Acta Biochim Biophys Sin 2008, 40: 651-662
Epac and PKA: a tale of two intracellular cAMP receptors
Xiaodong Cheng*, Zhenyu Ji, Tamara Tsalkova, and Fang Mei
Department of Pharmacology and Toxicology, Sealy Center for Cancer Cell Biology and Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-1031, USA
Received: May 6, 2008
Accepted: May 21, 2008
This work was supported by grants from the National Institutes of Health (No. GM061770) and the American Heart Association (No. 0755049Y)
*corresponding author: Tel, 1-409-772-9656; Fax, 1-409-772-9642; E-mail, email@example.com
cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions, including diabetes, heart failure and cancer. In eukaryotic cells, the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by camp (Epac)/cAMP-regulated guanine nucleotide exchange factors. Like PKA, Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions. The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner. Depending upon the specific cellular environments as well as their relative abundance, distribution and localization, Epac and PKA may act independently, converge synergistically or oppose each other in regulating a specific cellular function.
Keywords cAMP; exchange protein directly activated by cAMP (Epac)/cAMP-regulated guanine exchange factor; protein kinase A (PKA)/cAMP-dependent protein kinase; signal transduction.
Overview of the cAMP second messenger system
Eukaryotic cells respond to a wide range of extracellular signals, including hormones, growth factors and neurotransmitters, by eliciting the generation of intracellular second messengers. Second messengers in turn trigger a myriad of cellular reactions by orchestrating a network of intracellular signaling events. The discovery of cAMP 50 years ago marked the birth of second messenger theory and the age of signal transduction. cAMP regulates many physiological processes ranging from learning and memory in the brain, as well as contractility and relaxation in the heart, to water uptake in the gut and kidney. At the cellular level, cAMP plays an important role in virtually every known function, such as metabolism, gene expression, cell division and growth, cell differentiation, apoptosis, secretion and neurotransmission. In addition to regulating many important cellular processes directly, cAMP is also implicated in an array of cross-talks between intracellular signaling pathways. For example, cAMP exerts its growth effects through interactions with the Ras-mediated mitogen-activated protein kinase pathways [1-5]. There is evidence that suggests that cAMP cross talks with the Ca2+-dependent signaling pathway [6,7]. It has also been reported that cAMP can potentially modulate cytokine signaling through inhibiting the Jak/STAT pathway . cAMP signaling is closely interwoven with the phosphatidylinositol-3 kinase/protein kinase B (PKB) pathway [9,10].
For many years, the consensus was that the cAMP-mediated signaling in eukaryotic cells, which involves the sequential activation of a series of signaling molecules consisting of both plasma membrane and intracellular components, existed as a linear pathway. Upon binding of ligand, the G-protein-coupled receptor at the cell surface transduces the extracellular signal across the cell membrane via stimulatory or inhibitory heterotrimeric G-proteins that interact with the membrane-bound adenylate cyclase to regulate cAMP production inside the cell.
It was believed until recently that the major effects of cAMP in mammalian cells, with the exception of cyclic nucleotide-gated ion channels in photoreceptor cells, olfactory sensory neurons and cardiac sinoatrial node cells , were mediated intracellularly by protein kinase A (PKA), also known as cAMP-dependent protein kinase.
Protein kinase A
PKA was one of the first protein kinases to be discovered . Unlike most eukaryotic protein kinases, PKA is composed of two separate subunits: the catalytic (C) and regulatory (R) subunits. The C subunit is initially phosphorylated by phosphoinositide-dependent protein kinase at an essential phosphorylation site Threonine 197 (T197) [13,14]. Phosphorylation of T197 in the activation loop is necessary for the maturation and optimal biological activity of PKA [15,16]. However, once phosphorylated, the C subunit of PKA is fully active, and the T197 phosphate does not turn over readily . The C subunit of PKA is then regulated via interaction with the inhibitory R subunit, a major intracellular cAMP receptor that sequesters the C subunit in an inactive heterotetrameric holoenzyme, R2C2. The activating ligand cAMP binds to the R subunit and induces conformational changes that lead to the dissociation of the holoenzyme into its constituent C and R subunits . The free active C subunit can then affect a range of diverse cellular events by phosphorylating an array of cytoplasmic and nuclear protein substrates, including enzymes and transcriptional factors .
There are two general classes of PKA, designated as PKA(I) and PKA(II), that are distinguished by differences in the R subunits, RI and RII, which interact with an identical C subunit . Four different R subunit genes, RIa , RIb , RIIa , and RIIb  have been identified. Three C subunit genes, Ca, Cb, and Cg have also been discovered. However, preferential expression of any of these C subunits with either RI or RII has not been found . While both RI and RII contain two tandem and highly conserved cAMP binding domains (CBD) at the C-terminus , RI and RII differ significantly at their amino terminus, especially at the proteolytically sensitive hinge region that binds to the peptide recognition site of the C subunit. The hinge region of the RII subunits contains a serine at the P site that can be auto-phosphorylated by the C subunit , whereas RI contains a pseudo-phosphorylation site.
R isoforms are differentially expressed in tissues [27-29], and their subcellular distribution also appears to be distinct [30-33]. The existence of a family of A-kinase anchoring proteins (AKAP) that tether RII subunits to specific subcellular structures has been well documented , and the majority of AKAPs preferentially bind RII subunits. However, AKAPs specific to both RI and RII have also been identified recently . These kinase anchoring proteins interact exclusively with the dimerization domain of the R subunits, and only the first 50 N-terminal amino acid residues of the R subunits are required for binding of AKAPs . The extensive sequence diversity at this region between RI and RII may account for the difference in their AKAP binding affinities. Large numbers of AKAPs have been identified. Compartmentalization of PKA molecules to discrete intracellular locations through association with anchoring proteins may ensure specificity in signal transduction by placing the kinase close to its appropriate effectors or substrates .
While the ratio of the total R subunits:C subunits in normal tissue was found to be relatively constant at around 1:1, the relative amount of RI and RII varies and depends highly on physiological conditions and the hormonal status of the tissue [28,29,37,38]. One study showed that, in knockout mice lacking the gene encoding RIIb, an increased level of RIa compensates for the loss of RIIb in brown fat cells. The switching of PKA isoform from PKA(IIb) to PK(Ia) results in an elevated basal level of PKA activity and increased energy expenditure. The RIIb knockout mice are leaner and protected against diet-induced obesity . These results clearly demonstrate that RIa and RIIb are functionally distinct. Although many of the physiologic effects of cAMP can be ascribed to the action of one or more of the PKA isoforms, some of the cAMP-dependent effects can not be explained based on the functions of PKA. For example, the ability of cAMP to enhance the secretion of insulin from pancreatic beta cells is not affected by specific inhibitors of PKA . Many similar experimental observations have hinted at the existence of "PKA-independent" mechanisms of cAMP action.
Epac, a new intracellular cAMP receptor
Recently, a family of novel cAMP sensor proteins, named exchange protein directly activated by camp (Epac) or cAMP-regulated guanine exchange factor (cAMP-GEF), was identified [41,42]. These proteins contain a CBD that is homologous to that of PKA R subunits and the prokaryotic transcription regulator, cAMP receptor protein (CRP) (Fig. 1). Epac proteins bind to cAMP with high affinity and activate the Ras superfamily small GTPases Rap1 and Rap2. Rap1 was initially identified as an antagonist for the transforming function of Ras . It can be activated in response to a variety of second messengers, including cAMP . Although PKA can phosphorylate Rap1 at its C-terminus, PKA phosphorylation is not required for cAMP-dependent activation of Rap1 .
There are two isoforms of Epac, Epac1 and Epac2, which are products of independent genes in mammals. While Epac1 is ubiquitously expressed in all tissues, Epac2 has a more limited distribution [41,42]. Epac1 and Epac2 share extensive sequence homology, and both contain an N-terminal regulatory region and a C-terminal catalytic region. The catalytic region of Epac1 consists of a Ras exchange motif domain, Ras association domain and a classic CDC25-homology domain responsible for nucleotide exchange activity. Whereas the regulatory region of Epac1 and Epac2 shares a Dishevelled/Egl-10/pleckstrin (DEP) domain followed by a CBD domain that is evolutionally conserved to the CBD of PKA and the bacterial transcriptional factor CRP, an additional CBD N-terminal to the DEP domain is presented in Epac2 (Fig. 1). The function of this extra CBD domain is not clear, as it binds cAMP with low affinity and does not seem to be essential for Epac2 regulation by cAMP .
Cellular functions regulated by Epac
The discovery of Epac proteins as a new family of intracellular cAMP receptors suggests that the cAMP-mediated signaling mechanism is much more complex than what was believed earlier. Many cAMP-mediated effects that were previously thought to act through PKA alone may also be transduced by Epac. Extensive studies have so far established that Epac proteins are involved in a host of cAMP-related cellular functions, such as cell adhesion [46,47], cell-cell junction [48,49], exocytosis/secretion [50-53], cell differentiation  and proliferation, gene expression, apoptosis, cardiac hypertrophy and phagocytosis. With the exception of a few preliminary reports of Epac knockout in fly and worm models, so far no detailed in vivo genetic and function analyses of either Epac isoform in an animal model system have been reported. Our discussion of Epac抯 biological functions will mainly be based on ex vivo studies in cell culture models.
Epac and cell adhesion
One of the first cellular functions attributed to Epac is its ability to enhance cell adhesion. When Epac is ectopically overexpressed in HEK293 cells, it induces flattened cell morphology and increases cell adhesion . This is not surprising since one of the major functions of Rap1, a downstream effector of Epac, is control of cell morphology/adhesion [56,57]. A study using an Epac-selective cAMP analog, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate , suggests that activation of Epac induces Rap-dependent integrin-mediated cell adhesion to fibronectin in Ovcar3, a human ovarian carcinoma cell line . Subsequent analysis further revealed that the cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the a3b1 integrin but not through the a6b4 integrin . Interaction between Epac1 and light chain 2 of the microtubule-associated protein 1A enhances Rap1-dependent cell adhesion to laminin . Activation of Epac1 increases the b2-integrin-dependent adhesion of human endothelial progenitor cells to endothelial cell monolayers and to ICAM-1, as well as the b1-integrin-dependent adhesion of human endothelial progenitor cells and mesenchymal stem cells to the matrix protein fibronectin . These results demonstrate Epac's therapeutic potential via enhancing integrin-dependent homing functions of progenitor cells.
Interestingly, cAMP-Epac1-Rap1 signaling also stimulates sickle red blood cells adhesion to lammin. However, the adhesion of sickle red blood cells to lammin promoted by Epac-Rap1 is not dependent on integrin, but it is mediated by the cell adhesion molecule/Lutheran receptor, a member of the Ig superfamily of receptors . Consistent with the stimulatory effect of Epac1-Rap1 on cell adhesion, activation of Epac1 inhibits epithelial cell migration, which requires the disruption of cell-cell adhesion, in response to both hepatocyte growth factor and transforming growth factor b (TGFb) . Direct interaction between Epac1 and type I TGFb receptor has been reported and may be responsible for the observed inhibitory effect of Epac1 on TGFb-mediated cell migration .
While the effects of cAMP on cell adhesion are reported to be PKA independent, cAMP-regulated integrin-dependent adhesions of vascular endothelial cells to extracellular matrix proteins are coordinated by both PKA and Epac . In human primary monocytes and in monocytic U937 cells, Epac1-Rap1 has been shown to regulate b1-integrin-dependent cell adhesion, cell polarization and chemotaxis . However, a similar study showed that, although Epac1 is expressed in human peripheral monocytes and activates Rap1, cAMP modulates most monocyte immune functions through PKA and not Epac1-Rap1 . Therefore, it appears that the roles of Epac1 and PKA in monocytes also remain unsettled.
Epac and cell junctions
In addition to its effects on integrin-mediated adhesion, Epac1/Rap1 signaling has also shown to contribute to E-cadherin-mediated adhesion . This is consistent with the fact that Rap1 plays an important role in the formation of cell-cell junctions . Stable cell-cell contacts are critical for the barrier function of epithelial and endothelial cells. Endothelial cell junctions are of central importance for regulating vascular permeability. It is well established that cAMP enhances the formation of cell junctions and endothelial barrier function. cAMP decreases basal permeability and reverse vascular leakage induced by inflammatory mediators. Previously, it was believed that cAMP exerted its effects through activation of PKA. However, inhibition of PKA activity does not block cAMP-enhanced endothelial cell barrier function, suggesting the existence of PKA-independent pathways.
Several studies in human umbilical vein endothelial cells now show that Epac1 induces junction formation and actin remodeling, and reduces endothelial permeability through activating Rap1, which is enriched at endothelial cell-cell contacts [48,49,69]. Activation of Epac leads to enhanced basal endothelial barrier function by increasing cortical actin and redistributing adherens and tight junctional molecules to cell-cell contacts. Moreover, activation of Epac offsets thrombin-induced hyperpermeability through down-regulation of Rho GTPase activation . Using VE-cadherin null mouse cells immortalized with polyoma mT, Kooistra et al demonstrated that regulation of endothelial permeability by Epac1 requires VE-cadherin and that Epac-specific cAMP analog-induced actin rearrangements are independent of cell junction formation . Recently, it was shown that Epac1 can directly promote microtubule (MT) growth independent of Rap1 activation  and that Epac activation reverses MT-dependent increases in vascular permeability induced by tumor necrosis factor-a and TGFb. Therefore, it appears that Epac1 promotes endothelial barrier function through a two-leg strategy: a Rap1-depedent increase in cortical actin and a Rap-independent regulation of MTs .
Studies using human pulmonary artery endothelial cells show that barrier-protective effects of cAMP, downstream of Prostaglandin E2, prostacyclin and atrial natriuretic peptide, on pulmonary endothelial cells are mediated by both PKA and Epac pathways. Activation of PKA and Epac/Rap1 converges on Rac activation via stimulation of Rac-specific GEFs Tiam1 and Vav2, leading to the enhancement of peripheral actin cytoskeleton and adherens junctions [72,73]. In rat venular microvessels, activation of the Epac/Rap1 pathway significantly attenuates the platelet-activating factor-induced increase in microvessel permeability, as measured by hydraulic conductivity, and completely prevents the platelet-activating factor-induced rearrangement of VE-cadherin . Collectively, these results suggest that Epac/Rap1 signaling plays an important role in maintaining endothelial barrier function and vascular integrity.
Epac and secretion
While regulated exocytoses are mainly triggered by the elevation of intracellular Ca2+, second messenger cAMP also plays a role in modulating exocytosis in a variety of secretory cells. Epac has been implicated in stimulating numerous secretory pathways, including insulin secretion in pancreatic b cells [75,76], the release of the non-amyloidogenic soluble form of amyloid precursor protein [53,77,78], progesterone secretion by luteinizing human granulosa cells , secretory activity in mouse melanotrophs  and rat chromaffin cells [81,82], neurotensin secretion in human endocrine cells , acrosomal exocytosis in sperm , and apical exocytotic insertion of aquaporin-2 in the inner medullary collecting duct . As cAMP-regulated exocytosis has been reviewed in detail , we will focus on some of the most recent advances in the area of Epac-mediated insulin secretion.
A recent study investigated the effect of PKA and Epac on two types of secretory vesicles in mouse pancreatic -cells: large dense-core vesicles (LVs) and small vesicles (SVs). By directly visualizing Ca2+-dependent exocytosis of both LVs and SVs with two-photon imaging, it was revealed that Epac and PKA selectively regulate exocytosis of SVs and LVs, respectively . In a similar study, FM1-43 epifluorescence imaging was used to dissect the distinct contributions of Epac and PKA in regulating the number of plasma membrane (PM) exocytic sites and insulin secretory granule (SG)-to-granule fusions in these exocytic events. Again, Epac and PKA modulate both distinct and common exocytic steps to potentiate insulin exocytosis. Whereas Epac activation mobilizes SGs to fuse at the PM and thereby increase the number of PM exocytic sites, PKA and Epac activation synergistically increases both the number of exocytic sites at the PM and SG-SG fusions . Lastly, a study using primary cultured pancreatic b-cells isolated from wild-type and mutant mice lacking Epac2 suggests that, although activation of cAMP signaling alone does not cause either significant docking or fusion events of insulin granules, it substantially potentiates both the first phase (a prompt, marked and transient increase) and the second phase (a moderate and sustained increase) of glucose-induced fusion events. Moreover, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in b-cells isolated from Epac2 null mice. The data indicates that Epac2 signaling is important in cAMP-regulated insulin secretion because it controls insulin granule density near the PM .
Epac and differentiation
cAMP has been implicated in regulating differentiation in a variety of cell systems, such as neurite outgrowth in the neuroendocrine model cell line PC12  and adipocyte formation from mouse 3T3-L1 fibroblasts . The role that PKA plays in these processes is controversial, and it has been speculated that a PKA-independent cAMP signaling component may be involved. Indeed, several studies have revealed that Epac plays an important role in mediating the effects of pituitary adenylate cyclase-activating polypeptide in inducing neurite outgrowths in PC12 cells [54,90] and human neuroblastoma SH-SY5Y cells . However, as summarized in a recent Science STKE perspective , the detailed signal transduction pathways that mediate the neurotrophic effects of cAMP are not clear, and the involvement of PKA remains contentious .
Intracellular second messenger cAMP is essential for the induction of adipocyte differentiation in the mouse 3T3-L1 preadipocyte cell line. Again, it is generally believed that cAMP exerts its effects through activation of PKA. However, our recent studies suggest that PKA catalytic activity is not required for cAMP-mediated adipocyte differentiation in 3T3-L1 preadipocyte cells, as IBMX- or forskolin-induced 3T3-L1 adipocyte differentiation is not sensitive to two mechanistically distinct PKA inhibitors, H89 and PKI. On the other hand, selectively suppressing Epac1 expression using short hairpin RNAs substantially reduces the efficiency of IBMX- or forskolin-induced 3T3-L1 adipocyte differentiation.
Interestingly, while Epac1 is required for cAMP-mediated 3T3-L1 adipocyte differentiation, Epac-selective cAMP analog, 8-CPT-2'-O-Me-cAMP, is not sufficient to replace IBMX or forskolin to induce 3T3-L1 adipocyte differentiation, nor are cAMP analogs selective for PKA RI or RII. 3T3-L1 adipocyte differentiation requires the combination treatment of cAMP analogs selective for Epac, PKA RI and RII (Cheng et al, unpublished data). We are currently investigating the signaling mechanism of cAMP/Epac-mediated adipocyte differentiation.
Epac and cardiomyocyte hypertrophy
cAMP is the main second messenger in cardiomyocytes, which can be activated by the sympathetic and parasympathetic systems, cardioactive hormones and drugs . cAMP regulates many important processes, such as contractility and relaxation, in both normal and failing hearts . Traditionally, these effects have been attributed to the classic intracellular cAMP receptor, PKA . For example, PKA has been shown to phosphorylate key Ca2+-handling proteins, such as voltage-gated L-type Ca2+ channel , ryanodine receptor , and phospholamban [99,100]. The net result in increase in the sarcoplasmic reticulum Ca2+ release via ryanodine receptor 2 and enhanced uptake by SR Ca2+ pump results in larger intracellular Ca2+ transients. Increased Ca2+ transients significantly enhance contractility. However, emerging evidence suggests that Epac may also play an important role in many cellular functions, particularly cardiac hypertrophy, as a new mediator of cAMP signaling in the cardiovascular system .
Recent studies have shown that the expression of both Epac1 and Epac2 are developmentally increased in the heart from neonatal stages to adulthood, and Epac levels are significantly up-regulated in mouse hearts with myocardial hypertrophy induced by chronic isoproterenol infusion or with pressure overload by transverse aortic banding . In cardiomyocytes, Epac is involved in the formation of gap junctions, which are essential for gating ions and small molecules to coordinate cardiac contractions . Epac also enhances intracellular Ca2+ release during cardiac excitation-contraction coupling in cardiac myocytes by activating calcium-calmodulin-dependent protein kinase II  or activation of phospholipase Ce , which is known to associate with cardiac hypertrophy. Interestingly, activation of Epac leads to induction of hypertrophic program based on morphological changes, cytoskeletal reorganization, increase in protein synthesis and induction of cardiac hypertrophic markers. This effect is mediated by a Ca2+-dependent activation of Rac, calcineurin and its primary downstream effector, NFAT . It has been reported that Epac1 is the major Epac isoform expressed in the human heart, and its level increases during heart failure. Knockdown of Epac1 strongly suppresses beta-adrenergic receptor-induced hypertrophic program. Surprisingly, Epac1's hypertrophic effects are mediated by the small GTPase Ras, the phosphatase calcineurin and Ca(2+)/calmodulin-dependent protein kinase II, independent of Rap1, a canonical Epac effector .
Cross-talk between Epac and PKA
The discovery of second intracellular cAMP receptor raises many questions regarding the mechanism of cAMP-mediated signaling. The existence of two highly coordinated cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner. Since both PKA and Epac are ubiquitously expressed in all tissues, an increase in intracellular cAMP levels will lead to the activation of both PKA and Epac, and possibly other potential cAMP effectors as well. Therefore, the net cellular effects of cAMP are not just dictated by PKA or Epac alone, but by the sum of all the relevant pathways. As such, it is critical to consider which cAMP effects are mediated by Epac and which by PKA, as well as whether there is cross-talk between Epac and PKA.
Our earlier studies demonstrated that although PKA and Epac are activated by the same second messenger cAMP, they can exert opposing effects on the regulation of the PKB/AKT pathway. While PKA suppresses PKB phosphorylation and activity, activation of Epac leads to increased PKB phosphorylation . Since our initial report, many studies have shown that Epac and PKA can act antagonistically in controlling various cellular functions, such as insulin-stimulated PKB phosphorylation , proliferation and differentiation , myelin phagocytosis , regulation of hedgehog signaling and glucocorticoid sensitivity in acute lymphoblastic leukemia cells , and expression of high affinity choline transporter and the cholinergic locus . In contrast, we and others have shown that Epac and PKA, depending upon the specific cellular context, can exert synergistic effects on downstream signaling, such as stimulation of neurotensin secretion , promotion of PC12 cell neurite extension , regulation of sodium-proton exchanger isoform 3 , and attenuation of cAMP signaling through phosphodiesterases . While a model of synergistic activation of Rap1 by Epac and PKA has been proposed by Stork et al , the origin and causes of antagonism between Epac and PKA is not understood. It is very likely that antagonism between Epac and PKA involves complex mechanisms, and understanding the basis of Epac and PKA cross-talk may represent a major research interest for future cAMP-mediated signaling studies.
Mechanism of cAMP-mediated Activation
Both Epac and PKA are regulated by a CBD, which is a compact and evolutionally conserved structural/signaling motif that controls a set of diverse functionalities when linked to other structural domains [115,116]. CBD, the only common structural module between PKA and Epac, acts as a molecular switch for sensing intracellular second messenger cAMP levels. X-ray crystal structures and in-depth biochemical/biophysical analyses of PKA holoenzyme complex and individual subunits reveal a molecular mechanism for cAMP-mediated activation of PKA [117-120]. The R and C subunits form a large interface in the PKA holoenzyme complex with several key residues (Y247 and W196) of the C subunit binding directly to the phosphate binding cassette of the first CBD in the R subunit . cAMP not only competes directly with the C subunit for these interactions, but it also induces major conformational changes in the R subunit, particularly the helical subdomain of CBD, the inhibitor sequence and the linker region [118-120]. Binding of the cAMP results in the retraction of the phosphate binding cassette in the direction of cAMP-binding pocket and global reorientation of the subhelical domain of CBD. The pivot motion around the hydrophobic hinge dislodges the single extended B/C helix and, subsequently, the inhibitor sequence from the docking site on the C subunit. In the absence of the C subunit's stabilizing/anchoring effects, the B/C helix bends in the middle to form two individual helices, with the C helix portion folded back onto the b barrel to form the "lid" of the cAMP-binding pocket. These extensive cAMP-induced conformation changes eventually lead to the activation of PKA.
The CBD in Epac is covalently connected to the catalytic GEF domain as a single polypeptide chain, and the intramolecular interaction between the CBD and GEF domains sterically blocks the access of downstream effector Rap. Recently, the crystal structure of Epac2 was solved in the absence of cAMP . In this autoinhibited Epac2 structure, the second CBD of Epac2, which is common in both Epac1 and Epac2, is anchored to the catalytic core indirectly by the Ras exchange motif domain through the so called "switchboard". One major structural difference between the CBD in Epac and PKA is located in the lid region. The lid in CBD of PKA is a helix that covers the cAMP-binding pocket, whereas the corresponding region in Epac points away from the cAMP binding-packet in a two-strand b-sheet that forms the first part of the five-strand -sheet-like "switchboard" structure. In addition, unlike the extensive interface between the R and C subunits of PKA holoenzyme, the intramolecular interaction between the regulatory and catalytic regions in Epac2 is surprisingly brief. There is only one direct contact point between the CBD and catalytic core of Epac, described as the "ionic latch". These major differences suggest that although it is likely that Epac and PKA activations share the same underlying principal, the detailed mechanisms of PKA and Epac activation by cAMP will most likely be different at the structural level.
Since the crystal structure of cAMP-bound Epac in its active state is not currently available, the mechanism of Epac activation is not clear. Extensive biochemical and structural studies by the Bos and Wittinghofer groups suggest that the lid region of the C-terminus of CBD in Epac plays an important role in communicating between the regulatory and catalytic domains and is pivotal for the activation of Epac by cAMP [45,122-125]. To further probe the mechanism of Epac activation, we used amide H/D exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and mass spectrometry to examine the conformation and structural dynamics of Epac1 in the presence and absence of cAMP. Our studies show that binding of cAMP to Epac1 does not induce significant changes in overall secondary structure and structural dynamics, suggesting that conformational changes induced by cAMP in Epac1 are most likely local motion, such as hinge movements [126,127]. Hinge prediction based on Gaussian Network Model first normal model displacement analysis revealed a major hinge in Epac1 between residues 310 and 345 . Indeed, our amide H/D exchange mass spectrometry study reveals that the solvent accessibility of this hinge region decreases upon cAMP binding, indicating conformational changes . Based on the cAMP-free Epac2 structure and our in-depth H/D exchange and comparative sequence/structure analyses of Epac and PKA, we propose a model of Epac activation (Fig. 2). In this model, binding of cAMP induces an allosteric switch manifested by a hinge motion that bends the extended C helix lid toward the b-barrel of the CBD. This hinge movement pulls the b-strands S1 and S2 away from the five-strand b-sheet-like switchboard to form the base of the cAMP-binding pocket. The conformational changes induced upon cAMP binding result in a closed CBD conformation and reorientation of the CBD/DEP domains relative to the rest of the molecule, which releases the catalytic core from the inhibitory contact imposed by the CBD. This structural transition allows Epac, albeit with a completely different lid conformation in the inactive Epac structure, to utilize the same underlying principal to bind cAMP in almost exactly the same manner as PKA and other CBD-containing proteins [118,119,129,130]. Although final validation of the model requires the three-dimensional structure of an Epac-cAMP complex, our earlier studies using Epac-based fluorescence resonance energy transfer indicators suggest that binding of cAMP leads to a more extended Epac conformation , an observation in agreement with our model.
Since the discovery of Epac proteins a decade ago, the cAMP research area has undergone a renaissance. It is now well recognized that eukaryotic cAMP signaling is much more complex than it was initially believed and that the classic PKA pathway is only part of the story. The net physiological effects of cAMP necessitate the integration of Epac- and PKA-dependent pathways in a spatial and temporal manner, which dramatically increases the complexity and, consequently, the possible readouts of cAMP signaling. Depending upon the precise cellular environment as well as their relative abundance, distribution and localization, the two intracellular cAMP receptors may act independently, converge synergistically or oppose each other in regulating a specific cellular function. Therefore, careful dissection of the individual role and relative contribution of Epac and PKA within the overall cAMP signaling in various model systems will continue to be an important part of future research activity. In addition, although we have learned a great deal about the structure and functions of Epac, much remains to be discovered. Important future research in the area includes but is not limited to understanding the physiological roles of Epac isoforms using animal models, elucidating the mechanism of cross-talk between Epac and PKA, and mapping the conformational cAMP-induced changes during Epac activation.
The authors wish to apologize to the investigators whose outstanding work was not cited here because of space limitations. The authors would also like to thank Ms. Betty Redd and Mr. John Helms for assisting in manuscript preparation.
1 Frodin M, Peraldi P, Van Obberghen E. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells. J Biol Chem 1994, 269: 6207-6214
2 Burgering BM, Pronk GJ, van Weeren PC, Chardin P, Bos JL. cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J 1993, 12: 4211-4220
3 Wu J, Dent P, Jelinek T, Wolfman A, Weber MJ, Sturgill TW. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science 1993, 262: 1065-1069
4 Graves LM, Bornfeldt KE, Raines EW, Potts BC, Macdonald SG, Ross R, Krebs EG. Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci USA 1993, 90: 10300-10304
5 Cook SJ, McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science 1993, 262: 1069-1072
6 DeBernardi MA, Brooker G. Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging. Proc Natl Acad Sci USA 1996, 93: 4577-4582
7 Rogue PJ, Humbert JP, Meyer A, Freyermuth S, Krady MM, Malviya AN. cAMP-dependent protein kinase phosphorylates and activates nuclear Ca2+-ATPase. Proc Natl Acad Sci USA 1998, 95: 9178-9183
8 David M, Petricoin E, III, Larner AC. Activation of protein kinase A inhibits interferon induction of the Jak/STAT pathway in U266 cells. J Biol Chem 1996, 271: 4585-4588
9 Monfar M, Lemon KP, Grammer TC, Cheatham L, Chung J, Vlahos CJ, Blenis J. Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP. Mol Cell Biol 1995, 15: 326-337
10 Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem 1998, 273: 32377-32379
11 Zufall F, Shepherd GM, Barnstable CJ. Cyclic nucleotide gated channels as regulators of CNS development and plasticity. Curr Opin Neurobiol 1997, 7: 404-412
12 Walsh DA, Perkins JP, Krebs EG. An adenosine 3',5'-monophosphate-dependant protein kinase from rabbit skeletal muscle. J Biol Chem 1968, 243: 3763-3765
13 Cheng X, Ma Y, Moore M, Hemmings BA, Taylor SS. Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase. Proc Natl Acad Sci USA 1998, 95: 9849-9854
14 Cauthron RD, Carter KB, Liauw S, Steinberg RA. Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase. Mol Cell Biol 1998, 18: 1416-1423
15 Steinberg RA, Cauthron RD, Symcox MM, Shuntoh H. Autoactivation of catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by phosphorylation of threonine 197. Mol Cell Biol 1993, 13: 2332-2341
16 Adams JA, McGlone ML, Gibson R, Taylor SS. Phosphorylation modulates catalytic function and regulation in the cAMP- dependent protein kinase. Biochemistry 1995, 34: 2447-2454
17 Shoji S, Titani K, Demaille JG, Fischer EH. Sequence of two phosphorylated sites in the catalytic subunit of bovine cardiac muscle adenosine 3',5'-monophosphate-dependent protein kinase. J Biol Chem 1979, 254: 6211-6214
18 Taylor SS, Buechler JA, Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem 1990, 59: 971-1005
19 Zetterqvist OZ, Ragnarsson U, Engstrom L. In: Kemp BE, ed. Peptides and protein phosphorylation. Boca Raton: CRC Press Inc., 1991
20 Lee DC, Carmichael DF, Krebs EG, McKnight GS. Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase. Proc Natl Acad Sci USA 1983, 80: 3608-3612
21 Clegg CH, Cadd GG, McKnight GS. Genetic characterization of a brain-specific form of the type I regulatory subunit of cAMP-dependent protein kinase. Proc Natl Acad Sci USA 1988, 85: 3703-3707
22 Scott JD, Glaccum MB, Zoller MJ, Uhler MD, Helfman DM, McKnight GS, Krebs EG. The molecular cloning of a type II regulatory subunit of the cAMP-dependent protein kinase from rat skeletal muscle and mouse brain. Proc Natl Acad Sci USA 1987, 84: 5192-5196
23 Jahnsen T, Hedin L, Kidd VJ, Beattie WG, Lohmann SM, Walter U, Durica J et al. Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells. J Biol Chem 1986, 261: 12352-12361
24 Beebe SJ, Oyen O, Sandberg M, Froysa A, Hansson V, Jahnsen T. Molecular cloning of a tissue-specific protein kinase (C gamma) from human testis—representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase. Mol Endocrinol 1990, 4: 465-475
25 Weber IT, Steitz TA, Bubis J, Taylor SS. Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase. Biochemistry 1987, 26: 343-351
26 Rosen OM, Erlichman J. Reversible autophosphorylation of a cyclic 3',5'-AMP-dependent protein kinase from bovine cardiac muscle. J Biol Chem 1975, 250: 7788-7794
27 Corbin JD, Keely SL, Park CR. The distribution and dissociation of cyclic adenosine 3',5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues. J Biol Chem 1975, 250: 218-225
28 Hofmann F, Bechtel PJ, Krebs EG. Concentrations of cyclic AMP-dependent protein kinase subunits in various tissues. J Biol Chem 1977, 252: 1441-1447
29 Doskeland SO, Maronde E, Gjertsen BT. The genetic subtypes of cAMP-dependent protein kinase: functionally different or redundant? Biochim Biophys Acta 1993, 1178: 249-258
30 Joachim S, Schwoch G. Localization of cAMP-dependent protein kinase subunits along the secretory pathway in pancreatic and parotid acinar cells and accumulation of the catalytic subunit in parotid secretory granules following beta-adrenergic stimulation. Eur J Cell Biol 1990, 51: 76-84
31 Pariset C, Feinberg J, Dacheux JL, Oyen O, Jahnsen T, Weinman S. Differential expression and subcellular localization for subunits of cAMP-dependent protein kinase during ram spermatogenesis. J Cell Biol 1989, 109: 1195-1205
32 De Camilli P, Moretti M, Donini SD, Walter U, Lohmann SM. Heterogeneous distribution of the cAMP receptor protein RII in the nervous system: evidence for its intracellular accumulation on microtubules, microtubule-organizing centers, and in the area of the Golgi complex. J Cell Biol 1986, 103: 189-203
33 Skalhegg BS, Tasken K, Hansson V, Huitfeldt HS, Jahnsen T, Lea T. Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex. Science 1994, 263: 84-87
34 Scott JD, McCartney S. Localization of A-kinase through anchoring proteins. Mol Endocrinol 1994, 8: 5-11
35 Huang LJ, Durick K, Weiner JA, Chun J, Taylor SS. Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits. J Biol Chem 1997, 272: 8057-8064
36 Newlon MG, Roy M, Morikis D, Hausken ZE, Coghlan V, Scott JD, Jennings PA. The molecular basis for protein kinase A anchoring revealed by solution NMR. Nat Struct Biol 1999, 6: 222-227
37 Lohmann SM, Walter U. Regulation of the cellular and subcellular concentrations and distribution of cyclic nucleotide-dependent protein kinases. Adv Cyclic Nucleotide Protein Phosphorylation Res 1984, 18: 63-117
38 Sugden PH, Corbin JD. Adenosine 3',5'-cyclic monophosphate-binding proteins in bovine and rat tissues. Biochem J 1976, 159: 423-427
39 Cummings DE, Brandon EP, Planas JV, Motamed K, Idzerda RL, McKnight GS. Genetically lean mice result from targeted disruption of the RII beta subunit of protein kinase A. Nature 1996, 382: 622-626
40 Renstrom E, Eliasson L, Rorsman P. Protein kinase A-dependent and -independent stimulation of exocytosis by cAMP in mouse pancreatic B-cells. J Physiol 1997, 502: 105-118
41 de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 1998, 396: 474-477
42 Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE et al. A family of cAMP-binding proteins that directly activate Rap1. Science 1998, 282: 2275-2279
43 Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M. A ras-related gene with transformation suppressor activity. Cell 1989, 56: 77-84
44 Wang Z, Dillon TJ, Pokala V, Mishra S, Labudda K, Hunter B, Stork PJ. Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation. Mol Cell Biol 2006, 26: 2130-2145
45 de Rooij J, Rehmann H, van Triest M, Cool RH, Wittinghofer A, Bos JL. Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs. J Biol Chem 2000, 275: 20829-20836
46 Rangarajan S, Enserink JM, Kuiperij HB, de Rooij J, Price LS, Schwede F, Bos JL. Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor. J Cell Biol 2003, 160: 487-493
47 Enserink JM, Price LS, Methi T, Mahic M, Sonnenberg A, Bos JL, Taskén K. The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin. J Biol Chem 2004, 279: 44889-44896
48 Cullere X, Shaw SK, Andersson L, Hirahashi J, Luscinskas FW, Mayadas TN. Regulation of vascular endothelial barrier function by Epac, a cAMP-activated exchange factor for Rap GTPase. Blood 2005, 105: 1950-1955
49 Kooistra MR, Corada M, Dejana E, Bos JL. Epac1 regulates integrity of endothelial cell junctions through VE-cadherin. FEBS Lett 2005, 579: 4966-4972
50 Ozaki N, Shibasaki T, Kashima Y, Miki T, Takahashi K, Ueno H, Sunaga Y et al. cAMP-GEFII is a direct target of cAMP in regulated exocytosis. Nat Cell Biol 2000, 2: 805-811
51 Li J, O’Connor KL, Cheng X, Mei FC, Uchida T, Townsend CM Jr, Evers BM. Cyclic adenosine 5'-monophosphate-stimulated neurotensin secretion is mediated through Rap1 downstream of both Epac and protein kinase A signaling pathways. Mol Endocrinol 2007, 21: 159-171
52 Seino S, Shibasaki T. PKA-dependent and PKA-independent pathways for cAMP-regulated exocytosis. Physiol Rev 2005, 85: 1303-1342
53 Maillet M, Robert SJ, Cacquevel M, Gastineau M, Vivien D, Bertoglio J, Zugaza JL et al. Crosstalk between Rap1 and Rac regulates secretion of sAPPalpha. Nat Cell Biol 2003, 5: 633-639
54 Kiermayer S, Biondi RM, Imig J, Plotz G, Haupenthal J, Zeuzem S, Piiper A. Epac activation converts cAMP from a proliferative into a differentiation signal in PC12 cells. Mol Biol Cell 2005, 16: 5639-5648
55 Qiao J, Mei FC, Popov VL, Vergara LA, Cheng X. Cell cycle-dependent subcellular localization of exchange factor directly activated by cAMP. J Biol Chem 2002, 277: 26581-26586
56 Zwartkruis FJ, Bos JL. Ras and Rap1: two highly related small GTPases with distinct function. Exp Cell Res 1999, 253: 157-165
57 Bos JL, de Bruyn K, Enserink J, Kuiperij B, Rangarajan S, Rehmann H, Riedl J et al. The role of Rap1 in integrin-mediated cell adhesion. Biochem Soc Trans 2003, 31: 83-86
58 Enserink JM, Christensen AE, de Rooij J, van Triest M, Schwede F, Genieser HG, Døskeland SO et al. A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK. Nat Cell Biol 2002, 4: 901-906
59 Gupta M, Yarwood SJ. MAP1A light chain 2 interacts with exchange protein activated by cyclic AMP 1 (Epac1) to enhance Rap1 GTPase activity and cell adhesion. J Biol Chem 2005, 280: 8109-8116
60 Carmona G, Chavakis E, Koehl U, Zeiher AM, Dimmeler S. Activation of Epac stimulates integrin-dependent homing of progenitor cells. Blood 2008, 111: 2640-2646
61 Murphy MM, Zayed MA, Evans A, Parker CE, Ataga KI, Telen MJ, Parise LV. Role of Rap1 in promoting sickle red blood cell adhesion to laminin via BCAM/LU. Blood 2005, 105: 3322-3329
62 Lyle KS, Raaijmakers JH, Bruinsma W, Bos JL, de Rooij J. cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edge dynamics. Cell Signal 2008, 20: 1104-1116
63 Conrotto P, Yakymovych I, Yakymovych M, Souchelnytskyi S. Interactome of transforming growth factor-beta type I receptor (TbetaRI): inhibition of TGFbeta signaling by Epac1. J Proteome Res 2007, 6: 287-297
64 Netherton SJ, Sutton JA, Wilson LS, Carter RL, Maurice DH. Both protein kinase A and exchange protein activated by cAMP coordinate adhesion of human vascular endothelial cells. Circ Res 2007, 101: 768-776
65 Lorenowicz MJ, van Gils J, de Boer M, Hordijk PL, Fernandez-Borja M. Epac1-Rap1 signaling regulates monocyte adhesion and chemotaxis. J Leukoc Biol 2006, 80: 1542-1552
66 Bryn T, Mahic M, Enserink JM, Schwede F, Aandahl EM, Tasken K. The cyclic AMP-Epac1-Rap1 pathway is dissociated from regulation of effector functions in monocytes but acquires immunoregulatory function in mature macrophages. J Immunol 2006, 176: 7361-7370
67 Price LS, Hajdo-Milasinovic A, Zhao J, Zwartkruis FJ, Collard JG, Bos JL. Rap1 regulates E-cadherin-mediated cell-cell adhesion. J Biol Chem 2004, 279: 35127-35132
68 Kooistra MR, Dube N, Bos JL. Rap1: a key regulator in cell-cell junction formation. J Cell Sci 2007, 120: 17-22
69 Fukuhara S, Sakurai A, Sano H, Yamagishi A, Somekawa S, Takakura N, Saito Y et al. Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway. Mol Cell Biol 2005, 25: 136-146
70 Mei FC, Cheng XD. Interplay between exchange protein directly activated by cAMP (Epac) and microtubule cytoskeleton. Mol Biosyst 2005, 1: 325-331
71 Sehrawat S, Cullere X, Patel S, Italiano J Jr, Mayadas TN. Role of Epac1, an exchange factor for Rap GTPases, in endothelial microtubule dynamics and barrier function. Mol Biol Cell 2008, 19: 1261-1270
72 Birukova AA, Zagranichnaya T, Fu P, Alekseeva E, Chen W, Jacobson JR Birukov KG. Prostaglandins PGE(2) and PGI(2) promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation. Exp Cell Res 2007, 313: 2504-2520
73 Birukova AA, Zagranichnaya T, Alekseeva E, Bokoch GM, Birukov KG. Epac/Rap and PKA are novel mechanisms of ANP-induced Rac-mediated pulmonary endothelial barrier protection. J Cell Physiol 2008, 215: 715-724
74 Adamson RH, Ly JC, Sarai RK, Lenz JF, Altangerel A, Drenckhahn D, Curry FE. Epac/Rap1 pathway regulates microvascular hyperpermeability induced by PAF in rat mesentery. Am J Physiol Heart Circ Physiol 2008, 294: H1188-H1196
75 Kashima Y, Miki T, Shibasaki T, Ozaki N, Miyazaki M, Yano H, Seino S. Critical role of cAMP-GEFII—Rim2 complex in incretin-potentiated insulin secretion. J Biol Chem 2001, 276: 46046-4653
76 Kang G, Chepurny OG, Holz GG. cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic beta-cells. J Physiol 2001, 536:375-385
77 Robert S, Maillet M, Morel E, Launay JM, Fischmeister R, Mercken L, Lezoualc'h F. Regulation of the amyloid precursor protein ectodomain shedding by the 5-HT4 receptor and Epac. FEBS Lett 2005, 579: 1136-1142
78 Zaldua N, Gastineau M, Hoshino M, Lezoualc’h F, Zugaza JL. Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing. FEBS Lett 2007, 581: 5814-5818
79 Chin EC, Abayasekara DR. Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation. J Endocrinol 2004, 183: 51-60
80 Sedej S, Rose T, Rupnik M. cAMP increases Ca2+-dependent exocytosis through both PKA and Epac2 in mouse melanotrophs from pituitary tissue slices. J Physiol 2005, 567: 799-813
81 Novara M, Baldelli P, Cavallari D, Carabelli V, Giancippoli A, Carbone E. Exposure to cAMP and beta-adrenergic stimulation recruits Ca(V)3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins. J Physiol 2004, 558: 433-449
82 Giancippoli A, Novara M, de Luca A, Baldelli P, Marcantoni A, Carbone E, Carabelli V. Low-threshold exocytosis induced by cAMP-recruited CaV3.2 (alpha1H) channels in rat chromaffin cells. Biophys J 2006, 90: 1830-1841
83 Branham MT, Mayorga LS, Tomes CN. Calcium induced acrosomal exocytosis requires cAMP acting through a PKA-independent, Epac-mediated pathway. J Biol Chem 2006, 281: 8656-8666
84 Yip KP. Epac-mediated Ca(2+) mobilization and exocytosis in inner medullary collecting duct. Am J Physiol Renal Physiol 2006, 291: F882-F890
85 Hatakeyama H, Takahashi N, Kishimoto T, Nemoto T, Kasai H. Two cAMP-dependent pathways differentially regulate exocytosis of large dense-core and small vesicles in mouse beta-cells. J Physiol 2007, 582: 1087-1098
86 Kwan EP, Gao X, Leung YM, Gaisano HY. Activation of exchange protein directly activated by cyclic adenosine monophosphate and protein kinase A regulate common and distinct steps in promoting plasma membrane exocytic and granule-to-granule fusions in rat islet beta cells. Pancreas 2007, 35: e45-e54
87 Shibasaki T, Takahashi H, Miki T, Sunaga Y, Matsumura K, Yamanaka M, Zhang C et al. Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP. Proc Natl Acad Sci U S A 2007, 104: 19333-19338
88 Gunning PW, Landreth GE, Bothwell MA, Shooter EM. Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells. J Cell Biol 1981, 89: 240-245
89 Williams IH, Polakis SE. Differentiation of 3T3-L1 fibroblasts to adipocytes. The effect of indomethacin, prostaglandin E1 and cyclic AMP on the process of differentiation. Biochem Biophys Res Commun 1977, 77: 175-186
90 Shi GX, Rehmann H, Andres DA. A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation. Mol Cell Biol 2006, 26: 9136-9147
91 Monaghan TK, Mackenzie CJ, Plevin R, Lutz EM. PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases. J Neurochem 2008, 104: 74-88
92 Gerdin MJ, Eiden LE. Regulation of PC12 cell differentiation by cAMP signaling to ERK independent of PKA: do all the connections add up? Sci STKE 2007, 2007: pe15
93 Christensen AE, Selheim F, Rooij JJ, Dremier S, Schwede F, Dao KK, Martinez A et al. cAMP analog mapping of Epac1 and cAMP-kinase. Discriminating analogs demonstrate that Epac and cAMP-kinase act synergistically to promote PC12 cell neurite extension. J Biol Chem 2003, 278: 35394-35402
94 Vandecasteele G, Rochais F, Abi-Gerges A, Fischmeister R. Functional localization of cAMP signalling in cardiac myocytes. Biochem Soc Trans 2006, 34: 484-488
95 Hunter JJ, Chien KR. Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 1999, 341: 1276-1283
96 Kopperud R, Krakstad C, Selheim F, Doskeland SO. cAMP effector mechanisms. Novel twists for an "old" signaling system. FEBS Lett 2003, 546: 121-126
97 Kamp TJ, Hell JW. Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res 2000, 87: 1095-1102
98 Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 2000, 101: 365-376
99 Kiss E, Edes I, Sato Y, Luo W, Liggett SB, Kranias EG. beta-Adrenergic regulation of cAMP and protein phosphorylation in phospholamban-knockout mouse hearts. Am J Physiol 1997, 272: H785-H790
100 Simmerman HK, Jones LR. Phospholamban: protein structure, mechanism of action, and role in cardiac function. Physiol Rev 1998, 78: 921-947
101 Schmidt M, Sand C, Jakobs KH, Michel MC, Weernink PA. Epac and the cardiovascular system. Curr Opin Pharmacol 2007, 7: 193-200
102 Ulucan C, Wang X, Baljinnyam E, Bai Y, Okumura S, Sato M, Minamisawa S et al. Developmental changes in gene expression of Epac and its up-regulation in myocardial hypertrophy. Am J Physiol Heart Circ Physiol 2007, 293: H1662-H1672
103 Somekawa S, Fukuhara S, Nakaoka Y, Fujita H, Saito Y, Mochizuki N. Enhanced functional gap junction neoformation by protein kinase A-dependent and Epac-dependent signals downstream of cAMP in cardiac myocytes. Circ Res 2005, 97: 655-662
104 Pereira L, Métrich M, Fernández-Velasco M, Lucas A, Leroy J, Perrier R, Morel E et al. The cAMP binding protein Epac modulates Ca2+ sparks by a Ca2+/calmodulin kinase signalling pathway in rat cardiac myocytes. J Physiol 2007, 583: 685-694
105 Oestreich EA, Wang H, Malik S, Kaproth-Joslin KA, Blaxall BC, Kelley GG, Dirksen RT et al. Epac-mediated activation of phospholipase C(epsilon) plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes. J Biol Chem 2007, 282: 5488-5495
106 Morel E, Marcantoni A, Gastineau M, Birkedal R, Rochais F, Garnier A, Lompré AM et al. cAMP-binding protein Epac induces cardiomyocyte hypertrophy. Circ Res 2005, 97: 1296-1304
107 Metrich M, Lucas A, Gastineau M, Samuel JL, Heymes C, Morel E, Lezoualc’h F. Epac mediates beta-adrenergic receptor-induced cardiomyocyte hypertrophy. Circ Res 2008, 102: 959-965
108 Mei FC, Qiao J, Tsygankova OM, Meinkoth JL, Quilliam LA, Cheng X. Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation. J Biol Chem 2002, 277: 11497-11504
109 Brennesvik EO, Ktori C, Ruzzin J, Jebens E, Shepherd PR, Jensen J. Adrenaline potentiates insulin-stimulated PKB activation via cAMP and Epac: implications for cross-talk between insulin and adrenaline. Cell Signal 2005, 17: 1551-1559
110 Makranz C, Cohen G, Reichert F, Kodama T, Rotshenker S. cAMP cascade (PKA, Epac, adenylyl cyclase, Gi, and phosphodiesterases) regulates myelin phagocytosis mediated by complement receptor-3 and scavenger receptor-AI/II in microglia and macrophages. Glia 2006, 53: 441-448
111 Ji Z, Mei FC, Johnson BH, Thompson EB, Cheng X. Protein kinase A, not Epac, suppresses hedgehog activity and regulates glucocorticoid sensitivity in acute lymphoblastic leukemia cells. J Biol Chem 2007, 282: 37370-37377
112 Brock M, Nickel AC, Madziar B, Blusztajn JK, Berse B. Differential regulation of the high affinity choline transporter and the cholinergic locus by cAMP signaling pathways. Brain Res 2007, 1145: 1-10
113 Honegger KJ, Capuano P, Winter C, Bacic D, Stange G, Wagner CA, Biber J et al. Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (Epac). Proc Natl Acad Sci U S A 2006, 103: 803-808
114 Dodge-Kafka KL, Soughayer J, Pare GC, Carlisle Michel JJ, Langeberg LK, Kapiloff MS, Scott JD. The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways. Nature 2005, 437: 574-578
115 Berman HM, Ten Eyck LF, Goodsell DS, Haste NM, Kornev A, Taylor SS. The cAMP binding domain: an ancient signaling module. Proc Natl Acad Sci U S A 2005, 102: 45-50
116 Kannan N, Wu J, Anand GS, Yooseph S, Neuwald AF, Venter JC, Taylor SS. Evolution of allostery in the cyclic nucleotide binding module. Genome Biol 2007, 8: R264
117 Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 1991, 253: 407-414
118 Su Y, Dostmann WR, Herberg FW, Durick K, Xuong NH, Ten Eyck L, Taylor SS et al. Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains. Science 1995, 269: 807-813
119 Kim C, Xuong NH, Taylor SS. Crystal structure of a complex between the catalytic and regulatory (RIalpha) subunits of PKA. Science 2005, 307: 690-696
120 Wu J, Brown SH, von Daake S, Taylor SS. PKA type IIalpha holoenzyme reveals a combinatorial strategy for isoform diversity. Science 2007, 318: 274-279
121 Rehmann H, Das J, Knipscheer P, Wittinghofer A, Bos JL. Structure of the cyclic-AMP-responsive exchange factor Epac2 in its auto-inhibited state. Nature 2006, 439: 625-628
122 Rehmann H, Rueppel A, Bos JL, Wittinghofer A. Communication between the regulatory and the catalytic region of the cAMP-responsive guanine nucleotide exchange factor Epac. J Biol Chem 2003, 278: 23508-23514
123 Kraemer A, Rehmann HR, Cool RH, Theiss C, de Rooij J, Bos JL, Wittinghofer A. Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1. J Mol Biol 2001, 306: 1167-1177
124 Rehmann H, Schwede F, Doskeland SO, Wittinghofer A, Bos JL. Ligand-mediated activation of the cAMP-responsive guanine nucleotide exchange factor Epac. J Biol Chem 2003, 278: 38548-38556
125 Rehmann H, Prakash B, Wolf E, Rueppel A, de Rooij J, Bos JL, Wittinghofer A. Structure and regulation of the cAMP binding domains of Epac2. Nat Struct Biol 2003, 10: 26-32
126 Yu S, Mei FC, Lee JC, Cheng X. Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting. Biochemistry 2004, 43: 1908-1920
127 Yu S, Fan F, Flores SC, Mei F, Cheng X. Dissecting the mechanism of Epac activation via hydrogen-deuterium exchange FT-IR and structural modeling. Biochemistry 2006, 45: 15318-15326
128 Brock M, Fan F, Mei FC, Li S, Gessner C, Woods VL, Jr., Cheng X. Conformational analysis of Epac activation using amide hydrogen/deuterium exchange mass spectrometry. J Biol Chem 2007, 282: 32256-32263
129 McKay DB, Steitz TA. Structure of catabolite gene activator protein at 2.9 Å resolution suggests binding to left-handed B-DNA. Nature 1981, 290: 744-749
130 Clayton GM, Silverman WR, Heginbotham L, Morais-Cabral JH. Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel. Cell 2004, 119: 615-627
131 DiPilato LM, Cheng X, Zhang J. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl Acad Sci USA 2004, 101: 16513-16518