RESUMO
BACKGROUND: Emerging studies have proved that colonic inflammation caused by refractory inflammatory bowel disease (IBD) can initiate the colitis-associated cancer (CAC), but the transition from inflammation to carcinoma is still largely unknown. METHODS: In this study, mouse colitis and CAC models were established, and the RNA-seq by circRNA microarray was employed to identify the differentially expressed circRNAs and mRNAs in different comparisons (DSS vs. NC and AOM/DSS vs. DSS). The bioinformatics analyses were used to search the common characteristics in mouse colitis and CAC. RESULTS: The K-means clustering algorithm packaged these differential expressed circRNAs into subgroup analysis, and the data strongly implied that mmu_circ_0001109 closely correlated to the pro-inflammatory signals, while mmu_circ_0001845 was significantly associated with the Wnt signalling pathway. Our subsequent data in vivo and in vitro confirmed that mmu_circ_0001109 could exacerbate the colitis by up-regulating the Jak-STAT3 and NF-kappa B signalling pathways, and mmu_circ_0001845 promoted the CAC transformation through the Wnt signalling pathway. By RNA blasting between mice and humans, the human RTEL1- and PRKAR2A-derived circRNAs, which might be considered as homeotic circRNAs of mmu_circ_0001109 and mmu_circ_0001845, respectively, were identified. The clinical data revealed that RTEL1-derived circRNAs had no clinical significance in human IBD and CAC. However, three PRKAR2A-derived circRNAs, which had the high RNA similarities to mmu_circ_0001845, were remarkably up-regulated in CAC tissue samples and promoted the transition from colitis to CAC. CONCLUSIONS: Our results suggested that these human PRKAR2A-derived circRNAs could be novel candidates for distinguishing CAC patients and predicted the prognosis of CAC.
Assuntos
Colite/complicações , Neoplasias Colorretais/classificação , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/efeitos adversos , Neoplasias/classificação , Animais , Colite/genética , Neoplasias Colorretais/etiologia , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Camundongos , Neoplasias/etiologia , RNA CircularRESUMO
Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Glioblastoma/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Glioblastoma/genética , Glioblastoma/fisiopatologia , Glioma/genética , Glioma/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução Genética/métodosRESUMO
Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level.
Assuntos
Metabolismo Energético/genética , Glucose/farmacologia , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/genética , Linhagem Celular , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pró-Proteína Convertase 1/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
The habenula (Hb) is a bilateral, evolutionarily conserved epithalamic structure connecting forebrain and midbrain structures that has gained attention for its roles in depression, addiction, rewards processing, and motivation. Of its 2 major subdivisions, the medial Hb (MHb) and lateral Hb (LHb), MHb circuitry and function are poorly understood relative to those of the LHb. Prkar2a codes for cAMP-dependent protein kinase (PKA) regulatory subunit IIα (RIIα), a component of the PKA holoenzyme at the center of one of the major cell-signaling pathways conserved across systems and species. Type 2 regulatory subunits (RIIα, RIIß) determine the subcellular localization of PKA, and unlike other PKA subunits, Prkar2a has minimal brain expression except in the MHb. We previously showed that RIIα-knockout (RIIα-KO) mice resist diet-induced obesity. In the present study, we report that RIIα-KO mice have decreased consumption of palatable, "rewarding" foods and increased motivation for voluntary exercise. Prkar2a deficiency led to decreased habenular PKA enzymatic activity and impaired dendritic localization of PKA catalytic subunits in MHb neurons. Reexpression of Prkar2a in the Hb rescued this phenotype, confirming differential roles for Prkar2a in regulating the drives for palatable foods and voluntary exercise. Our findings show that in the MHb decreased PKA signaling and dendritic PKA activity decrease motivation for palatable foods, while enhancing the motivation for exercise, a desirable combination of behaviors.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Comportamento Alimentar/fisiologia , Habenula/metabolismo , Animais , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Habenula/fisiologia , Holoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Motivação/genética , Neurônios/metabolismo , Fenótipo , Condicionamento Físico Animal/fisiologiaRESUMO
Type II isoforms of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA-II) contain a phosphorylatable epitope within the inhibitory domain of RII subunits (pRII) with still unclear function. In vitro, RII phosphorylation occurs in the absence of cAMP, whereas staining of cells with pRII-specific antibodies revealed a cAMP-dependent pattern. In sensory neurons, we found that increased pRII immunoreactivity reflects increased accessibility of the already phosphorylated RII epitope during cAMP-induced opening of the tetrameric RII2:C2 holoenzyme. Accordingly, induction of pRII by cAMP was sensitive to novel inhibitors of dissociation, whereas blocking catalytic activity was ineffective. Also in vitro, cAMP increased the binding of pRII antibodies to RII2:C2 holoenzymes. Identification of an antibody specific for the glycine-rich loop of catalytic subunits facing the pRII-epitope confirmed activity-dependent binding with similar kinetics, proving that the reassociation is rapid and precisely controlled. Mechanistic modeling further supported that RII phosphorylation precedes cAMP binding and controls the inactivation by modulating the reassociation involving the coordinated action of phosphodiesterases and phosphatases.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Subunidades Proteicas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Anticorpos/farmacologia , Extratos Celulares , Permeabilidade da Membrana Celular/efeitos dos fármacos , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Isoenzimas/metabolismo , Masculino , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The main function of the A kinase-anchoring proteins (AKAPs) is to target the cyclic AMP-dependent protein kinase A (PKA) to its cellular substrates through the interaction with its regulatory subunits. Besides anchoring of PKA, AKAP8 participates in regulating the histone H3 lysine 4 (H3K4) histone methyltransferase (HMT) complexes. It is also involved in DNA replication, apoptosis, transcriptional silencing of rRNA genes, alternative splicing, and chromatin condensation during mitosis. In this study, we focused on the interaction between AKAP8 and the core subunit of all known H3K4 HMT complexes-DPY30 protein. Here, we demonstrate that the PKA-binding domain of AKAP8 and the C-terminal domain of DPY30, also called Dpy-30 motif, are crucial for the interaction between these proteins. We show that a single amino acid substitution in DPY30 L69D affects its dimerization and completely abolishes its interaction with AKAP8 and another DPY30-binding partner brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1), which is also AKAP domain-containing protein. We further demonstrate that AKAP8 interacts with DPY30 and the RII alpha regulatory subunit of PKA both in the interphase and in mitotic cells, and we show evidences that AKAP8L, a homologue of AKAP8, interacts with core subunits of the H3K4 HMT complexes, which suggests its role as a potential regulator of these complexes. The results presented here reinforce the analogy between AKAP8-RII alpha and AKAP8-DPY30 interactions, postulated before, and improve our understanding of the complexity of the cellular functions of the AKAP8 protein.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ancoragem à Quinase A/química , Ciclo Celular , Nucléolo Celular/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Dimerização , Genes Reporter , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Código das Histonas , Histona Metiltransferases/metabolismo , Humanos , Metilação , Modelos Moleculares , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TranscriçãoRESUMO
Pseudomonas aeruginosa is an opportunistic bacterium which induces some complications in immunocompromised patients. Pseudomonas aeruginosa is a quorum-sensing using bacterium which regulates its genes expression. The bacterium uses two famous pathways for quorum sensing entitled LasI/LasR and RhlI/RhlR systems. It has been documented that the bacteria which use quorum sensing are able to overcome immune responses. This review article aims to present recent information regarding the effects of Pseudomonas aeruginosa quorum sensing systems on the host immune responses.
Assuntos
Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Percepção de Quorum , Animais , Proteínas de Bactérias/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunidade Inata , Transativadores/metabolismoRESUMO
BACKGROUND: Ageing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast-twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow-twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre-type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle-specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown. METHODS: Studies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real-time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ. RESULTS: Levels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region-but mainly in the fast-twitch, not the slow-twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIß are enriched at, the NMJ-again, preferentially in fast-twitch but not slow-twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii) decreased the levels of gene expression of muscle denervation markers; and (iii) enhanced neurotransmission efficiency at NMJ. CONCLUSIONS: Cardiac troponin T at the NMJ region contributes to NMJ functional decline with ageing mainly in the fast-twitch skeletal muscle through interfering with PKA signalling. This knowledge could inform useful targets for prevention and therapy of age-related decline in muscle function.
Assuntos
Envelhecimento , Denervação Muscular , Fibras Musculares de Contração Rápida/metabolismo , Troponina T/metabolismo , Potenciais de Ação , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Chlorocebus aethiops , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Contração Muscular , Junção Neuromuscular/fisiologia , Transmissão Sináptica , Troponina T/genéticaRESUMO
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.
Assuntos
Sinalização do Cálcio , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Histona Desacetilases/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas S100/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Ativação Enzimática , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/enzimologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas S100/genéticaRESUMO
The kinetic participation of macrophages is critical for inflammatory resolution and recovery from myocardial infarction (MI), particularly with respect to the transition from the M1 to the M2 phenotype; however, the underlying mechanisms are poorly understood. In this study, we found that the deletion of prostaglandin (PG) D2 receptor subtype 1 (DP1) in macrophages retarded M2 polarization, antiinflammatory cytokine production, and resolution in different inflammatory models, including the MI model. DP1 deletion up-regulated proinflammatory genes expression via JAK2/STAT1 signaling in macrophages, whereas its activation facilitated binding of the separated PKA regulatory IIα subunit (PRKAR2A) to the transmembrane domain of IFN-γ receptor, suppressed JAK2-STAT1 axis-mediated M1 polarization, and promoted resolution. PRKAR2A deficiency attenuated DP1 activation-mediated M2 polarization and resolution of inflammation. Collectively, PGD2-DP1 axis-induced M2 polarization facilitates resolution of inflammation through the PRKAR2A-mediated suppression of JAK2/STAT1 signaling. These observations indicate that macrophage DP1 activation represents a promising strategy in the management of inflammation-associated diseases, including post-MI healing.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Prostaglandina D2/metabolismo , Subunidades Proteicas/metabolismo , Animais , Ceco/patologia , Polaridade Celular , Modelos Animais de Doenças , Feminino , Deleção de Genes , Hidantoínas , Janus Quinase 2/metabolismo , Ligadura , Macrófagos/citologia , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Isquemia Miocárdica/patologia , Peritonite/patologia , Ligação Proteica , Punções , Receptores Imunológicos , Receptores de Interferon , Receptores de Prostaglandina/deficiência , Receptores de Prostaglandina/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Cicatrização , ZimosanRESUMO
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.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/deficiência , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Neoplasias Hematológicas/genética , Animais , Transformação Celular Neoplásica/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/deficiência , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/deficiência , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Neoplasias Hematológicas/patologia , Imunofenotipagem , Camundongos , Camundongos Knockout , Fenótipo , Fatores de TempoRESUMO
Protein kinase activity is regulated not only by direct strategies affecting activity but also by spatial and temporal regulatory mechanisms. Kinase signaling pathways are coordinated by scaffolding proteins that orchestrate the assembly of multi-protein complexes. One family of such scaffolding proteins are the A-kinase anchoring proteins (AKAPs). AKAPs share the commonality of binding cAMP-dependent protein kinase (PKA). In addition, they bind further signaling proteins and kinase substrates and tether such multi-protein complexes to subcellular locations. The A-kinase binding (AKB) domain of AKAPs typically contains a conserved helical motif that interacts directly with the dimerization/docking (D/D) domain of the regulatory subunits of PKA. Based on a pull-down proteomics approach, we identified neurochondrin (neurite-outgrowth promoting protein) as a previously unidentified AKAP. Here, we show that neurochondrin interacts directly with PKA through a novel mechanism that involves two distinct binding regions. In addition, we demonstrate that neurochondrin has strong isoform selectivity towards the RIIα subunit of PKA with nanomolar affinity. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ancoragem à Quinase A/química , Sequência de Aminoácidos , Sítios de Ligação , AMP Cíclico/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Humanos , Complexos Multiproteicos , Proteínas do Tecido Nervoso/química , Ligação Proteica , Transdução de SinaisRESUMO
Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC). However, the cellular functions of RAI16 are still unclear. In this study, by immunoprecipitation and tandem (MS/MS) mass spectrometry analysis, we identified that RAI16 interacted with the type II regulatory subunit of PKA (PKA-RIIα), acting as a novel protein kinase A anchoring protein (AKAP). In addition, RAI16 also interacted with heat shock protein 70 (HSP70) and 14-3-3θ. Further studies indicated that RAI16 mediated PKA phosphorylation of HSP70 at serine 486, resulting in anti-apoptosis events. RAI16 was also phosphorylated by the anchored PKA at serine 325, which promoted the recruitment of 14-3-3θ, which, in turn, inhibited RAI16 mediated PKA phosphorylation of HSP70. These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas/metabolismo , Apoptose/fisiologia , Carcinoma Hepatocelular/patologia , Linhagem Celular , Sobrevivência Celular , Transformação Celular Neoplásica , Células HEK293 , Humanos , Neoplasias Hepáticas/patologia , Fosforilação , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
There is increasing evidence for a role of cytidine 3',5'-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin(-) cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N(4)-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N(4)-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Animais , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , CMP Cíclico/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Linfoma/metabolismo , Camundongos , Modelos Moleculares , Fosforilação/efeitos dos fármacosRESUMO
PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser(265) and Ser(296) by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser(1928) induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canais de Cálcio Tipo L/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Células HEK293 , Humanos , Immunoblotting , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutação , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Domínios PDZ/genética , Fosforilação , Ligação Proteica , Ratos , Sarcômeros/metabolismo , Serina/química , Serina/genética , Serina/metabolismoRESUMO
Ejaculated spermatozoa must undergo a series of biochemical modifications called capacitation, prior to fertilization. Protein-kinase A (PKA) mediates sperm capacitation, although its regulation is not fully understood. Sperm contain several A-kinase anchoring proteins (AKAPs), which are scaffold proteins that anchor PKA. In this study, we show that AKAP3 is degraded in bovine sperm incubated under capacitation conditions. The degradation rate is variable in sperm from different bulls and is correlated with the capacitation ability. The degradation of AKAP3 was significantly inhibited by MG-132, a proteasome inhibitor, indicating that AKAP3 degradation occurs via the proteasomal machinery. Treatment with Ca(2+)-ionophore induced further degradation of AKAP3; however, this effect was found to be enhanced in the absence of Ca(2+) in the medium or when intracellular Ca(2+) was chelated the degradation rate of AKAP3 was significantly enhanced when intracellular space was alkalized using NH4Cl, or when sperm were treated with Ht31, a peptide that contains the PKA-binding domain of AKAPs. Moreover, inhibition of PKA activity by H89, or its activation using 8Br-cAMP, increased AKAP3 degradation rate. This apparent contradiction could be explained by assuming that binding of PKA to AKAP3 protects AKAP3 from degradation. We conclude that AKAP3 degradation is regulated by intracellular alkalization and PKARII anchoring during sperm capacitation.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Capacitação Espermática/fisiologia , Animais , Cálcio/metabolismo , Bovinos , Immunoblotting , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Espermatozoides/metabolismoRESUMO
In mammalian oocytes, cAMP-dependent protein kinase (PKA) has critical functions in meiotic arrest and meiotic maturation. Although subcellular localization of PKA is regulated by A-kinase anchor proteins (AKAPs) and PKA compartmentalization is essential for PKA functions, the role of AKAPs in meiotic regulation has not been fully elucidated. In the present study, we performed far-Western blot analysis using porcine PRKAR2A for detection of AKAPs and found, to our knowledge, several novel signals in porcine oocytes. Among these signals, a 150-kDa AKAP showed the major expression and was the product of porcine AKAP1. Overexpression of AKAP1 changed the PKA localization and promoted meiotic resumption of porcine oocytes even in the presence of a high concentration of cAMP, which inhibits meiotic resumption by inducing high PKA activity. On the contrary, knockdown of AKAP1 showed inhibitory effects on meiotic resumption and oocyte maturation. In addition, the expression level of AKAP1 in porcine growing oocytes, which show meiotic incompetence and PKA mislocalization, was significantly lower than that in fully grown oocytes. However, AKAP1 insufficiency was not the primary cause of the meiotic incompetence of the growing oocytes. These results suggest that the regulation of PKA localization by AKAP1 may be involved in meiotic resumption and oocyte maturation but not in meiotic incompetence of porcine growing oocytes.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Meiose , Oócitos/fisiologia , Suínos , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Células Cultivadas , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Meiose/efeitos dos fármacos , Meiose/genética , Meiose/fisiologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Oogênese/genética , Oogênese/fisiologia , Suínos/genética , Suínos/metabolismo , Suínos/fisiologia , Distribuição Tecidual/efeitos dos fármacos , TransfecçãoRESUMO
It is known that the activity of AMP-activated protein kinase (AMPKα2) was depressed under high glucose conditions. However, whether protein expression of AMPKα2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPKα2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPKα2 down-regulation. Endogenous AMPKα2 ubiquitination was detected by immunoprecipitation of AMPKα2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPKα2-interacting protein in skeletal muscle cells. Interaction between AMPKα2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPKα2 down-regulation. The overexpression of WWP1 down-regulated AMPKα2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPKα2 was down-regulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPKα2 under high glucose culture conditions via the ubiquitin-proteasome pathway.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Músculo Esquelético/metabolismo , Ubiquitina-Proteína Ligases/química , Animais , Regulação para Baixo , Inativação Gênica , Glucose/metabolismo , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Transforming growth factor ß (TGFß) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFß signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFß activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFß-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFß-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Movimento Celular , AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/fisiologia , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ativação Enzimática , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Vison , Transplante de Neoplasias , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Deleção de Sequência , Transdução de Sinais , Proteína Smad4/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
A-kinase anchoring proteins (AKAPs) are a family of scaffolding proteins that target PKA and other signaling molecules to cellular compartments and thereby spatiotemporally define cellular signaling events. The AKAP18 family comprises AKAP18α, AKAP18ß, AKAP18γ, and AKAP18δ. The δ isoform targets PKA and phosphodiesterase PDE4D to AQP2 (aquaporin-2)-bearing vesicles to orchestrate the acute regulation of body water balance. Therefore, AKAP18δ must adopt a membrane localization that seems at odds with (i) its lack of palmitoylation or myristoylation sites that tailor its isoforms AKAP18α and AKAP18ß to membrane compartments and (ii) the high sequence identity to the preferentially cytoplasmic AKAP18γ. Here, we show that the electrostatic attraction of the positively charged amino acids of AKAP18δ to negatively charged lipids explains its membrane targeting. As revealed by fluorescence correlation spectroscopy, the binding constant of purified AKAP18δ fragments to large unilamellar vesicles correlates (i) with the fraction of net negatively charged lipids in the bilayer and (ii) with the total amount of basic residues in the protein. Although distantly located on the sequence, these positively charged residues concentrate in the tertiary structure and form a clear binding surface. Thus, specific recruitment of the AKAP18δ-based signaling module to membranes such as those of AQP2-bearing vesicles must be achieved by additional mechanisms, most likely compartment-specific protein-protein interactions.