Search for new cyclic AMP-binding proteins

Today, there is evidence that the cAMP-dependent kinases (PKA) are not the only intracellular receptors involved in intracellular cAMP signalling in eukaryotes. Other cAMP-binding proteins have been recently identified, including some cyclic nucleotide-gated channels and Epac (exchange protein directly activated by cAMP) proteins. All these proteins bind cAMP through conserved cyclic nucleotide monophosphate-binding domains. However, all putative cAMP-binding proteins having such domains, as revealed by computer analysis, do not necessarily bind cAMP, indicating that their presence is not a sufficient criteria to predict cAMP-binding property for a protein.     

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.     

Dissecting the mechanism of Epac activation via hydrogen-deuterium exchange FT-IR and structural modeling

Exchange proteins directly activated by cAMP (Epac) make up a family of cAMP binding domain-containing proteins that play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Ras-proximate proteins. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and structural modeling. Our studies show that unlike that of cAMP-dependent protein kinase (PKA), the classic intracellular cAMP receptor, binding of cAMP to Epac does not induce significant changes in overall secondary structure and structural dynamics, as measured by FT-IR and the rate of H-D exchange, respectively. These results suggest that Epac activation does not involve significant changes in the amount of exposed surface areas as in the case of PKA activation, and conformational changes induced by cAMP in Epac are most likely confined to small local regions. Homology modeling and comparative structural analyses of the CBDs of Epac and PKA lead us to propose a model of Epac activation. On the basis of our model, Epac activation by cAMP employs the same underlying structural principal utilized by PKA, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different. In addition, we predict that during Epac activation the first beta-strand of the switchboard switches its conformation to a alpha-helix, which folds back to the beta-barrel core of the CBD and interacts directly with cAMP to form the base of the cAMP-binding pocket.     

Interrogating cyclic AMP signaling using optical approaches

Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.     

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.     

Conformational analysis of Epac activation using amide hydrogen/deuterium exchange mass spectrometry

Exchange proteins directly activated by cAMP (Epac) play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Rap. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen/deuterium exchange and structural modeling. Our studies show that cAMP induces significant conformational changes that lead to a spatial rearrangement of the regulatory components of Epac and allows the exposure of the catalytic core for effector binding without imposing significant conformational change on the catalytic core. Homology modeling and comparative structural analyses of the cAMP binding domains of Epac and cAMP-dependent protein kinase (PKA) lead to a model of Epac activation, in which Epac and PKA activation by cAMP employs the same underlying principle, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different.     

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.     

cAMP-dependent allostery and dynamics in Epac: an NMR view

Epac (exchange protein directly activated by cAMP) is a critical cAMP receptor, which senses cAMP and couples the cAMP signal to the catalysis of guanine exchange in the Rap substrate. In the present paper, we review the NMR studies that we have undertaken on the CBD (cyclic-nucleotide-binding domain) of Epac1. Our NMR investigations have shown that cAMP controls distal autoinhibitory interactions through long-range modulations in dynamics. Such dynamically mediated allosteric effects contribute not only to the cAMP-dependent activation of Epac, but also to the selectivity of Epac for cAMP in contrast with cGMP. In addition, we have mapped the interaction networks that couple the cAMP-binding site to the sites involved in the autoinhibitory interactions, using a method based on the covariance analysis of NMR chemical shifts. We anticipate that this approach is generally applicable to dissect allosteric networks in signalling domains.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.     

Cytoplasmic cAMP concentrations in intact cardiac myocytes

In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both beta(1)-adrenergic and M(2) muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer-based biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is approximately 1.2 muM, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both beta(1) and M(2) receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though beta(1) and M(2) receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors.     

Communication between the regulatory and the catalytic region of the cAMP-responsive guanine nucleotide exchange factor Epac

Epac1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1 that is directly activated by cAMP. This protein consists of a regulatory region with a cAMP-binding domain and a catalytic region that mediates the GEF activity. Epac is inhibited by an intramolecular interaction between the cAMP-binding domain and the catalytic region in the absence of cAMP. cAMP binding is proposed to induce a conformational change, which allows a LID, an alpha-helix at the C-terminal end of the cAMP-binding site, to cover the cAMP-binding site (Rehmann, H., Prakash, B., Wolf, E., Rueppel, A., de Rooij, J., Bos, J. L., and Wittinghofer, A. (2003) Nat. Struct. Biol. 10, 26-32). Here we show that mutations of conserved residues in the LID region affect cAMP binding only marginally but have a drastic effect on cAMP-induced GEF activity. Surprisingly, some of the mutants have an increased maximal GEF activity compared with wild type. Furthermore, mutation of the conserved VLVLE sequence at the C-terminal end of the LID into five alanine residues makes Epac constitutively active. From these results we conclude that the LID region plays a pivotal role in the communication between the regulatory and catalytic part of Epac.     

Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.     

Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation

In diverse neuronal processes ranging from neuronal survival to synaptic plasticity cyclic adenosine monophosphate (cAMP)-dependent signaling is tightly connected with the protein kinase B (PKB)/Akt pathway but the precise nature of this connection remains unknown. In the current study we investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons and HT-4 cells. We demonstrate that PKA activation leads to a reduction of PKB/Akt phosphorylation, whereas activation of Epac has the opposite effect. This effect of Epac on PKB/Akt phosphorylation was mediated by Rap activation. The increase in PKB/Akt phosphorylation after Epac activation could be blocked by pretreatment with Epac2 siRNA and to a somewhat smaller extent by Epac1 siRNA. PKA, PKB/Akt and Epac were all shown to establish complexes with neuronal A-kinase anchoring protein150 (AKAP150). Interestingly, activation of Epac increased phosphorylation of PKB/Akt complexed to AKAP150. From experiments using PKA-binding deficient AKAP150 and peptides disrupting PKA anchoring to AKAPs, we conclude that AKAP150 acts as a key regulator in the two cAMP pathways to control PKB/Akt phosphorylation.     

Prostaglandin E2-activated Epac promotes neointimal formation of the rat ductus arteriosus by a process distinct from that of cAMP-dependent protein kinase A

We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2'-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA.     

The cyclic AMP-Epac1-Rap1 pathway is dissociated from regulation of effector functions in monocytes but acquires immunoregulatory function in mature macrophages

cAMP mediates its intracellular effects through activation of protein kinase A (PKA), nucleotide-gated ion channels, or exchange protein directly activated by cAMP (Epac). Although elevation of cAMP in lymphocytes leads to suppression of immune functions by a PKA-dependent mechanism, the effector mechanisms for cAMP regulation of immune functions in monocytes and macrophages are not fully understood. In this study, we demonstrate the presence of Epac1 in human peripheral blood monocytes and activation of Rap1 in response to cAMP. However, by using an Epac-specific cAMP analog (8-CPT-2'-O-Me-cAMP), we show that monocyte activation parameters such as synthesis and release of cytokines, stimulation of cell adhesion, chemotaxis, phagocytosis, and respiratory burst are not regulated by the Epac1-Rap1 pathway. In contrast, activation of PKA by a PKA-specific compound (6-Bnz-cAMP) or physiological cAMP-elevating stimuli like PGE(2) inhibits monocyte immune functions. Furthermore, we show that the level of Epac1 increases 3-fold during differentiation of monocytes into macrophages, and in monocyte-derived macrophages cAMP inhibits FcR-mediated phagocytosis via both PKA and the Epac1-Rap1 pathway. However, LPS-induced TNF-alpha production is only inhibited through the PKA pathway in these cells. In conclusion, the Epac1-Rap1 pathway is present in both monocytes and macrophages, but only regulates specific immune effector functions in macrophages.     

Epac1 is upregulated during neointima formation and promotes vascular smooth muscle cell migration

Vascular remodeling after mechanoinjury largely depends on the migration of smooth muscle cells, an initial key step to wound healing. However, the role of the second messenger system, in particular, the cAMP signal, in regulating such remodeling remains controversial. Exchange protein activated by cAMP (Epac) has been identified as a new target molecule of the cAMP signal, which is independent from PKA. We thus examined whether Epac plays a distinct role from PKA in vascular remodeling. To examine the role of Epac and PKA in migration, we used primary culture smooth muscle cells from both the fetal and adult rat aorta. A cAMP analog selective to PKA, 8-(4-parachlorophenylthio)-cAMP (pCPT-cAMP), decreased cell migration, whereas an Epac-selective analog, 8-pCPT-2'-O-Me-cAMP, enhanced migration. Adenovirus-mediated gene transfer of PKA decreased cell migration, whereas that of Epac1 significantly enhanced cell migration. Striking morphological differences were observed between pCPT-cAMP- and 8-pCPT-2'-O-Me-cAMP-treated aortic smooth muscle cells. Furthermore, overexpression of Epac1 enhanced the development of neointimal formation in fetal rat aortic tissues in organ culture. When the mouse femoral artery was injured mechanically in vivo, we found that the expression of Epac1 was upregulated in vascular smooth muscle cells, whereas that of PKA was downregulated with the progress of neointimal thickening. Our findings suggest that Epac1, in opposition to PKA, increases vascular smooth muscle cell migration. Epac may thus play an important role in advancing vascular remodeling and restenosis upon vascular injury.     

Epac1 and cAMP-dependent protein kinase holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclic nucleotide recognition

The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K(d) value (2.9 microM) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K(d) values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIalpha led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.     

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.     

Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation

Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.     

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1-GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the G alpha s-coupled beta 2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.