Cyclic AMP-dependent protein kinase and Epac mediate cyclic AMP responses in pancreatic acini

The pancreatic acinar cell has several phenotypic responses to cAMP agonists. At physiological concentrations of the muscarinic agonist carbachol (1 microM) or the CCK analog caerulein (100 pM), ligands that increase cytosolic Ca(2+), cAMP acts synergistically to enhance secretion. Supraphysiological concentrations of carbachol (1 mM) or caerulein (100 nM) suppress secretion and cause intracellular zymogen activation; cAMP enhances both zymogen activation and reverses the suppression of secretion. In addition to stimulating cAMP-dependent protein kinase (PKA), recent studies using cAMP analogs that lack a PKA response have shown that cAMP can also act through the cAMP-binding protein, Epac (exchange protein directly activated by cyclic AMP). The roles of PKA and Epac in cAMP responses were examined in isolated pancreatic acini. The activation of both cAMP-dependent pathways or the selective activation of Epac was found to enhance amylase secretion induced by physiological and supraphysiological concentrations of the muscarinic agonist carbachol. Similarly, activation of both PKA or the specific activation of Epac enhanced carbachol-induced activation of trypsinogen and chymotrypsinogen. Disorganization of the apical actin cytoskeleton has been linked to the decreased secretion observed with supraphysiological concentrations of carbachol and caerulein. Although stimulation of PKA and Epac or Epac alone could largely overcome the decreased secretion observed with either supraphysiological carbachol or caerulein, stimulation of cAMP pathways did not reduce the disorganization of the apical cytoskeleton. These studies demonstrate that PKA and Epac pathways are coupled to both secretion and zymogen activation in the pancreatic acinar cell.     

Identification and Validation of Modulators of Exchange Protein Activated by cAMP (Epac) Activity: STRUCTURE-FUNCTION IMPLICATIONS FOR Epac ACTIVATION AND INHIBITION*

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. (R_p)-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.     

Epac proteins: multi-purpose cAMP targets

Epac1 and Epac2 are cAMP-dependent guanine-nucleotide-exchange factors for the small GTPases Rap1 and Rap2, and are known to be important mediators of cAMP signaling. The recent determination of the crystal structure of Epac2 has indicated a mechanism for the activation of the multi-domain Epac proteins. In addition, these proteins have been implicated in various cellular processes such as integrin-mediated cell adhesion and cell-cell junction formation, the control of insulin secretion and neurotransmitter release. In most of these processes, cAMP signaling through protein kinase A (PKA) is also involved, stressing the interconnectivity between Epac- and PKA-mediated signaling.     

cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells

Pulsatile insulin release from glucose-stimulated β-cells is driven by oscillations of the Ca²⁺ and cAMP concentrations in the subplasma membrane space ([Ca²⁺]_pm and [cAMP]_pm). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP]_pm, [Ca²⁺]_pm, and phosphatidylinositol 3,4,5-trisphosphate (PIP₃) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 β-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca²⁺]_pm, [cAMP]_pm, and PIP₃. Inhibitors of protein kinase A (PKA) markedly diminished the PIP₃ response when applied before glucose stimulation, but did not affect already manifested PIP₃ oscillations. The reduced PIP₃ response could be attributed to accelerated depolarization causing early rise of [Ca²⁺]_pm that preceded the elevation of [cAMP]_pm. However, the amplitude of the PIP₃ response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP₃ oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP₃ oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca²⁺]_pm and amplifying [cAMP]_pm signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.     

Critical role of the N‐terminal cyclic AMP‐binding domain of Epac2 in its subcellular localization and function

cAMP is a well‐known regulator of exocytosis, and cAMP‐GEFII (Epac2) is involved in the potentiation of cAMP‐dependent, PKA‐independent regulated exocytosis in secretory cells. However, the mechanisms of its action are not fully understood. In the course of our study of Epac2 knockout mice, we identified a novel splicing variant of Epac2, which we designate Epac2B, while renaming the previously identified Epac2 Epac2A. Epac2B, which lacks the first cAMP‐binding domain A in the N‐terminus but has the second cAMP‐binding domain B of Epac2A, possesses GEF activity towards Rap1, as was found for Epac2A. Immunocytochemical analysis revealed that exogenously introduced Epac2A into insulin‐secreting MIN6 cells was localized near the plasma membrane, while Epac2B was found primarily in the cytoplasm. Interestingly, cAMP‐binding domain A alone introduced into MIN6 cells was also localized near the plasma membrane. In MIN6 cells, Epac2A was involved in triggering hormone secretion by stimulation with 5.6 mM glucose plus 1 mM 8‐Bromo‐cAMP, but Epac2B was not. The addition of a membrane‐targeting signal to the N‐terminus of Epac2B was able to mimic the effect of Epac2A on hormone secretion. Thus, the present study indicates that the N‐terminal cAMP‐binding domain A of Epac2A plays a critical role in determining its subcellular localization and potentiating insulin secretion by cAMP. J. Cell. Physiol. 219: 652–658, 2009. © 2009 Wiley‐Liss, Inc.     

Ras is required for the cyclic AMP-dependent activation of Rap1 via Epac2

Exchange proteins activated by cAMP (cyclic AMP) 2 (Epac2) is a guanine nucleotide exchange factor for Rap1, a small G protein involved in many cellular functions, including cell adhesion, differentiation, and exocytosis. Epac2 interacts with Ras-GTP via a Ras association (RA) domain. Previous studies have suggested that the RA domain was dispensable for Epac2 function. Here we show for the first time that Ras and cAMP regulate Epac2 function in a parallel fashion and the Ras-Epac2 interaction is required for the cAMP-dependent activation of endogenous Rap1 by Epac2. The mechanism for this requirement is not allosteric activation of Epac2 by Ras but the compartmentalization of Epac2 on the Ras-containing membranes. A computational modeling is consistent with this compartmentalization being a function of both the level of Ras activation and the affinity between Ras and Epac2. In PC12 cells, a well-established model for sympathetic neurons, the Epac2 signaling is coupled to activation of mitogen-activated protein kinases and contributes to neurite outgrowth. Taken together, the evidence shows that Epac2 is not only a cAMP sensor but also a bona fide Ras effector. Coincident detection of both cAMP and Ras signals is essential for Epac2 to activate Rap1 in a temporally and spatially controlled manner.     

Ligand-mediated activation of the cAMP-responsive guanine nucleotide exchange factor Epac

Epac is a cAMP-dependent exchange factor for the small GTP-binding protein Rap. The activity of Epac is inhibited by a direct interaction between the C-terminal helical part of the cAMP-binding domain, called the lid, and the catalytic region, which is released after binding of cAMP. Herein, we show that the activation properties are very sensitive to modifications of the cyclic nucleotide. Some analogues are inhibitory and others are stimulatory; some are characterized by a much higher activation potential than normal cAMP. Mutational analysis of Epac allows insights into a network of interactions between the cyclic nucleotides and Epac. Mutations in the lid region are able to amplify or to attenuate selectively the activation potency of cAMP analogues. The properties of cAMP analogues previously used for the activation of the cAMP responsive protein kinase A and of 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclicmonophosphate, an analogue highly selective for activation of Epac were investigated in detail.     

cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension

Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAPKII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N6-benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2'-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2'-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10-100-fold. Phenylthio substituents in position 8, particularly with MeO- or Cl- in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT- and 2'-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2'-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.     

The (R)-enantiomer of CE3F4 is a preferential inhibitor of human exchange protein directly activated by cyclic AMP isoform 1 (Epac1)

Isoform 1 and isoform 2 of exchange protein directly activated by cAMP (Epac1 and Epac2) contribute to cAMP signaling in numerous cellular processes. Their guanine-nucleotide exchange factor (GEF) activity toward the small GTP-binding protein Rap1 is stimulated by the agonist cAMP. CE3F4, a tetrahydroquinoline analog, prevents Epac1 activation in vitro and in living cultured cells by inhibiting the GEF activity of Epac1. However, the activity of the (R)- and (S)-enantiomers of CE3F4, as well as the ability of CE3F4 and its analogs to inhibit Epac2 GEF activity, have not yet been investigated. In this study, we report that (R)-CE3F4 is a more potent cAMP antagonist than racemic CE3F4 and (S)-CE3F4, inhibiting the GEF activity of Epac1 with 10-times more efficiency than (S)-CE3F4. Epac2, in contrast to Epac1, is activated more efficiently by cAMP than by 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (007), an Epac-selective cAMP analog. (R)-CE3F4 displays Epac isoform preference, with 10-fold selectivity for Epac1 over Epac2. Deletion of the N-terminal cyclic nucleotide-binding domain of Epac2 does not affect the characteristics of activation of Epac2 by cAMP and by 007, nor its inhibition by CE3F4. Finally, the evaluation of a series of CE3F4 structural analogs as GEF inhibitors allowed identifying structural features that are important for high Epac1 inhibitory activity of CE3F4. We conclude that the (R)-enantiomer of CE3F4 is a preferential inhibitor of Epac1 with high potency in the low micromolar range, and we suggest that this compound may be a useful pharmacological tool for investigating the functional role of Epac1 in cAMP signaling.     

Off-target effect of the Epac agonist 8-pCPT-2′-O-Me-cAMP on P2Y12 receptors in blood platelets

The primary target of the cAMP analogue 8-pCPT-2′-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2′-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2′-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2′O-Me-cAMP binding into the purinergic platelet receptor P2Y₁₂ revealed that the analogue docks similar to the P2Y₁₂ antagonist 2MeSAMP. The 8-pCPT-2′-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2′-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2′-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y₁₂ receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2′-O-Me-cAMP did not affect platelet activation at similar concentrations.     

Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1

Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.     

Glucocorticoid-induced changes in insulin secretion related to the metabolism and ultrastructure of pancreatic islets

The effect of adrenalectomy and dexamethasone-treatment on insulin secretion was studied and related to the changes observed in the glucose oxidation, calcium uptake, cAMP and insulin content, as well as the ultrastructure of pancreatic rat islets. It was found that adrenalectomy was followed by a decreased glucose-induced insulin secretion, glucose oxidation, calcium uptake, cAMP and insulin content without any remarkable change observed at the ultrastructural level. Conversely, adrenalectomized-rats supplemented with dexamethasone showed an increased glucose-induced insulin secretion, glucose oxidation, calcium uptake and cAMP content but a diminished islet insulin content. At the ultrastructural level, a clear picture of increased secretory activity was found, with diminished number of mature B granules and greater number of pale granules, while rough endoplasmic reticulum and Golgi complex frequently appeared hypertrophic. These changes were only observed in the B cells. On account of our results, we might suggest that insulin secretion is partially controlled by glucocorticoid circulating levels throughout their effect on pancreatic islet metabolism.     

Mechanism of Epac Activation: STRUCTURAL AND FUNCTIONAL ANALYSES OF Epac2 HINGE MUTANTS WITH CONSTITUTIVE AND REDUCED ACTIVITIES*

Epac2 is a member of the family of exchange proteins directly activated by cAMP (Epac). Our previous studies suggest a model of Epac activation in which cAMP binding to the enzyme induces a localized “hinge” motion that reorients the regulatory lobe relative to the catalytic lobe without inducing large conformational changes within individual lobes. In this study, we identified the location of the major hinge in Epac2 by normal mode motion correlation and structural alignment analyses. Targeted mutagenesis was then performed to test the functional importance of hinge bending for Epac activation. We show that substitution of the conserved residue phenylalanine 435 with glycine (F435G) facilitates the hinge bending and leads to a constitutively active Epac2 capable of stimulating nucleotide exchange in the absence of cAMP. In contrast, substitution of the same residue with a bulkier side chain, tryptophan (F435W), impedes the hinge motion and results in a dramatic decrease in Epac2 catalytic activity. Structural parameters determined by small angle x-ray scattering further reveal that whereas the F435G mutant assumes a more extended conformation in the absence of cAMP, the F435W mutant is incapable of adopting the fully extended and active conformation in the presence of cAMP. These findings demonstrate the importance of hinge motion in Epac activation. Our study also suggests that phenylalanine at position 435 is the optimal size side chain to keep Epac closed and inactive in the absence of cAMP while still allowing the proper hinge motion for full Epac extension and activation in the presence of cAMP.Exchange proteins directly activated by cAMP (Epac)² are a family of novel intracellular sensors for the second messenger cAMP (1, 2). Unlike the classic eukaryotic cAMP receptor, cAMP-dependent protein kinase (protein kinase A; PKA), Epac proteins do not function as protein kinases that phosphorylate downstream substrates. Instead, they act as guanine nucleotide exchange factors and exert their functions by activating small GTP-binding proteins, Rap1 and Rap2. At the cellular level, Epac proteins assume distinct subcellular localization (3), and depending upon the specific cellular environment, Epac and PKA may act independently, converge synergistically, or oppose each other in regulating a specific cellular function (4, 5).Both Epac and PKA share a common cyclic nucleotide binding domain (CBD), a compact and evolutionarily conserved structural motif found in a variety of proteins with diverse cellular functions (6). CBDs act as molecular switches for sensing intracellular second messenger cAMP levels and regulate the functionality of the domain(s) to which they are linked (6, 7). In depth structural and biophysical analyses of CBDs in several CBD-containing families, including cAMP receptor proteins, PKAs, and cyclic nucleotide-gated ion channels, have revealed a conserved structural core as well as functional motifs important for cyclic nucleotide-induced activation (8–11). The CBD is composed of an eight-strand β-barrel core that forms the base of the nucleotide binding pocket and a lateral α-helical bundle subdomain. Although the β-barrel core remains relatively constant between the ligand-free and nucleotide-bound states, binding of cAMP to a CBD leads to significant conformational changes in the overall arrangement of the α-helical bundle subdomain. A general mechanism of cyclic nucleotide-induced activation of CBD-containing proteins has been recently proposed (12). In this model, binding of cAMP leads to the retraction of the phosphate-binding cassette toward the cyclic nucleotide binding pocket and consequently releases the steric hindrance imposed on the α-helix C-terminal to the β-barrel, termed the CBD lid, by a conserved hydrophobic residue within the phosphate-binding cassette. These structural changes result in a hinge motion that allows the lid to move closer to the β-barrel core and to interact with the base of the cyclic nucleotide.The recently solved crystal structure of Epac2 reveals that, unlike other CBD-containing proteins, the corresponding lid region in Epac points away from the cAMP binding pocket as a two-strand β-sheet that forms the first part of the five-strand β-sheet “switchboard” structure (13). Although this major structural difference suggests that the detailed mechanisms of PKA and Epac activation by cAMP will most likely be different at the atomic level, it is not clear if the aforementioned general mechanism, namely the hinge motion, is conserved during Epac activation. To address this important question, we determined the effects of targeted hinge perturbations on Epac activation using site-directed mutagenesis that specifically targeted a key residue in the hinge region of Epac2.     

Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator

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.     

Cyclic adenosine 5'-monophosphate-stimulated neurotensin secretion is mediated through Rap1 downstream of both Epac and protein kinase A signaling pathways

Neurotensin (NT), a gut peptide, plays important roles in gastrointestinal secretion, inflammation, and growth of normal and neoplastic tissues. cAMP regulates the secretion of hormones via its effector proteins protein kinase A (PKA) or Epac (exchange protein directly activated by cAMP). The small GTPase Rap1 can be activated by both PKA and Epac; however, the role of Rap1 in hormone secretion is unknown. Here, using the BON human endocrine cell line, we found that forskolin (FSK)-stimulated NT secretion was reduced by inhibition of Rap1 expression and activity. FSK-stimulated NT secretion was enhanced by overexpression of either wild-type or constitutively active Rap1. Epac activators and wild-type Epac enhanced NT release and Rap1 activity. In contrast, overexpression of a cAMP binding mutant, EpacR279E, decreased NT release and Rap1 activity. PKA activation increased NT release and Rap1 activity. FSK-stimulated NT release was reduced by PKA inhibition and the dominant negative Rap1N17. NT secretion, stimulated by Epac activation, was reduced by PKA inhibition; NT release, stimulated by PKA activation, was enhanced by wild-type Epac but reduced by the mutant EpacR279E. Finally, prostaglandin E2 (PGE2), a physiological agent that increases cAMP, stimulated NT secretion via cAMP/PKA/Rap1. Importantly, we demonstrate that PKA and Epac mediate the cAMP-induced NT secretion synergistically by converging at the common downstream target protein Rap1. Moreover, PGE2, a potent mediator of inflammation and associated with colorectal carcinogenesis, stimulates NT release suggesting a possible link between PGE2 and NT on intestinal inflammatory disorders and colorectal cancers.     

Cyclic AMP-binding protein Epac1 acts as a metabolic sensor to promote cardiomyocyte lipotoxicity

Cyclic adenosine monophosphate (cAMP) is a master regulator of mitochondrial metabolism but its precise mechanism of action yet remains unclear. Here, we found that a dietary saturated fatty acid (FA), palmitate increased intracellular cAMP synthesis through the palmitoylation of soluble adenylyl cyclase in cardiomyocytes. cAMP further induced exchange protein directly activated by cyclic AMP 1 (Epac1) activation, which was upregulated in the myocardium of obese patients. Epac1 enhanced the activity of a key enzyme regulating mitochondrial FA uptake, carnitine palmitoyltransferase 1. Consistently, pharmacological or genetic Epac1 inhibition prevented lipid overload, increased FA oxidation (FAO), and protected against mitochondrial dysfunction in cardiomyocytes. In addition, analysis of Epac1 phosphoproteome led us to identify two key mitochondrial enzymes of the the β-oxidation cycle as targets of Epac1, the long-chain FA acyl-CoA dehydrogenase (ACADL) and the 3-ketoacyl-CoA thiolase (3-KAT). Epac1 formed molecular complexes with the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), which phosphorylated ACADL and 3-KAT at specific amino acid residues to decrease lipid oxidation. The Epac1-CaMKII axis also interacted with the α subunit of ATP synthase, thereby further impairing mitochondrial energetics. Altogether, these findings indicate that Epac1 disrupts the balance between mitochondrial FA uptake and oxidation leading to lipid accumulation and mitochondrial dysfunction, and ultimately cardiomyocyte death.Subject terms: Mechanisms of disease, Cardiomyopathies     

Critical role of cAMP-GEFII--Rim2 complex in incretin-potentiated insulin secretion

Incretins such as glucagon-like peptide-1 and gastric inhibitory polypeptide/glucose-dependent insulinotropic peptide are known to potentiate insulin secretion mainly through a cAMP/protein kinase A (PKA) signaling pathway in pancreatic beta-cells, but the mechanism is not clear. We recently found that the cAMP-binding protein cAMP-GEFII (or Epac 2), interacting with Rim2, a target of the small G protein Rab3, mediates cAMP-dependent, PKA-independent exocytosis in a reconstituted system. In the present study, we investigated the role of the cAMP-GEFII--Rim2 pathway in incretin-potentiated insulin secretion in native pancreatic beta-cells. Treatment of pancreatic islets with antisense oligodeoxynucleotides (ODNs) against cAMP-GEFII alone or with the PKA inhibitor H-89 alone inhibited incretin-potentiated insulin secretion approximately 50%, while a combination of antisense ODNs and H-89 inhibited the secretion approximately 80-90%. The effect of cAMP-GEFII on insulin secretion is mediated by Rim2 and depends on intracellular calcium as well as on cAMP. Treatment of the islets with antisense ODNs attenuated both the first and second phases of insulin secretion potentiated by the cAMP analog 8-bromo-cAMP. These results indicate that the PKA-independent mechanism involving the cAMP-GEFII--Rim2 pathway is critical in the potentiation of insulin secretion by incretins.     

Direct Spatial Control of Epac1 by Cyclic AMP

Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.Cyclic AMP (cAMP) is an important second messenger that mediates many cellular hormone responses. It has become more and more appreciated that, along with the cAMP effector protein kinase A (PKA), Epac proteins also play pivotal roles in many cAMP-controlled processes, including insulin secretion (23, 39), cell adhesion (9, 17, 25, 49, 60), neurotransmitter release (22, 53, 63), heart function (13, 35, 54), and circadian rhythm (38). Epac1 and Epac2 are cAMP-dependent guanine nucleotide exchange factors (GEFs) for the small G proteins Rap1 and Rap2 (12, 24). They contain a regulatory region with one (Epac1) or two (Epac2) cAMP-binding domains, a Dishevelled, Egl-10, Pleckstrin (DEP) domain, and a catalytic region for GEF activity (11). The binding of cAMP is a prerequisite for catalytic activity in vitro and in vivo (11). Recently, the structures of both the inactive and active conformations of Epac2 were solved (51, 52). This revealed that in the inactive conformation, the regulatory region occludes the Rap binding site, which is relieved by a conformational change induced by cAMP binding.Like all G proteins of the Ras superfamily, Rap cycles between an inactive GDP-bound and active GTP-bound state in an equilibrium that is tightly regulated by specific GEFs and GTPase-activating proteins (GAPs). The GEF-induced dissociation of GDP results in the binding of the cellularly abundant GTP, whereas GAPs enhance the intrinsic GTPase activity of the G protein, thereby inducing the inactive GDP-bound state. Besides Epac, several other GEFs for Rap have been identified, including C3G, PDZ-GEF, and RasGRP, and these act downstream of different signaling pathways (7). Since Rap localizes to several membrane compartments, including the Golgi network, vesicular membranes, and the plasma membrane (PM) (2-4, 37, 42, 48), the spatial regulation of its activity is expected to be established by the differential distributions of its upstream GEFs, each activating distinct pools of Rap on specific intracellular locations.Similarly to Rap, Epac1 also is observed at many locations in the cell, including the cytosol, the nucleus, the nuclear envelope, endomembranes, and the PM (5, 11, 14, 21, 29, 47). These various locations may reflect the many different functions assigned to Epac1, such as the regulation of cell adhesion, cell junction formation, secretion, the regulation of DNA-dependent protein kinase by nuclear Epac1, and the regulation of the Na⁺/H⁺ exchanger NHE3 at the brush borders of kidney epithelium (19, 21, 26). Apparently, specific anchors are responsible for this spatial regulation of Epac1. Indeed, Epac1 was found to associate with phosphodiesterase 4 (PDE4) in a complex with mAKAP in cardiomyocytes (13), with MAP-LC bound to microtubules (62), and with Ezrin at the brush borders of polarized cells (M. Gloerich, J. Zhao, and J. L. Bos, unpublished data).In this study, we report the unexpected observation that, in addition to the temporal control of Epac1 activity, cAMP also induces the translocation of Epac1 toward the plasma membrane. Using confocal fluorescence microscopy, total internal reflection fluorescence (TIRF) microscopy, and fluorescence resonance energy transfer (FRET)-based assays for high spatial and temporal resolution, we observed that the translocation of Epac1 is immediate and that Epac1 approaches the PM to within ∼7 nm. In line with this, Epac1-induced Rap activation was registered predominantly on this compartment. Epac1 translocation results directly from the cAMP-induced conformational change and depends on the integrity of its DEP domain. We further show that Epac1 translocation is a prerequisite for cAMP-induced Rap activation at the PM and enhances Rap-mediated cell adhesion. Thus, cAMP exerts dual regulation on Epac1 for the activation of Rap, controlling both its GEF activity and targeting to the PM.