RESUMEN
The RIIß subunit of cAMP-dependent protein kinase A (PKA) is expressed in the brain and adipose tissue. RIIß-knockout mice show leanness and increased UCP1 in brown adipose tissue. The authors have previously reported that RIIß reexpression in hypothalamic GABAergic neurons rescues the leanness. However, whether white adipose tissue (WAT) browning contributes to the leanness and whether RIIß-PKA in these neurons governs WAT browning are unknown. Here, this work reports that RIIß-KO mice exhibit a robust WAT browning. RIIß reexpression in dorsal median hypothalamic GABAergic neurons (DMH GABAergic neurons) abrogates WAT browning. Single-cell sequencing, transcriptome sequencing, and electrophysiological studies show increased GABAergic activity in DMH GABAergic neurons of RIIß-KO mice. Activation of DMH GABAergic neurons or inhibition of PKA in these neurons elicits WAT browning and thus lowers body weight. These findings reveal that RIIß-PKA in DMH GABAergic neurons regulates WAT browning. Targeting RIIß-PKA in DMH GABAergic neurons may offer a clinically useful way to promote WAT browning for treating obesity and other metabolic disorders.
Asunto(s)
Tejido Adiposo Pardo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Hipotálamo , Animales , Ratones , Tejido Adiposo Pardo/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuronas GABAérgicas/metabolismo , Hipotálamo/metabolismo , Obesidad/metabolismo , Delgadez/metabolismoRESUMEN
Temporal lobe epilepsy (TLE) is one of the most common and intractable neurological disorders in adults. Dysfunctional PKA signaling is causally linked to the TLE. However, the mechanism underlying PKA involves in epileptogenesis is still poorly understood. In the present study, we found the autophosphorylation level at serine 114 site (serine 112 site in mice) of PKA-RIIß subunit was robustly decreased in the epileptic foci obtained from both surgical specimens of TLE patients and seizure model mice. The p-RIIß level was negatively correlated with the activities of PKA. Notably, by using a P-site mutant that cannot be autophosphorylated and thus results in the released catalytic subunit to exert persistent phosphorylation, an increase in PKA activities through transduction with AAV-RIIß-S112A in hippocampal DG granule cells decreased mIPSC frequency but not mEPSC, enhanced neuronal intrinsic excitability and seizure susceptibility. In contrast, a reduction of PKA activities by RIIß knockout led to an increased mIPSC frequency, a reduction in neuronal excitability, and mice less prone to experimental seizure onset. Collectively, our data demonstrated that the autophosphorylation of RIIß subunit plays a critical role in controlling neuronal and network excitabilities by regulating the activities of PKA, providing a potential therapeutic target for TLE.
Asunto(s)
Ondas Encefálicas , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Epilepsia del Lóbulo Temporal/enzimología , Hipocampo/enzimología , Adulto , Animales , Estudios de Casos y Controles , Preescolar , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/fisiopatología , Epilepsia del Lóbulo Temporal/prevención & control , Femenino , Hipocampo/fisiopatología , Humanos , Potenciales Postsinápticos Inhibidores , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , FosforilaciónRESUMEN
When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIß, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIß:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIß2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIß holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIß holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Carcinoma Hepatocelular/metabolismo , Microscopía por Crioelectrón/métodos , AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/ultraestructura , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/ultraestructura , Holoenzimas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Simulación de Dinámica Molecular , Unión Proteica , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/genética , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodosRESUMEN
OBJECTIVES: Reprogramming of cellular metabolism is profoundly implicated in tumorigenesis and can be exploited to cancer treatment. Cancer cells are known for their propensity to use glucose-dependent glycolytic pathway instead of mitochondrial oxidative phosphorylation for energy generation even in the presence of oxygen, a phenomenon known as Warburg effect. The type II beta regulatory subunit of protein kinase A (PKA), PRKAR2B, is highly expressed in castration-resistant prostate cancer (CRPC) and contributes to tumour growth and metastasis. However, whether PRKAR2B regulates glucose metabolism in prostate cancer remains largely unknown. MATERIALS AND METHODS: Loss-of-function and gain-of-function studies were used to investigate the regulatory role of PRKAR2B in aerobic glycolysis. Real-time qPCR, Western blotting, luciferase reporter assay and chromatin immunoprecipitation were employed to determine the underlying mechanisms. RESULTS: PRKAR2B was sufficient to enhance the Warburg effect as demonstrated by glucose consumption, lactate production and extracellular acidification rate. Mechanistically, loss-of-function and gain-of-function studies showed that PRKAR2B was critically involved in the tumour growth of prostate cancer. PRKAR2B was able to increase the expression level of hypoxia-inducible factor 1α (HIF-1α), which is a key mediator of the Warburg effect. Moreover, we uncovered that HIF-1α is a key transcription factor responsible for inducing PRKAR2B expression in prostate cancer. Importantly, inhibition of glycolysis by the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) or replacement of glucose in the culture medium with galactose (which has a much lower rate than glucose entry into glycolysis) largely compromised PRKAR2B-mediated tumour-promoting effect. Similar phenomenon was noticed by genetic silencing of HIF-1α. CONCLUSIONS: Our study identified that PRKAR2B-HIF-1α loop enhances the Warburg effect to enable growth advantage in prostate cancer.
Asunto(s)
Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Regulación Neoplásica de la Expresión Génica , Glucosa/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
Rett syndrome is a complex neurodevelopmental disorder that is mainly caused by mutations in MECP2. However, mutations in FOXG1 cause a less frequent form of atypical Rett syndrome, called FOXG1 syndrome. FOXG1 is a key transcription factor crucial for forebrain development, where it maintains the balance between progenitor proliferation and neuronal differentiation. Using genome-wide small RNA sequencing and quantitative proteomics, we identified that FOXG1 affects the biogenesis of miR200b/a/429 and interacts with the ATP-dependent RNA helicase, DDX5/p68. Both FOXG1 and DDX5 associate with the microprocessor complex, whereby DDX5 recruits FOXG1 to DROSHA. RNA-Seq analyses of Foxg1cre/+ hippocampi and N2a cells overexpressing miR200 family members identified cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) as a target of miR200 in neural cells. PRKAR2B inhibits postsynaptic functions by attenuating protein kinase A (PKA) activity; thus, increased PRKAR2B levels may contribute to neuronal dysfunctions in FOXG1 syndrome. Our data suggest that FOXG1 regulates PRKAR2B expression both on transcriptional and posttranscriptional levels.
Asunto(s)
Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Factores de Transcripción Forkhead/metabolismo , Hipocampo/metabolismo , MicroARNs/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transcripción Genética/fisiología , Factores de Edad , Animales , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Factores de Transcripción Forkhead/genética , Hipocampo/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , Proteínas del Tejido Nervioso/genéticaRESUMEN
Castration-resistant prostate cancers (CRPC) that occur after the failure of androgen-blocking therapies cause most of the deaths in prostate cancer (PCa) patients. In a previous study we identified that PRKAR2B expression is upregulated in CRPC and possesses potentials to develop CRPC. Here we further investigated the underlying mechanism of PRKAR2B in regulating prostate cancer metastasis. We established an androgen-independent LNCaPcell line (LNCaP-AI), and investigated the function of PRKAR2B on regulating cell invasion in vitro and in vivo. We found that PRKAR2B expression was markedly increased in LNCaP-AI cells and metastatic CRPC (mCRPC) tissues compared to LNCaP cells and primary PCa specimens, respectively. PRKAR2B level was significantly correlated with the Gleason score and lymph nodes metastasis in PCa. In vitro, PRKAR2B overexpression promoted cell invasion, whereas knockdown of PRKAR2B in CRPC cells inhibited cell invasion. PRKAR2B overexpression also promoted tumor metastasis in vivo. PRKAR2B resulted in a decreased expression of E-cadherin and an increased expression of Vimentin, N-cadherin, Fibronectin, indicating that PRKAR2B induced epithelial-mesenchymal transition (EMT). PRKAR2B activated Wnt/ß-catenin signaling in CRPC cells. More important, inhibition of Wnt/ß-catenin attenuated PRKAR2B-induced EMT and cancer cells invasion. Our results provided novel insights to PRKAR2B-driven CRPC cell invasion and indicated that PRKAR2B might be served as a potential target for CRPC therapy.
Asunto(s)
Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Transición Epitelial-Mesenquimal , Metástasis de la Neoplasia/fisiopatología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Vía de Señalización Wnt , Anciano , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , China , Modelos Animales de Enfermedad , Fibronectinas/metabolismo , Hospitales Universitarios , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica/fisiopatología , Células PC-3 , Regulación hacia Arriba , Vimentina/metabolismoRESUMEN
BACKGROUND/AIMS: Cyclic adenosine monophosphate (cAMP)-dependent type 2 regulatory subunit beta (Prkar2b) is a regulatory isoform of cAMP-dependent protein kinase (PKA), which is the primary target for cAMP actions. In oocytes, PKA and the pentose phosphate pathway (PPP) have important roles during the germinal vesicle (GV) stage arrest of development. Although the roles of the PKA signal pathway have been studied in the development of oocyte, there has been no report on the function of PRKAR2B, a key regulator of PKA. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), immunohistochemistry, and immunofluorescence, we determined the relative expression of Prkar2b in various tissues, including ovarian follicles, during oocyte maturation. Prkar2b-interfering RNA (RNAi) microinjection was conducted to confirm the effect of Prkar2b knockdown, and immunofluorescence, qRT-PCR, and time-lapse video microscopy were used to analyze Prkar2b-deficient oocytes. RESULTS: Prkar2b is strongly expressed in the ovarian tissues, particularly in the growing follicle. During oocyte maturation, the highest expression of Prkar2b was during metaphase I (MI), with a significant decrease at metaphase II (MII). RNAi-mediated Prkar2b suppression resulted in MI-stage arrest during oocyte development, and these oocytes exhibited abnormal spindle formation and chromosome aggregation. Expression of other members of the PKA family (except for Prkaca) were decreased, and the majority of the PPP factors were also reduced in Prkar2b-deficient oocytes. CONCLUSION: These results suggest that Prkar2b is closely involved in the maturation of oocytes by controlling spindle formation and PPP-mediated metabolism.
Asunto(s)
Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Interferencia de ARN , Animales , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/antagonistas & inhibidores , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Metafase , Ratones , Ratones Endogámicos ICR , Microscopía Fluorescente , Microscopía por Video , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Oogénesis , Folículo Ovárico/metabolismo , Folículo Ovárico/patología , ARN Bicatenario/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Imagen de Lapso de TiempoRESUMEN
Biochemical and structural studies demonstrate that S100A1 is involved in a Ca2+-dependent interaction with the type 2α and type 2ß regulatory subunits of protein kinase A (PKA) (RIIα and RIIß) to activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca2+-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca2+ and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.
Asunto(s)
Señalización del Calcio , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Histona Desacetilasas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas S100/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células Cultivadas , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Activación Enzimática , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Histona Desacetilasas/genética , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/enzimología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas S100/genéticaRESUMEN
Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.
Asunto(s)
Técnicas Biosensibles/métodos , AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Isoproterenol/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Ratas Sprague-Dawley , Sarcómeros/metabolismo , Sarcómeros/fisiología , Homología de Secuencia de AminoácidoRESUMEN
Genetic loss of the voltage-gated sodium channel Nav1.7 (Nav1.7-/-) results in lifelong insensitivity to pain in mice and humans. One underlying cause is an increase in the production of endogenous opioids in sensory neurons. We analyzed whether Nav1.7 deficiency altered nociceptive heterotrimeric guanine nucleotide-binding protein-coupled receptor (GPCR) signaling, such as initiated by GPCRs that respond to serotonin (pronociceptive) or opioids (antinociceptive), in sensory neurons. We found that the nociceptive neurons of Nav1.7 knockout (Nav1.7-/-) mice, but not those of Nav1.8 knockout (Nav1.8-/-) mice, exhibited decreased pronociceptive serotonergic signaling through the 5-HT4 receptors, which are Gαs-coupled GPCRs that stimulate the production of cyclic adenosine monophosphate resulting in protein kinase A (PKA) activity, as well as reduced abundance of the RIIß regulatory subunit of PKA. Simultaneously, the efficacy of antinociceptive opioid signaling mediated by the Gαi-coupled mu opioid receptors was increased. Consequently, opioids inhibited more efficiently tetrodotoxin-resistant sodium currents, which are important for pain-initiating neuronal activity in nociceptive neurons. Thus, Nav1.7 controls the efficacy and balance of GPCR-mediated pro- and antinociceptive intracellular signaling, such that without Nav1.7, the balance is shifted toward antinociception, resulting in lifelong endogenous analgesia.
Asunto(s)
Analgésicos Opioides/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Dolor/metabolismo , Serotonina/metabolismo , Transducción de Señal , Potenciales de Acción/efectos de los fármacos , Analgésicos Opioides/farmacología , Animales , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Ganglios Espinales/metabolismo , Indoles/farmacología , Masculino , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.7/genética , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Dolor/genética , Técnicas de Placa-Clamp , Ratas Sprague-Dawley , Receptores Opioides mu/metabolismo , Receptores de Serotonina 5-HT4/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Serotonina/farmacología , Antagonistas de la Serotonina/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Sulfonamidas/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIß via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Metabolismo de los Lípidos/fisiología , Células 3T3-L1 , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , RatonesRESUMEN
BACKGROUND: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunits and two inactive catalytic subunits that bind to the regulatory subunit dimer. Four regulatory subunits (RIα, RIß, RIIα, RIIß) and four catalytic subunits (Cα, Cß, Cγ, Prkx) have been described in the human and mouse genomes. Previous studies showed that complete inactivation of the Prkar1a subunit (coding for RIα) in the germline leads to embryonic lethality, while Prkar1a-deficient mice are viable and develop schwannomas, thyroid, and bone neoplasms, and rarely lymphomas and sarcomas. Mice with inactivation of the Prkar2a and Prkar2b genes (coding for RIIα and RIIß, respectively) are also viable but have not been studied for their susceptibility to any tumors. METHODS: Cohorts of Prkar1a (+/-) , Prkar2a (+/-) , Prkar2a (-/-) , Prkar2b (+/-) and wild type (WT) mice have been observed between 5 and 25 months of age for the development of hematologic malignancies. Tissues were studied by immunohistochemistry; tumor-specific markers were also used as indicated. Cell sorting and protein studies were also performed. RESULTS: Both Prkar2a (-/-) and Prkar2a (+/-) mice frequently developed hematopoietic neoplasms dominated by histiocytic sarcomas (HS) with rare diffuse large B cell lymphomas (DLBCL). Southern blot analysis confirmed that the tumors diagnosed histologically as DLBCL were clonal B cell neoplasms. Mice with other genotypes did not develop a significant number of similar neoplasms. CONCLUSIONS: Prkar2a deficiency predisposes to hematopoietic malignancies in vivo. RIIα's likely association with HS and DLBCL was hitherto unrecognized and may lead to better understanding of these rare neoplasms.
Asunto(s)
Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/deficiencia , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Neoplasias Hematológicas/genética , Animales , Transformación Celular Neoplásica/genética , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/deficiencia , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/deficiencia , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Neoplasias Hematológicas/patología , Inmunofenotipificación , Ratones , Ratones Noqueados , Fenotipo , Factores de TiempoRESUMEN
Mice lacking the RIIß regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) display reduced adiposity and resistance to diet-induced obesity. Here we show that RIIß knockout (KO) mice have enhanced sensitivity to leptin's effects on both feeding and energy metabolism. After administration of a low dose of leptin, the duration of hypothalamic JAK/STAT3 signalling is increased, resulting in enhanced POMC mRNA induction. Consistent with the extended JAK/STAT3 activation, we find that the negative feedback regulator of leptin receptor signalling, Socs3, is inhibited in the hypothalamus of RIIß KO mice. During fasting, RIIß-PKA is activated and this correlates with an increase in CREB phosphorylation. The increase in CREB phosphorylation is absent in the fasted RIIß KO hypothalamus. Selective inhibition of PKA activity in AgRP neurons partially recapitulates the leanness and resistance to diet-induced obesity of RIIß KO mice. Our findings suggest that RIIß-PKA modulates the duration of leptin receptor signalling and therefore the magnitude of the catabolic response to leptin.
Asunto(s)
Adiposidad/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Hipotálamo/metabolismo , Leptina/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Receptores de Leptina/metabolismo , Proteína Relacionada con Agouti/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dieta Alta en Grasa , Retroalimentación Fisiológica , Quinasas Janus/metabolismo , Leptina/farmacología , Ratones , Ratones Noqueados , Neuropéptido Y/metabolismo , Obesidad/genética , Fosforilación , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Cyclic AMP-dependent protein kinase (PKA) is regulated in part by N-terminal myristylation of its catalytic (C) subunit. Structural information about the role of myristylation in membrane targeting of PKA has been limited. In mammalian cells there are four functionally non-redundant PKA regulatory subunits (RIα, RIß, RIIα, and RIIß). PKA is assembled as an inactive R2C2 holoenzyme in cells. To explore the role of N-myristylation in membrane targeting of PKA holoenzymes, we solved crystal structures of RIα:myrC and RIIß2:myrC2, and showed that the N-terminal myristylation site in the myrC serves as a flexible "switch" that can potentially be mobilized for membrane anchoring of RII, but not RI, holoenzymes. Furthermore, we synthesized nanodiscs and showed by electron microscopy that membrane targeting through the myristic acid is specific for the RII holoenzyme. This membrane-anchoring myristylation switch is independent of A Kinase Anchoring Proteins (AKAPs) that target PKA to membranes by other mechanisms.
Asunto(s)
Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/química , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Ácido Mirístico/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Secundaria de ProteínaRESUMEN
To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme typically nucleates a macromolecular complex or a "PKA signalosome." Using the RIIß holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIß subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIß holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIß holoenzyme since the RIIß holoenzyme is located close to ion channels. The 2.8Å structure of an RIIßp2:C2:(Ca2ADP)2 holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-(ß,γ-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The holoenzyme serves as a "product trap" because of the slow off-rate of the pRIIß subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIß signaling cycle, we show that release of pRIIß in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site) inhibits holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RIIß holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II holoenzymes.
Asunto(s)
Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Catálisis , AMP Cíclico/metabolismo , Escherichia coli , Células HeLa , Holoenzimas/metabolismo , Humanos , Magnesio/metabolismo , Ratones , Células 3T3 NIH , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , RatasRESUMEN
We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/enzimología , Adenoma Corticosuprarrenal/enzimología , Síndrome de Cushing/enzimología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/genética , Adenoma Corticosuprarrenal/patología , Dominio Catalítico , Línea Celular Tumoral , Síndrome de Cushing/genética , Síndrome de Cushing/patología , AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/química , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Mutación , Unión Proteica , Estabilidad ProteicaRESUMEN
Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIß is structurally unique in that the type IIß holoenzyme is much more compact than the free RIIß homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIß C-terminal deletion mutant (RIIß(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIß holoenzyme and the RIIß domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIß holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIß homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIß isoform of PKA.
Asunto(s)
Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/química , AMP Cíclico/química , Holoenzimas/química , Animales , Dominio Catalítico , AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Holoenzimas/genética , Holoenzimas/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Modelos Moleculares , Mutación , Difracción de Neutrones , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Adolescent rats display reduced sensitivity to many dysphoria-related effects of alcohol (ethanol) including motor ataxia and sedative hypnosis, but the underlying neurobiological factors that contribute to these differences remain unknown. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway, particularly the type II regulatory subunit (RII), has been implicated in ethanol-induced molecular and behavioral responses in adults. Therefore, the current study examined cerebral cortical PKA in adolescent and adult ethanol responses. With the exception of early adolescence, PKA RIIα and RIIß subunit levels largely did not differ from adult levels in either whole cell lysate or P2 synaptosomal expression. However, following acute ethanol exposure, PKA RIIß P2 synaptosomal expression and activity were increased in adults, but not in adolescents. Behaviorally, intracerebroventricular administration of the PKA activator Sp-cAMP and inhibitor Rp-cAMP prior to ethanol administration increased adolescent sensitivity to the sedative-hypnotic effects of ethanol compared to controls. Sp-cAMP was ineffective in adults whereas Rp-cAMP suggestively reduced loss of righting reflex (LORR) with paralleled increases in blood ethanol concentrations. Overall, these data suggest that PKA activity modulates the sedative/hypnotic effects of ethanol and may potentially play a wider role in the differential ethanol responses observed between adolescents and adults.
Asunto(s)
Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Etanol/farmacología , Envejecimiento , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/biosíntesis , Masculino , Ratas Sprague-Dawley , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Leucine-rich repeat kinase 2 (LRRK2) is enriched in the striatal projection neurons (SPNs). We found that LRRK2 negatively regulates protein kinase A (PKA) activity in the SPNs during synaptogenesis and in response to dopamine receptor Drd1 activation. LRRK2 interacted with PKA regulatory subunit IIß (PKARIIß). A lack of LRRK2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of actin-disassembling enzyme cofilin and glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs. Furthermore, PKA-dependent phosphorylation of GluR1 was also aberrantly enhanced in the striatum of young and aged Lrrk2(-/-) mice after treatment with a Drd1 agonist. Notably, a Parkinson's disease-related Lrrk2 R1441C missense mutation that impaired the interaction of LRRK2 with PKARIIß also induced excessive PKA activity in the SPNs. Our findings reveal a previously unknown regulatory role for LRRK2 in PKA signaling and suggest a pathogenic mechanism of SPN dysfunction in Parkinson's disease.
Asunto(s)
Cuerpo Estriado/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Espinas Dendríticas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Receptores de Dopamina D1/metabolismo , Sinapsis/metabolismo , Animales , Cuerpo Estriado/patología , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/biosíntesis , Espinas Dendríticas/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Receptores de Dopamina D1/agonistas , Sinapsis/patología , Regulación hacia Arriba/genéticaRESUMEN
Knowledge about the molecular structure of protein kinase A (PKA) isoforms is substantial. In contrast, the dynamics of PKA isoform activity in living primary cells has not been investigated in detail. Using a high content screening microscopy approach, we identified the RIIß subunit of PKA-II to be predominantly expressed in a subgroup of sensory neurons. The RIIß-positive subgroup included most neurons expressing nociceptive markers (TRPV1, NaV1.8, CGRP, IB4) and responded to pain-eliciting capsaicin with calcium influx. Isoform-specific PKA reporters showed in sensory-neuron-derived F11 cells that the inflammatory mediator PGE2 specifically activated PKA-II but not PKA-I. Accordingly, pain-sensitizing inflammatory mediators and activators of PKA increased the phosphorylation of RII subunits (pRII) in subgroups of primary sensory neurons. Detailed analyses revealed basal pRII to be regulated by the phosphatase PP2A. Increase of pRII was followed by phosphorylation of CREB in a PKA-dependent manner. Thus, we propose RII phosphorylation to represent an isoform-specific readout for endogenous PKA-II activity in vivo, suggest RIIß as a novel nociceptive subgroup marker, and extend the current model of PKA-II activation by introducing a PP2A-dependent basal state.