RESUMO
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive decline. In hippocampal neurons, the pathological features of AD include the accumulation of extracellular amyloid-beta peptide (Aß) accompanied by oxidative stress, mitochondrial dysfunction, and neuron loss. A decrease in neuroprotective Protein Kinase A (PKA) signaling contributes to mitochondrial fragmentation and neurodegeneration in AD. By associating with the protein scaffold Dual-Specificity Anchoring Protein 1 (D-AKAP1), PKA is targeted to mitochondria to promote mitochondrial fusion by phosphorylating the fission modulator dynamin-related protein 1 (Drp1). We hypothesized that (1) a decrease in the endogenous level of endogenous D-AKAP1 contributes to decreased PKA signaling in mitochondria and that (2) restoring PKA signaling in mitochondria can reverse neurodegeneration and mitochondrial fragmentation in neurons in AD models. Through immunohistochemistry, we showed that endogenous D-AKAP1, but not other mitochondrial proteins, is significantly reduced in primary neurons treated with Aß42 peptide (10µM, 24 h), and in the hippocampus and cortex from asymptomatic and symptomatic AD mice (5X-FAD). Transiently expressing wild-type, but not a PKA-binding deficient mutant of D-AKAP1, was able to reduce mitochondrial fission, dendrite retraction, and apoptosis in primary neurons treated with Aß42. Mechanistically, the protective effects of D-AKAP1/PKA are moderated through PKA-mediated phosphorylation of Drp1, as transiently expressing a PKA phosphomimetic mutant of Drp1 (Drp1-S656D) phenocopies D-AKAP1's ability to reduce Aß42-mediated apoptosis and mitochondrial fission. Overall, our data suggest that a loss of D-AKAP1/PKA contributes to mitochondrial pathology and neurodegeneration in an in vitro cell culture model of AD.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/toxicidade , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neuroproteção/fisiologia , Fragmentos de Peptídeos/toxicidade , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neuroproteção/efeitos dos fármacos , Gravidez , RatosRESUMO
Alternative splicing has been shown to causally contribute to the epithelial-mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein-protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Processamento Alternativo/genética , Transição Epitelial-Mesenquimal/genética , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Células HCT116 , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Domínios e Motivos de Interação entre Proteínas , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Aldosterone (Aldo) contributes to mitochondrial dysfunction and cardiac oxidative stress. Using a proteomic approach, A-kinase anchor protein (AKAP)-12 has been identified as a down-regulated protein by Aldo in human cardiac fibroblasts. We aim to characterize whether AKAP-12 down-regulation could be a deleterious mechanism which induces mitochondrial dysfunction and oxidative stress in cardiac cells. Aldo down-regulated AKAP-12 via its mineralocorticoid receptor, increased oxidative stress and induced mitochondrial dysfunction characterized by decreased mitochondrial-DNA and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expressions in human cardiac fibroblasts. CRISPR/Cas9-mediated knock-down of AKAP-12 produced similar deleterious effects in human cardiac fibroblasts. CRISPR/Cas9-mediated activation of AKAP-12 blunted Aldo effects on mitochondrial dysfunction and oxidative stress in human cardiac fibroblasts. In Aldo-salt-treated rats, cardiac AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased and paralleled increased oxidative stress. In myocardial biopsies from patients with aortic stenosis (AS, n = 26), AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased as compared to Controls (n = 13). Circulating Aldo levels inversely correlated with cardiac AKAP-12. PGC-1α positively associated with AKAP-12 and with mitochondrial-DNA. Aldo decreased AKAP-12 expression, impairing mitochondrial biogenesis and increasing cardiac oxidative stress. AKAP-12 down-regulation triggered by Aldo may represent an important event in the development of mitochondrial dysfunction and cardiac oxidative stress.
Assuntos
Proteínas de Ancoragem à Quinase A/genética , Aldosterona/metabolismo , Estenose da Valva Aórtica/genética , Proteínas de Ciclo Celular/genética , Fibroblastos/metabolismo , Miocárdio/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/metabolismo , Idoso , Idoso de 80 Anos ou mais , Aldosterona/farmacologia , Animais , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/cirurgia , Sistemas CRISPR-Cas , Estudos de Casos e Controles , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Miocárdio/patologia , Biogênese de Organelas , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Congenital nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine. Congenital NDI is mainly caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R), leading to impaired aquaporin-2 (AQP2) water channel activity. So far, treatment options of congenital NDI either by rescuing mutant V2R with chemical chaperones or by elevating cyclic adenosine monophosphate (cAMP) levels have failed to yield effective therapies. Here we show that inhibition of A-kinase anchoring proteins (AKAPs) binding to PKA increases PKA activity and activates AQP2 channels in cortical collecting duct cells. In vivo, the low molecular weight compound 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives increase AQP2 activity to the same extent as vasopressin, and increase urine osmolality in the context of V2R inhibition. We therefore suggest that FMP-API-1 may constitute a promising lead compound for the treatment of congenital NDI caused by V2R mutations.
Assuntos
Proteínas de Ancoragem à Quinase A/genética , Aquaporina 2/genética , Compostos Benzidrílicos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Diabetes Insípido Nefrogênico/tratamento farmacológico , Fenóis/farmacologia , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/metabolismo , Sequência de Aminoácidos , Animais , Aquaporina 2/agonistas , Aquaporina 2/metabolismo , Arginina Vasopressina , Benzazepinas/antagonistas & inibidores , Benzazepinas/farmacologia , Linhagem Celular Transformada , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/metabolismo , Diabetes Insípido Nefrogênico/patologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Humanos , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , Masculino , Camundongos Endogâmicos C57BL , Concentração Osmolar , Ligação Proteica/efeitos dos fármacos , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Tolvaptan , Água/metabolismoRESUMO
Hypertension is a multifactorial chronic inflammatory disease that leads to cardiac remodeling. A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that has multiple functions in various biological events, including the regulation of vessel integrity and differentiation of neural barriers in blood. However, the role of AKAP12 in angiotensin II (Ang II)-induced cardiac injury remains unclear. In the present study, Ang II infusion reduced AKAP12 expressions in the hearts of wild-type (WT) mice, and AKAP12 knockout (KO) enhanced the infiltration of inflammatory cells. In addition, AKAP12 deletion accelerated Ang II-induced cardiac histologic alterations and dysfunction. Further, AKAP12-/- aggravated heart failure by promoting the inflammation, oxidative stress, cellular apoptosis, and autophagy induced by Ang II. Furthermore, AKAP12 KO elevated Ang II-induced cardiac fibrosis, as indicated by the following: (1) Masson trichrome staining showed that Ang II infusion markedly increased fibrotic areas of the WT mouse heart, which was greatly accelerated in AKAP12-/- mice; (2) immunohistochemistry analysis showed increased expression of transforming growth factor ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) in the AKAP12-/- mouse heart; (3) reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed increased expression of fibrosis-related molecules in the AKAP12-deficient mouse heart; and (4) Western blot analysis indicated significantly higher upregulation of p-SMAD2/3 in the AKAP12-/- mouse heart. In vitro, AKAP12 knockdown in HL-1â¯cells was responsible for TGF-ß1-induced inflammation, the generation of reactive oxygen species (ROS), apoptosis, autophagy, and fibrosis. Furthermore, overexpression of AKAP12 reduced fibrosis triggered by TGF-ß1 in cells. Overall, our study suggests that fibrosis induced by Ang II may be alleviated by AKAP12 expression through inactivation of the TGF-ß1 pathway.
Assuntos
Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/patologia , Miocárdio/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Proteínas de Ancoragem à Quinase A/deficiência , Proteínas de Ancoragem à Quinase A/metabolismo , Angiotensina II , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Progressão da Doença , Fibrose , Técnicas de Silenciamento de Genes , Traumatismos Cardíacos/induzido quimicamente , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Generation of the second messenger molecule cAMP mediates a variety of cellular responses which are essential for critical cellular processes. In response to elevated cAMP levels, cAMP dependent protein kinase (PKA) phosphorylates serine and threonine residues on a wide variety of target substrates. In order to enhance the precision and directionality of these signaling events, PKA is localized to discrete locations within the cell by A-kinase anchoring proteins (AKAPs). The interaction between PKA and AKAPs is mediated via an amphipathic α-helix derived from AKAPs which binds to a stable hydrophobic groove formed in the dimerization/docking (D/D) domain of PKA-R in an isoform-specific fashion. Although numerous AKAP disruptors have previously been identified that can inhibit either RI- or RII-selective AKAPs, no AKAP disruptors have been identified that have isoform specificity for RIα versus RIß or RIIα versus RIIß. As a strategy to identify isoform-specific AKAP inhibitors, a library of chemically stapled protein-protein interaction (PPI) disruptors was developed based on the RII-selective AKAP disruptor, STAD-2. An alanine was substituted at each position in the sequence, and from this library it was possible to delineate the importance of longer aliphatic residues in the formation of a region which complements the hydrophobic cleft formed by the D/D domain. Interestingly, lysine residues that were added to both terminal ends of the peptide sequence to facilitate water solubility appear to contribute to isoform specificity for RIIα over RIIß while having only weak interaction with RI. This work supports current hypotheses on the mechanisms of AKAP binding and highlights the significance of particular residue positions that aid in distinguishing between the RII isoforms and may provide insight into future design of isoform-selective AKAP disruptors.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeos/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Sequência de Aminoácidos , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Polarização de Fluorescência , Humanos , Cinética , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , Mapas de Interação de Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/genética , Biomarcadores/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Centrossomo/ultraestrutura , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Humanos , Imageamento Tridimensional , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Centro Organizador dos Microtúbulos/ultraestrutura , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteômica/métodos , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mitochondria are the powerhouses of energy production and the sites where metabolic pathway and survival signals integrate and focus, promoting adaptive responses to hormone stimulation and nutrient availability. Increasing evidence suggests that mitochondrial bioenergetics, metabolism and signaling are linked to tumorigenesis. AKAP1 scaffolding protein integrates cAMP and src signaling on mitochondria, regulating organelle biogenesis, oxidative metabolism and cell survival. Here, we provide evidence that AKAP1 is a transcriptional target of Myc and supports the growth of cancer cells. We identify Sestrin2, a leucine sensor and inhibitor of the mammalian target of rapamycin (mTOR), as a novel component of the complex assembled by AKAP1 on mitochondria. Downregulation of AKAP1 impaired mTOR pathway and inhibited glioblastoma growth. Both effects were reversed by concomitant depletion of AKAP1 and sestrin2. High levels of AKAP1 were found in a wide variety of high-grade cancer tissues. In lung cancer, AKAP1 expression correlates with high levels of Myc, mTOR phosphorylation and reduced patient survival. Collectively, these data disclose a previously unrecognized role of AKAP1 in mTOR pathway regulation and cancer growth. AKAP1/mTOR signal integration on mitochondria may provide a new target for cancer therapy.
Assuntos
Proteínas de Ancoragem à Quinase A/genética , Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Mitocôndrias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Serina-Treonina Quinases TOR/genética , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Mitocôndrias/metabolismo , Transplante de Neoplasias , Neuroglia/metabolismo , Neuroglia/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo , Transcrição GênicaRESUMO
TRPV4 ion channels have a broad expression profile and were shown to contribute to enhanced pain sensation in inflammation. Directly blocking TRPV4 might run the risk of interfering with normal physiology, and has prompted to explore the interaction with the scaffolding protein AKAP79, an approach successfully used for TRPV1 channels. HEK293t cells express AKAP79, additional transfection did not sensitize human TRPV4. Application of trypsin facilitated responses to TRPV4 agonist GSK1016790A. Using a specific protease-activated receptor 2 agonist, involvement of an A-kinase anchoring protein in TRPV4 activation was demonstrated by inhibition with AKAP inhibitor peptide Ht31. TRPV4 has substantial sequence similarity to TRPV1 in the range interacting with AKAP79. A synthetic peptide, resembling these amino acids and extended by a positive region for transmembrane uptake, was tested. Sensitization of TRPV4 responses could be reduced after exposure to this 771-781::TAT peptide but not by a scrambled control peptide. This validates the concept of targeting the interaction between TRPV4 and AKAP79 and controlling increased TRPV4 activity.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Canais de Cátion TRPV/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Oligopeptídeos/farmacologia , Sulfonamidas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Transfecção , Tripsina/farmacologiaRESUMO
G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of Gi/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that Gi/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two Gi/o-mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/agonistas , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Fosfatidilinositol 3-Quinase/metabolismo , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Substituição de Aminoácidos , Ciclopentanos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/genética , Mutação , Domínios PDZ , Fosfatidilinositol 3-Quinase/química , Fosfatidilinositol 3-Quinase/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Quinolinas/farmacologia , Interferência de RNA , Ensaio Radioligante , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The cardiac IK1 current stabilizes the resting membrane potential of cardiomyocytes. Protein kinase A (PKA) induces an inhibition of IK1 current which strongly promotes focal arrhythmogenesis. The molecular mechanisms underlying this regulation have only partially been elucidated yet. Furthermore, the role of A-kinase anchoring proteins (AKAPs) in this regulation has not been examined to date. The objective of this project was to elucidate the molecular mechanisms underlying the inhibition of IK1 by PKA and to identify novel molecular targets for antiarrhythmic therapy downstream ß-adrenoreceptors. Patch clamp and voltage clamp experiments were used to record currents and co-immunoprecipitation, and co-localization experiments were performed to show spatial and functional coupling. Activation of PKA inhibited IK1 current in rat cardiomyocytes. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide inhibitor AKAP-IS. We observed functional and spatial coupling of the plasma membrane-associated AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channel subunits, but not to Kir2.3 channels. In contrast, AKAPyotiao had no functional effect on the PKA regulation of Kir channels. AKAP15 and AKAP79 co-immunoprecipitated with and co-localized to Kir2.1 and Kir2.2 channel subunits in ventricular cardiomyocytes. In this study, we provide evidence for coupling of cardiac Kir2.1 and Kir2.2 subunits with the plasma membrane-bound AKAPs 15 and 79. Cardiac membrane-associated AKAPs are a functionally essential part of the regulatory cascade determining IK1 current function and may be novel molecular targets for antiarrhythmic therapy downstream from ß-adrenoreceptors.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Membrana Celular/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/enzimologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Cricetulus , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Células HEK293 , Humanos , Imunoprecipitação , Ativação do Canal Iônico , Potenciais da Membrana , Microinjeções , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ligação Proteica , Ratos , Transfecção , XenopusRESUMO
The rational design of small molecules that mimic key residues at the interface of interacting proteins can be a successful approach to target certain biological signaling cascades causing pathophysiological outcome. The A-Kinase Anchoring Protein, i.e. AKAP-Lbc, catalyses nucleotide exchange on RhoA and is involved in cardiac repolarization. The oncogenic AKAP-Lbc induces the RhoA GTPase hyperactivity and aberrantly amplifies the signaling pathway leading to hypertrophic cardiomyocytes. We took advantage of the AKAP-Lbc-RhoA complex crystal structure to design in silico small molecules predicted to inhibit the associated pathological signaling cascade. We adopted the strategies of pharmacophore building, virtual screening and molecular docking to identify the small molecules capable to target AKAP-Lbc and RhoA interactions. The pharmacophore model based virtual screening unveils two lead compounds from the TIMBAL database of small molecules modulating the targeted protein-protein interactions. The molecular docking analysis revealed the lead compounds' potentialities to establish the essential chemical interactions with the key interactive residues of the complex. These features provided a road map for designing additional potent chemical derivatives and fragments of the original lead compounds to perturb the AKAP-Lbc and RhoA interactions. Experimental validations may elucidate the therapeutic potential of these lead chemical scaffolds to deal with aberrant AKAP-Lbc signaling based cardiac hypertrophy.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Inibidores Enzimáticos/química , Antígenos de Histocompatibilidade Menor/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/química , Desenho de Fármacos , Humanos , Antígenos de Histocompatibilidade Menor/química , Simulação de Acoplamento Molecular , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/química , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/químicaRESUMO
Uncontrolled activation of Rho signaling by RhoGEFs, in particular AKAP13 (Lbc) and its close homologs, is implicated in a number of human tumors with poor prognosis and resistance to therapy. Structure predictions and alanine scanning mutagenesis of Lbc identified a circumscribed hot region for RhoA recognition and activation. Virtual screening targeting that region led to the discovery of an inhibitor of Lbc-RhoA interaction inside cells. By interacting with the DH domain, the compound inhibits the catalytic activity of Lbc, halts cellular responses to activation of oncogenic Lbc pathways, and reverses a number of prostate cancer cell phenotypes such as proliferation, migration, and invasiveness. This study provides insights into the structural determinants of Lbc-RhoA recognition. This is a successful example of structure-based discovery of a small protein-protein interaction inhibitor able to halt oncogenic Rho signaling in cancer cells with therapeutic implications.
Assuntos
Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor/metabolismo , Modelos Moleculares , Estrutura Molecular , Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.
Assuntos
Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteína Quinase Tipo II Dependente de AMP Cíclico/antagonistas & inibidores , Proteína Quinase Tipo I Dependente de AMP Cíclico/antagonistas & inibidores , Peptídeos/química , Inibidores de Proteínas Quinases/química , Subunidades Proteicas/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proteína Quinase Tipo I Dependente de AMP Cíclico/química , Proteína Quinase Tipo I Dependente de AMP Cíclico/metabolismo , Proteína Quinase Tipo II Dependente de AMP Cíclico/química , Proteína Quinase Tipo II Dependente de AMP Cíclico/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Peptídeos/farmacologia , Fosforilação , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoAssuntos
Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Piridinas/química , Proteínas de Ancoragem à Quinase A/metabolismo , Sítios de Ligação , Ligação Competitiva , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Piridinas/síntese química , Piridinas/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
AIM: To determine the mechanism whereby transient stimulation of the ß-adrenergic receptor subtypes (ß-AR) elicit cardioprotection against subsequent ischaemia. METHODS: Isolated rat hearts were subjected to 35 min regional ischaemia (RI) and reperfusion and infarct size (IS) determined. Hearts were preconditioned with 5 min isoproterenol (ß1/ß2-AR agonist), denopamine (ß1-AR agonist), formoterol hemifumarate (ß2-AR agonist) or BRL37344 (ß3-AR agonist) and 5 min reperfusion. The roles of the ß-ARs, NO, PKA, and PI3-K were explored by using selective antagonists/blockers. Pertussis toxin was administered i.p., 48 h prior to experimentation. RESULTS: IS of hearts preconditioned with either isoproterenol, denopamine or formoterol (% of area at risk: 23.6 ± 1.26; 24.52 ± 0.89; 20.74 ± 0.85 respectively) were significantly smaller than that of non-preconditioned hearts (41.7 ± 1.65) and associated with improvement in postischaemic mechanical performance. The ß3-AR agonist BRL37344 could not reduce IS. The ß1- and ß2-AR blockers CGP-20712A and ICI-118551 abolished the reduction in IS and improvement in mechanical recovery during reperfusion induced by isoproterenol preconditioning, while the ß3-AR blocker SR59230A was without effect. Both Rp-8-CPT-cAMPs and wortmannin significantly increased IS when administered before and during ß1/ß2-AR preconditioning and reduced mechanical recovery. PTX pretreatment had no significant effect on the reduction in IS induced by ß1/ß2-AR or ß2-AR preconditioning, but reduced mechanical recovery in ß2-AR preconditioning. Similarly the NOS inhibitors L-NAME and LNNA had no effect on IS in ß1/ß2-AR preconditioning, but depressed mechanical recovery. CONCLUSION: Protection afforded by ß-ARs stimulation, depends on activation of both ß1-AR and ß2-ARs but not ß3-AR. With functional recovery as endpoint, results suggest involvement of NO in ß1/ß2-AR preconditioning and the Gi protein in ß2-AR preconditioning. Both PKA and PI3-K activation were essential for ß1/ß2-AR cardioprotection.
Assuntos
Coração/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Etanolaminas/farmacologia , Fumarato de Formoterol , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Precondicionamento Isquêmico Miocárdico/métodos , Isoproterenol/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos WistarRESUMO
During antigen recognition by T cells, membrane receptors and cytoskeletal molecules form a specialized structure at the T cell-antigen-presenting cell junction called the immune synapse (IS). We report a role for the scaffolding protein A-kinase anchoring protein-450 (AKAP450), a member of the A-kinase anchoring protein family, in IS formation and T-cell signaling in antigen- and superantigen-dependent T-cell activation. Suppression of AKAP450 by overexpression of a dominant-negative form or siRNA knockdown disrupted the positioning and conformational activation of lymphocyte function-associated antigen 1 at the IS and impaired associated signaling events, including phosphorylation of phospholipase C-gamma1 and protein kinase C-. AKAP450 was also required for correct activation and phosphorylation of CD3, LAT, and Vav1, key T-cell receptor-activated intracellular signaling molecules. Consistently, antigen-triggered reorientation of the microtubule-organizing center at the IS and interleukin-2 secretion were diminished in AKAP450-disrupted T cells. These results indicate key roles for AKAP450 in the organization and activation of receptor molecules at the IS during T-cell signaling events.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Complexo CD3/metabolismo , Proteínas do Citoesqueleto/metabolismo , Integrinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/genética , Apresentação de Antígeno , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Linhagem Celular , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Humanos , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/metabolismo , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Centro Organizador dos Microtúbulos/imunologia , Centro Organizador dos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.
Assuntos
Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Placa Motora/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/metabolismo , Actinas/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Denervação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Proteínas Motores Moleculares/metabolismo , Placa Motora/efeitos dos fármacos , Cadeias Pesadas de Miosina/antagonistas & inibidores , Miosina Tipo V/antagonistas & inibidores , Plasticidade Neuronal , Receptores Colinérgicos/metabolismo , Transdução de SinaisRESUMO
A fundamental biologic principle is that diverse biologic signals are channeled through shared signaling cascades to regulate development. Large scaffold proteins that bind multiple proteins are capable of coordinating shared signaling pathways to provide specificity to activation of key developmental genes. Although much is known about transcription factors and target genes that regulate cardiomyocyte differentiation, less is known about scaffold proteins that couple signals at the cell surface to differentiation factors in developing heart cells. Here we show that AKAP13 (also known as Brx-1, AKAP-Lbc, and proto-Lbc), a unique protein kinase A-anchoring protein (AKAP) guanine nucleotide exchange region belonging to the Dbl family of oncogenes, is essential for cardiac development. Cardiomyocytes of Akap13-null mice had deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5-11.0. Disruption of Akap13 was accompanied by reduced expression of Mef2C. Consistent with a role of AKAP13 upstream of MEF2C, Akap13 siRNA led to a reduction in Mef2C mRNA, and overexpression of AKAP13 augmented MEF2C-dependent reporter activity. The results suggest that AKAP13 coordinates Galpha(12) and Rho signaling to an essential transcription program in developing cardiomyocytes.