from our laboratory. Finally, immediately after voluntary consumption all mice were again tested for self-administration of 10% ethanol. Alterations in the reinforcement schedule were also explored as RIIbeta(+/+) and RIIbeta(-/-) mice were tested for self-administration of 10% ethanol at FR-3 and FR-5 schedules. RESULTS: The RIIbeta(-/-) mice displayed lower operant responding for ethanol and food reinforcement compared with RIIbeta(+/+) controls. However, this effect was driven by a significant increase in lever responses made by female RIIbeta(+/+) mice. When the excessive lever responses of the female RIIbeta(+/+) mice are accounted for, the RIIbeta(-/-) mice show ethanol lever responses comparable to controls. Following operant self-administration testing, RIIbeta(-/-) mice of both sexes consumed more ethanol solution compared with RIIbeta(+/+) mice during 2-bottle testing. CONCLUSIONS: Increased ingestion of ethanol by RIIbeta(-/-) mice is likely the result of altered PKA activity within neuronal pathways that control ethanol-consummatory behaviors. Conversely, the RIIbeta subunit of PKA appears not to play a critical role in neuronal pathways that regulate appetitive behaviors directed at obtaining ethanol. Finally, increased operant self-administration of food and ethanol by female wild-type mice was absent in female RIIbeta(-/-) mice, suggesting that normal PKA signaling may be part of a general, and sex-dependent, mechanism involved with reinforcement-seeking behavior.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Etanol/administración & dosificación , Consumo de Bebidas Alcohólicas , Animales , Conducta Apetitiva , Conducta Consumatoria , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Ingestión de Líquidos , Femenino , Alimentos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Autoadministración , Caracteres Sexuales , Soluciones , Sacarosa/administración & dosificaciónRESUMEN
RATIONALE: Mutant mice lacking the RIIbeta subunit of protein kinase A (regulatory subunit II beta(-/-)) show increased ethanol preference. Recent evidence suggests a relationship between heightened ethanol preference and susceptibility to ethanol-induced locomotor sensitization. It is currently unknown if protein kinase A signaling modulates the stimulant effects and/or behavioral sensitization caused by ethanol administration. To address this question, we examined the effects of repeated ethanol administration on locomotor activity RIIbeta(-/-) and littermate wild-type (RIIbeta(+/+)) mice on multiple genetic backgrounds. METHODS: Over three consecutive days, mice were given single i.p. saline injections and immediately placed in a locomotor activity apparatus to establish a composite baseline for locomotor activity. Next, mice maintained on a hybrid 129/SvEvxC57BL/6J or pure C57BL/6J genetic background were given 10 i.p. ethanol injections before being placed in the activity apparatus. Each ethanol injection was separated by 3-4 days. To determine if changes in behavior were specific to ethanol injection, naïve mice were tested following repeated daily saline injections. The effects of ethanol injection on locomotor behavior were also assessed using an alternate paradigm in which mice were given repeated ethanol injections in their home cage environment. RESULTS: Relative to RIIbeta(+/+) mice, RIIbeta(-/-) mice, regardless of genetic background, consistently showed significantly greater ethanol-induced locomotor activation. RIIbeta(-/-) mice also showed increased sensitivity to ethanol-induced locomotor sensitization resulting from repeated administration, an effect that was dependent on genetic background and testing paradigm. Increased locomotor activity by RIIbeta(-/-) mice was specific to ethanol injections, and was not related to altered blood ethanol levels. CONCLUSIONS: These data provide novel evidence implicating an influence of protein kinase A signaling on ethanol-induced locomotor activity and behavioral sensitization. The observation that RIIbeta(-/-) mice are more sensitive to the effects of repeated ethanol administration suggests that normal protein kinase A signaling limits, or is protective against, the stimulant effects of ethanol and the plastic alterations that underlie behavioral sensitization.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Etanol/farmacología , Actividad Motora/efectos de los fármacos , Análisis de Varianza , Animales , Conducta Animal , Depresores del Sistema Nervioso Central/sangre , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Esquema de Medicación , Etanol/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Factores de TiempoRESUMEN
Previous studies have shown that inhibition of L-type Ca(2+) current (I(Ca)) by cytosolic free Mg(2+) concentration ([Mg(2+)](i)) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I(Ca), rat cardiac myocytes and tsA201 cells expressing L-type Ca(2+) channels were whole cell voltage-clamped with patch pipettes in which [Mg(2+)] ([Mg(2+)](p)) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca(2+) channels (alpha(1C)/beta(2A)/alpha(2)delta), increasing [Mg(2+)](p) from 0.2 mM to 1.8 mM decreased peak I(Ca) by 76 +/- 4.5% (n = 7). Mg(2+)-dependent modulation of I(Ca) was also observed in cells loaded with ATP-gamma-S. With 0.2 mM [Mg(2+)](p), manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP(2A)) produced large changes in I(Ca) amplitude; however, with 1.8 mM [Mg(2+)](p), these same manipulations had no significant effect on I(Ca). With mutant channels lacking principal PKA phosphorylation sites (alpha(1C/S1928A)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) had only small effects on I(Ca). However, when channel open probability was increased by alpha(1C)-subunit truncation (alpha(1CDelta1905)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) greatly reduced peak I(Ca). Correspondingly, in myocytes voltage-clamped with pipette PP(2A) to minimize channel phosphorylation, increasing [Mg(2+)](p) produced a much larger reduction in I(Ca) when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg(2+) modulates the extent to which channel phosphorylation regulates I(Ca). This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Citosol/metabolismo , Cloruro de Magnesio/química , Animales , Bloqueadores de los Canales de Calcio/química , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Yoduros/química , Yoduros/metabolismo , Activación del Canal Iónico/fisiología , Cloruro de Magnesio/metabolismo , Compuestos de Magnesio/química , Compuestos de Magnesio/metabolismo , Masculino , Miocitos Cardíacos/metabolismo , Fosfatos/metabolismo , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIalpha regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIalpha regulatory subunit of PKA (-/-) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and -/- mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the -/- mice are similar to WT controls. In vitro, splenocytes from -/- mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8(+) IFN-gamma producing effector cells were stimulated upon infection of WT or -/- mice with Listeria monocytogenes. These data represent the first study of the role of RIIalpha in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIalpha -/- mice, suggesting either that RIIalpha is not required for normal immune function or that other proteins are able to compensate for RIIalpha function.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Bazo/enzimología , Bazo/inmunología , Timo/enzimología , Timo/inmunología , Proteínas de Anclaje a la Quinasa A , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Western Blotting , Antígenos CD28/inmunología , Complejo CD3/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sueros Inmunes/farmacología , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Listeriosis/enzimología , Listeriosis/genética , Listeriosis/inmunología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Bazo/citología , Células TH1/citología , Células TH1/enzimología , Células TH1/inmunología , Células Th2/citología , Células Th2/enzimología , Células Th2/inmunología , Timo/citologíaRESUMEN
Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1-125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2(+/-) mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2(+/-) mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2(+/-) animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2(+/-) mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2(+/-) animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.
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Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Encefalomielitis Autoinmune Experimental/enzimología , Esclerosis Múltiple/enzimología , Receptores Acoplados a Proteínas G/deficiencia , Animales , Estudios de Casos y Controles , Movimiento Celular/inmunología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Quinasa 2 del Receptor Acoplado a Proteína-G , Quinasa 3 del Receptor Acoplado a Proteína-G , Humanos , Inflamación/inmunología , Leucocitos/enzimología , Leucocitos/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Noqueados , Esclerosis Múltiple/etiología , Esclerosis Múltiple/inmunología , Receptores Acoplados a Proteínas G/fisiología , Recurrencia , Linfocitos T/inmunología , Quinasas de Receptores Adrenérgicos betaAsunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Desarrollo de Músculos , Transducción de Señal , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Unión al ADN/metabolismo , Ratones , Proteínas Musculares/metabolismo , Músculos/citología , Músculos/embriología , Músculos/metabolismo , Factor 5 Regulador Miogénico , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Receptores Acoplados a Proteínas G/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas WntRESUMEN
Select members of the Wnt family of secreted glycoproteins have been implicated in inducing the myogenic determinant genes Pax3, MyoD and Myf5 during mammalian embryogenesis, but the mechanism of induction has not been defined. We describe an unexpected role for protein kinase A (PKA) signalling via CREB in this induction. Using a combination of in vitro explant assays, mutant analysis and gene delivery into mouse embryos cultured ex vivo, we demonstrate that adenylyl cyclase signalling via PKA and its target transcription factor CREB are required for Wnt-directed myogenic gene expression. Wnt proteins can also stimulate CREB-mediated transcription, providing evidence for a Wnt signalling pathway involving PKA and CREB. Our findings raise the possibility that PKA/CREB signalling may also contribute to other Wnt-regulated processes in embryonic patterning, stem cell renewal and cancer.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Desarrollo de Músculos , Transducción de Señal , Adenilil Ciclasas/metabolismo , Animales , Línea Celular , Técnicas de Cocultivo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Unión al ADN/metabolismo , Técnicas de Cultivo de Embriones , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Proteínas Musculares/metabolismo , Músculos/embriología , Músculos/metabolismo , Mutación/genética , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , ARN Mensajero/genética , ARN Mensajero/metabolismo , Somitos/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas WntRESUMEN
BACKGROUND: Genetic and pharmacological evidence suggests that the cyclic adenosine monophosphate-dependent protein kinase A pathway modulates neurobiological responses to ethanol. Mutant mice lacking the RIIbeta subunit of protein kinase A (RIIbeta(-/-)) are resistant to ethanol-induced sedation and drink significantly more ethanol than littermate wild-type mice (RIIbeta(+/+)). We determined whether high ethanol intake by the RIIbeta(-/-) mice on alternate genetic backgrounds is reliably predicted by high basal levels of anxiety or resistance to the sedative effects of ethanol. METHODS: Two-bottle choice procedures and a battery of behavioral tests (elevated plus maze, open-field activity, and zero maze) were used to assess voluntary ethanol consumption and basal levels of anxiety in RIIbeta(-/-) and RIIbeta(+/+) mice on either a C57BL/6J or a 129/SvEv x C57BL/6J genetic background. Additionally, ethanol-induced sedation and blood ethanol levels were determined in RIIbeta(-/-) and RIIbeta(+/+) mice after intraperitoneal injection of ethanol (3.8 g/kg). RESULTS: RIIbeta(-/-) mice on both genetic backgrounds consumed more ethanol and had a greater preference for ethanol relative to RIIbeta(+/+) mice. However, RIIbeta(-/-) mice showed reduced basal levels of anxiety when maintained on the C57BL/6J background but showed increased anxiety when maintained on the 129/SvEv x C57BL/6J background. Consistent with prior research, RIIbeta(-/-) mice were resistant to the sedative effects of ethanol, regardless of the genetic background. Finally, RIIbeta(-/-) and RIIbeta(+/+) mice showed similar blood ethanol levels. CONCLUSIONS: These results indicate that high ethanol consumption is associated with resistance to the sedative effects of ethanol but that basal levels of anxiety, as well as ethanol metabolism, do not reliably predict high ethanol drinking by RIIbeta(-/-) mice.
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Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/metabolismo , Ansiedad/enzimología , Ansiedad/genética , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Etanol/farmacología , Animales , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Femenino , Hipnóticos y Sedantes/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Valor Predictivo de las Pruebas , Reflejo/efectos de los fármacos , Reflejo/fisiología , Sueño/efectos de los fármacos , Sueño/fisiologíaAsunto(s)
Anemia Hemolítica Congénita no Esferocítica/genética , Codón sin Sentido , Proteínas Quinasas Dependientes de AMP Cíclico/deficiencia , Anemia Hemolítica Congénita no Esferocítica/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Femenino , Humanos , Recién Nacido , Masculino , LinajeRESUMEN
An unusual cAMP signaling system mediates many of the events that prepare spermatozoa to meet the egg. Its components include the atypical, bicarbonate-stimulated, sperm adenylyl cyclase and a cAMP-dependent protein kinase (PKA) with the unique catalytic subunit termed Calpha(2) or C(s). We generated mice that lack Calpha(2) to determine its importance in the events downstream of cAMP production. Male Calpha(2) null mice produce normal numbers of sperm that swim spontaneously in vitro. Thus, Calpha(2) has no required role in formation of a functional flagellum or the initiation of motility. In contrast, we find that Calpha(2) is required for bicarbonate to speed the flagellar beat and facilitate Ca(2+) entry channels. In addition, Calpha(2) is needed for the protein tyrosine phosphorylation that occurs late in the sequence of sperm maturation and for a negative feedback control of cAMP production, revealed here. Consistent with these specific defects in several important sperm functions, Calpha(2) null males are infertile despite normal mating behavior. These results define several crucial roles of PKA in sperm cell biology, bringing together both known and unique PKA-mediated events that are necessary for male fertility.