Epac: new emerging cAMP-binding protein.
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ABSTRACT:
SUBMITTER: Lee K
PROVIDER: S-EPMC8016657 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In the heart, cAMP is a key regulator of excitation-contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) reduced Ca(2+) transient amplitude and increased cell shortening in intact cardiomyocytes and myofilament Ca(2+) sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca(2+) sensitivity compared to control cells infected with green fluorescent protein (GFP) alone. At the molecular level, Epac increased phosphorylation of 2 key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca(2+) sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA and were blocked by protein kinase C (PKC) and Ca(2+) calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.
| S-EPMC2729034 | biostudies-other
Project description:Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP-Epac-YFP fusion construct. When expressed in mammalian cells, CFP-Epac-YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. Thus, by undergoing a cAMP-induced conformational change, CFP-Epac-YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)-based sensor, Epac-based cAMP probes show an extended dynamic range and a better signal-to-noise ratio; furthermore, as a single polypeptide, CFP-Epac-YFP does not suffer from the technical problems encountered with multisubunit PKA-based sensors. These properties make Epac-based FRET probes the preferred indicators for monitoring cAMP levels in vivo.
| S-EPMC1299185 | biostudies-literature
Project description:The signaling molecule cAMP primarily mediates its effects by activating PKA and/or exchange protein activated by cAMP (Epac). Epac has been implicated in many responses in cells, but its precise roles have been difficult to define in the absence of Epac inhibitors. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Using a bioluminescence resonance energy transfer-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-chlorophenylthio-cAMP), and super agonists (8-chlorophenylthio-2'-O-Me-cAMP). The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g. (Rp)-cAMPS and CE3F4, respectively. To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. We used computational molecular modeling to analyze the interaction of analogs with Epac1. The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition.
| S-EPMC3961650 | biostudies-literature
Project description:It has now been over 10 years since efforts to completely understand the signalling actions of cAMP (3'-5'-cyclic adenosine monophosphate) led to the discovery of exchange protein directly activated by cAMP (EPAC) proteins. In the current review we will highlight important advances in the understanding of EPAC structure and function and demonstrate that EPAC proteins mediate multiple actions of cAMP in cells, revealing future targets for pharmaceutical intervention. It has been known for some time that drugs that elevate intracellular cAMP levels have proven therapeutic benefit for diseases ranging from depression to inflammation. The challenge now is to determine which of these positive actions of cAMP involve activation of EPAC-regulated signal transduction pathways. EPACs are specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, which they activate independently of the classical routes for cAMP signalling, cyclic nucleotide-gated ion channels and protein kinase A. Rather, EPAC activation is triggered by internal conformational changes induced by direct interaction with cAMP. Leading from this has been the development of EPAC-specific agonists, which has helped to delineate numerous cellular actions of cAMP that rely on subsequent activation of EPAC. These include regulation of exocytosis and the control of cell adhesion, growth, division and differentiation. Recent work also implicates EPAC in the regulation of anti-inflammatory signalling in the vascular endothelium, namely negative regulation of pro-inflammatory cytokine signalling and positive support of barrier function. Further elucidation of these important signalling mechanisms will no doubt support the development of the next generation of anti-inflammatory drugs.
| S-EPMC2795248 | biostudies-literature
Project description:The identification of 2'-O-methyl substituted adenosine-3',5'-cyclic monophosphate (cAMP) analogs that activate the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs, also known as Epac1 and Epac2), has ushered in a new era of cyclic nucleotide research in which previously unrecognized signalling properties of the second messenger cAMP have been revealed. These Epac-Selective Cyclic AMP Analogs (ESCAs) incorporate a 2'-O-methyl substitution on the ribose ring of cAMP, a modification that impairs their ability to activate protein kinase A (PKA), while leaving intact their ability to activate Epac (the Exchange Protein directly Activated by Cyclic AMP). One such ESCA in wide-spread use is 8-pCPT-2'-O-Me-cAMP. It is a cell-permeant derivative of 2'-O-Me-cAMP, and it is a super activator of Epac. A wealth of newly published studies demonstrate that 8-pCPT-2'-O-Me-cAMP is a unique tool with which to asses atypical actions of cAMP that are PKA-independent. Particularly intriguing are recent reports demonstrating that ESCAs reproduce the PKA-independent actions of ligands known to stimulate Class I (Family A) and Class II (Family B) GTP-binding protein-coupled receptors (GPCRs). This topical review summarizes the current state of knowledge regarding the molecular pharmacology and signal transduction properties of Epac-selective cAMP analogs. Special attention is focused on the rational drug design of ESCAs in order to improve their Epac selectivity, membrane permeability, and stability. Also emphasized is the usefulness of ESCAs as new tools with which to assess the role of Epac as a determinant of intracellular Ca2+ signalling, ion channel function, neurotransmitter release, and hormone secretion.
| S-EPMC2215344 | biostudies-literature
Project description:Fibrosis, the result of excess deposition of extracellular matrix (ECM), in particular collagen, leads to scarring and loss of function in tissues that include the heart, lung, kidney and liver. The second messenger cAMP can inhibit the formation and extent of ECM during this late phase of inflammation, but the mechanisms for these actions of cAMP and of agents that elevate tissue cAMP levels are not well understood. In this article, we review the fibrotic process and focus on two recently recognized aspects of actions of cAMP and its effector Epac (Exchange protein activated by cAMP): (a) blunting of epithelial-mesenchymal transformation (EMT) and (b) down-regulation of Epac expression by profibrotic agents (e.g. TGF-?, angiotensin II), which may promote tissue fibrosis by decreasing Epac-mediated antifibrotic actions. Pharmacological approaches that raise cAMP or blunt the decrease in Epac expression by profibrotic agents may thus be strategies to block or perhaps reverse tissue fibrosis. LINKED ARTICLES This article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-2.
| S-EPMC3417479 | biostudies-literature
Project description:This review focuses on one family of the known cAMP receptors, the exchange proteins directly activated by cAMP (EPACs), also known as the cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs). Although EPAC proteins are fairly new additions to the growing list of cAMP effectors, and relatively "young" in the cAMP discovery timeline, the significance of an EPAC presence in different cell systems is extraordinary. The study of EPACs has considerably expanded the diversity and adaptive nature of cAMP signaling associated with numerous physiological and pathophysiological responses. This review comprehensively covers EPAC protein functions at the molecular, cellular, physiological, and pathophysiological levels; and in turn, the applications of employing EPAC-based biosensors as detection tools for dissecting cAMP signaling and the implications for targeting EPAC proteins for therapeutic development are also discussed.
| S-EPMC6050347 | biostudies-literature
Project description:Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (DeltacAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with DeltacAMP-EPAC1 and DeltaDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.
| S-EPMC1133955 | biostudies-literature
Project description:Leptin regulates energy balance and glucose homeostasis. Shortly after leptin was identified, it was established that obesity is commonly associated with leptin resistance, though the molecular mechanisms remain to be identified. To explore potential mechanisms of leptin resistance, we employed organotypic brain slices to identify candidate signaling pathways that negatively regulate leptin sensitivity. We found that elevation of adenosine 3', 5'-monophosphate (cAMP) levels impairs multiple signaling cascades activated by leptin within the hypothalamus. Notably, this effect is independent of protein kinase A activation. In contrast, activation of Epac, a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap1, was sufficient to impair leptin signaling with concomitant induction of SOCS-3 expression. Epac activation also blunted leptin-induced depolarization of hypothalamic POMC neurons. Finally, central infusion of an Epac activator blunted the anorexigenic actions of leptin. Thus, activation of hypothalamic cAMP-Epac pathway is sufficient to induce multiple indices of leptin resistance.
| S-EPMC3747952 | biostudies-literature
Project description:Evidence collected over the last ten years indicates that Epac and cAMP scaffold proteins play a critical role in integrating and transducing multiple signaling pathways at the basis of cardiac and lung physiopathology. Some of the deleterious effects of Epac, such as cardiomyocyte hypertrophy and arrhythmia, initially described in vitro, have been confirmed in genetically modified mice for Epac1 and Epac2. Similar recent findings have been collected in the lung. The following sections will describe how Epac and cAMP signalosomes in different subcellular compartments may contribute to cardiac and lung diseases.
| S-EPMC5872357 | biostudies-literature