Frank Schwede

A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK

cAMP is involved in a wide variety of cellular processes that were thought to be mediated by protein kinase A (PKA). However, cAMP also directly regulates Epac1 and Epac2, guanine nucleotide-exchange factors (GEFs) for the small GTPases Rap1 and Rap2 (refs 2,3). Unfortunately, there is an absence of tools to discriminate between PKA- and Epac-mediated effects. Therefore, through rational drug design we have developed a novel cAMP analogue, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8CPT-2Me-cAMP), which activates Epac, but not PKA, both in vitro and in vivo. Using this analogue, we tested the widespread model that Rap1 mediates cAMP-induced regulation of the extracellular signal-regulated kinase (ERK). However, both in cell lines in which cAMP inhibits growth-factor-induced ERK activation and in which cAMP activates ERK, 8CPT-2Me-cAMP did not affect ERK activity. Moreover, in cell lines in which cAMP activates ERK, inhibition of PKA and Ras, but not Rap1, abolished cAMP-mediated ERK activation. We conclude that cAMP-induced regulation of ERK and activation of Rap1 are independent processes.

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the β2-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 Gαs-coupled β2-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.

Cyclic nucleotide analogs as probes of signaling pathways

To the editor: Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers that regulate multiple targets including different cAMPor cGMP-dependent protein kinases (PKAs, PKGs)1,2, exchange proteins directly activated by cAMP (Epacs)3, phosphodiesterases (PDEs)4 and cyclic nucleotide-gated ion channels. Cyclic nucleotide analogs are widely used to study specificity of cellular signaling mediated by these target proteins. However, the selectivities and stabilities of these analogs need to be fully understood to properly interpret results and rigorously assess the mechanisms by which these analogs work in the cell. To better understand the selectivity and cross-reactivity of these analogs, we measured the activation or inhibitory activity of 13 commonly used cyclic nucleotide analogs with isozymes of PKA, PKG and Epac (Table 1), and with 8 different PDEs (Table 2 and Supplementary Tables 1 and 2 online). To measure their stability against hydrolysis, we used isothermal microcalorimetry5, a method that allowed us to evaluate whether or not an analog can function as a substrate or inhibitor for PDEs. We found that indeed some of these analogs were hydrolyzed by multiple PDEs, and other analogs were competitive inhibitors of PDEs. Here we provide half-maximal inhibition constant (Ki) data for all of the non-hydrolyzable analogs, and MichaelisMenten constant (Km) and maximum velocity (Vmax) values for all of the hydrolyzable analogs. Each of these values as well as the analog’s mode of inhibition can be determined in a single experiment (Table 2, Supplementary Methods and Supplementary Figures 1–5 online). The data strongly implied that several of these analogs might, in addition to their primary effects, also cause elevation of cAMP or cGMP indirectly by inhibiting PDEs in the cell. Such an effect could cloud interpretation of the use of these analogs. Similarly, analogs that are PDE substrates also might have their duration of action substantially reduced. To illustrate this point we showed that Sp-8-pCPT-2′O-Me-cAMPS, a highly specific, non-hydrolyzable Epac activator in vitro, can under certain conditions enhance cGMP-PKG and cAMPPKA signaling pathways in intact platelets (Supplementary Fig. 1). Specifically, we observed enhanced phosphorylation of vasodialatorstimulated phosphoprotein (VASP) at both PKA and PKG phosphorylation sites after the addition of Sp-8-pCPT-2′-O-Me-cAMPS. These data indicate that this ‘selective Epac activator’ is able to indirectly activate the cAMP-PKA and cGMP-PKG signaling pathways presumably through inhibition of platelet PDE5 and/or PDE3 (Supplementary Methods and Supplementary Discussion online). We also list in vitro selectivity data for all of the presently available commonly used cyclic nucleotide analogs for different forms of PKA, PKG and Epac I (Table 1). Data for several of these analogs have not

Structure of Epac2 in complex with a cyclic AMP analogue and RAP1B

Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the cyclic nucleotide binding domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif domain.

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.

A Novel EPAC-Specific Inhibitor Suppresses Pancreatic Cancer Cell Migration and Invasion

Exchange protein directly activated by cAMP (EPAC) and cAMP-dependent protein kinase (PKA) are two intracellular receptors that mediate the effects of the prototypic second messenger cAMP. Identifying pharmacological probes for selectively modulating EPAC activity represents a significant unmet need within the research field. Herein, we report the identification and characterization of 3-(5-tert-butyl-isoxazol-3-yl)-2-[(3-chloro-phenyl)-hydrazono]-3-oxo-propionitrile (ESI-09), a novel noncyclic nucleotide EPAC antagonist that is capable of specifically blocking intracellular EPAC-mediated Rap1 activation and Akt phosphorylation, as well as EPAC-mediated insulin secretion in pancreatic β cells. Using this novel EPAC-specific inhibitor, we have probed the functional roles of overexpression of EPAC1 in pancreatic cancer cells. Our studies show that EPAC1 plays an important role in pancreatic cancer cell migration and invasion, and thus represents a potential target for developing novel therapeutic strategies for pancreatic cancer.

cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic β cells and rat INS-1 cells

The Epac family of cAMP‐regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic β cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide‐binding fold‐1 (NBF‐1) of the β‐cell sulphonylurea receptor‐1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit β‐cell ATP‐sensitive K+ channels (KATP channels; a hetero‐octomer of SUR1 and Kir6.2). If so, Epac‐mediated inhibition of KATP channels might explain prior reports that cAMP‐elevating agents promote β‐cell depolarization, Ca2+ influx and insulin secretion. Here we report that Epac‐selective cAMP analogues (2′‐O‐Me‐cAMP; 8‐pCPT‐2′‐O‐Me‐cAMP; 8‐pMeOPT‐2′‐O‐Me‐cAMP), but not a cGMP analogue (2′‐O‐Me‐cGMP), inhibit the function of KATP channels in human β cells and rat INS‐1 insulin‐secreting cells. Inhibition of KATP channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6‐Bnz‐cAMP, a cAMP analogue that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac‐selective cAMP analogues at KATP channels are mimicked by a cAMP agonist (8‐Bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Sp‐isomer, Sp‐8‐Br‐cAMPS), but not a cAMP antagonist (8‐Bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Rp‐isomer, Rp‐8‐Br‐cAMPS), and are abrogated following transfection of INS‐1 cells with a dominant‐negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 coimmunoprecipitate with full‐length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of KATP channel activity in pancreatic β cells.

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 Kd value (2.9 μm) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had Kd 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 RIα 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.

Relating ligand binding to activation gating in CNGA2 channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP < 1 μM, steady-state binding was larger than steady-state activation, whereas at higher concentrations it was smaller, generating a crossover of the steady-state relationships. Global analysis of these relationships together with multiple activation time courses following cGMP jumps showed that four ligands bind to the channels and that there is significant interaction between the binding sites. Among the binding steps, the second is most critical for channel opening: its association constant is three orders of magnitude smaller than the others and it triggers a switch from a mostly closed to a maximally open state. These results contribute to unravelling the role of the subunits in the cooperative mechanism of CNGA2 channel activation and could be of general relevance for the action of other ion channels and receptors.

8-pCPT-2′-O-Me-cAMP-AM: An Improved Epac-Selective cAMP Analogue

Cyclic adenosine monophosphate (cAMP) is a common second messenger involved in the regulation of many different cellular processes through the activation of protein kinase A (PKA), exchange protein directly activated by cAMP (Epac) and cyclicnucleotide-regulated ion channels. Adenylyl cyclases are ACHTUNGTRENNUNGresponsible for catalysing the formation of cAMP from ATP. Levels of cAMP can be raised in cells in response to a large variety of extracellular stimuli, which act via receptors coupled to heterotrimeric G proteins, which stimulate the activity of adenylyl cyclase. In addition, cAMP levels are controlled by phosphodiesterases (PDE), which catalyse the degradation of cAMP to AMP. In cells, cAMP levels can be artificially elevated by forskolin, which activates adenylyl cyclase directly. Furthermore, cAMP levels can be raised by inhibiting PDEs. These approaches are commonly used in tissue culture experiments, but, by generating cAMP, they do not discriminate between the various target proteins that are activated. Alternatively, membrane-permeable cAMP analogues, which selectively interact with particular receptor proteins, can be applied. For example, signalling pathways activated by Epac and PKA can be ACHTUNGTRENNUNGdistinguished by using 8-pCPT-2’-O-Me-cAMP and 6-Bnz-cAMP, respectively. Epac is a guanine nucleotide exchange factor for the small G protein Rap. Rap cycles between a signalling-inactive GDPbound state and a signalling-active GTP-bound state. cAMP-activated Epac catalyses the exchange of Rap-bound GDP for GTP. Epac and Rap function in a number of different cellular processes including insulin secretion, inhibition of cell scattering, neurotransmitter release and cAMP-induced barrier function in endothelial cells. Even though 8-pCPT-2’-O-Me-cAMP has become a widely used tool in Epac-related research, its biological application is limited by its low membrane permeability, caused by the negatively charged phosphate. However, the negatively charged singly bonded oxygen on the phosphate group can be masked by labile esters. Such a precursor is expected to enter the cell efficiently, where the ester is hydrolysed either directly by water or by cellular esterases to liberate the active compound. We therefore synthesised 8-pCPT-2’-O-Me-cAMP-AM from 8pCPT-2’-O-Me-cAMP, whereby acetoxymethyl bromide was used as a donor for the AM group. The product that was obtained had a purity exceeding 97% and consisted of a mixture of the equatorial and the axial isomers of the ester (Figure S1 in the Supporting Information, Scheme 1). Even though the isomers could be resolved by repetitive analytical HPLC runs, efficient separation on a preparative scale was not possible. Orange peel acetylesterase and esterase from porcine liver cleaved the equatorial isomer about five times more efficiently than the axial isomer within minutes (data not shown). The pharmacokinetics of both isomers are thus expected to be similar, justifying the application of a mixture of both isomers to cells. In any case, the isomeric ratio of an individual synthesis can be easily quality controlled by P NMR (Figure S1). To compare the efficiency of 8-pCPT-2’-O-Me-cAMP-AM and 8-pCPT-2’-O-Me-cAMP in activating Epac1 in vivo, an Epac1based fluorescence resonance energy transfer (FRET) probe was used. In this assay, activation of Epac1 by the binding of cAMP to the Epac1-FRET probe is measured as a reduction in the FRET signal. A431 cells transfected with the FRET probe were stimulated with 8-pCPT-2’-O-Me-cAMP-AM or 8-pCPT-2’O-Me-cAMP (Figure 1). Stimulation of cells with 100 mm 8pCPT-2’-O-Me-cAMP resulted in a decrease of the FRET signal that was approximately one order of magnitude slower than the decrease obtained upon stimulation with 1 mm 8-pCPT-2’O-Me-cAMP-AM. Furthermore, activation of Epac1 following stimulation with 100 mm 8-pCPT-2’-O-Me-cAMP could be further enhanced by the addition of forskolin, whereas 1 mm 8pCPT-2’-O-Me-cAMP-AM induced maximal activity of Epac1 under the given conditions. The activation of Epac by 8-pCPT2’-O-Me-cAMP-AM occurs within one minute after application. This is comparable with the kinetics of forskolin-induced Epac activation, and thus 8-pCPT-2’-O-Me-cAMP-AM mimics the “natural” response time of the signalling pathway. The activity of endogenous Epac can be monitored by isolating selectively Rap·GTP from cell lysates. Primary human umbilical vein endothelial cells (HUVEC) were stimulated with different concentrations of 8-pCPT-2’-O-Me-cAMP and 8-pCPT-2’-OMe-cAMP-AM (Figure 2A). Partial activation of Rap was induced by 10 mm 8-pCPT-2’-O-Me-cAMP, and full activation of the G protein was stimulated by 100 mm 8-pCPT-2’-O-Me-cAMP. In contrast, treatment of the cells with just 0.1 mm 8-pCPT-2’-OMe-cAMP-AM was sufficient to induce full Rap activation. [a] M. J. Vliem, W.-J. Pannekoek, Dr. J. Riedl, M. R. H. Kooistra, Prof. Dr. J. L. Bos, Dr. H. Rehmann Department of Physiological Chemistry Centre for Biomedical Genetics and Cancer Genomics Centre University Medical Center Utrecht Universiteitsweg 100, 3584CG Utrecht (The Netherlands) Fax: (+31)88-75-68101 E-mail : j.l.bos@umcutrecht.nl h.rehmann@umcutrecht.nl [b] B. Ponsioen, Dr. K. Jalink Division of Cell Biology, The Netherlands Cancer Institute Amsterdam (The Netherlands) [c] Dr. F. Schwede, Dr. H.-G. Genieser BIOLOG Life Science Institute Flughafendamm 9a, 28071 Bremen (Germany) [] These authors contribute equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.