cAMP Analog Mapping of Epac1 and cAMP KinaseDISCRIMINATING ANALOGS DEMONSTRATE THAT Epac AND cAMP KINASE ACT SYNERGISTICALLY TOPROMOTE PC-12 CELL NEURITE EXTENSION*

Received for publication, March 3, 2003, and in revised form, June 15, 2003Published, JBC Papers in Press, June 20, 2003, DOI 10.1074/jbc.M302179200

Anne E. Christensen‡, Frode Selheim‡, Johan de Rooij§, Sarah Dremier¶�, Frank Schwede**,Khanh K. Dao‡, Aurora Martinez‡‡, Carine Maenhaut¶, Johannes L. Bos§, H.-G. Genieser**,and Stein O. Døskeland‡§§

From the Departments of ‡Anatomy and Cell Biology and ‡‡Biochemistry and Molecular Biology, University of Bergen,5009 Bergen, Norway, §Department of Physiological Chemistry and Centre for Biomedical Genetics, University MedicalCenter Utrecht, 3584 CG Utrecht, The Netherlands, ¶Institute of Interdisciplinary Research, Free University of Brussels,Campus Erasme, B-1070 Brussels, Belgium, and **BIOLOG Life Science Institute, 28071 Bremen, Germany

Little is known about the relative role of cAMP-de-pendent protein kinase (cAPK) and guanine exchangefactor directly activated by cAMP (Epac) as mediators ofcAMP action. We tested cAMP analogs for ability to se-lectively activate Epac1 or cAPK and discriminate be-tween the binding sites of Epac and of cAPKI and cAP-KII. We found that commonly used cAMP analogs, like8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPKequally as well as cAMP, i.e. were full agonists. In con-trast, 6-modified cAMP analogs, like N6-benzoyl-cAMP,were inefficient Epac activators and full cAPK activa-tors. Analogs modified in the 2�-position of the riboseinduced stronger Epac1 activation than cAMP but wereonly partial agonists for cAPK. 2�-O-Alkyl substitution ofcAMP improved Epac/cAPK binding selectivity 10–100-fold. Phenylthio substituents in position 8, particularlywith MeO- or Cl- in p-position, enhanced the Epac/cAPKselectivity even more. The combination of 8-pCPT- and2�-O-methyl substitutions improved the Epac/cAPKbinding selectivity about three orders of magnitude. ThecAPK selectivity of 6-substituted cAMP analogs, thepreferential inhibition of cAPK by moderate concentra-tions of Rp-cAMPS analogs, and the Epac selectivity of8-pCPT-2�-O-methyl-cAMP was also demonstrated in in-tact cells. Using these compounds to selectively modu-late Epac and cAPK in PC-12 cells, we observed thatanalogs selectively activating Epac synergized stronglywith cAPK specific analogs to induce neurite outgrowth.We therefore conclude that cAMP-induced neurite out-growth is mediated by both Epac and cAPK.

With the exception of cyclic nucleotide gated channels in spe-cialized cells like olfactory neurons (1), the only known directcAMP effector in mammalian cells was, until recently, the cAMP-dependent protein kinase (cAPK),1 whose mechanism of activa-

tion and structure has been studied in detail (2–4).It has been questioned whether cAPK is the sole mediator of

cAMP action (5, 6). The discovery (7, 8) of Rap guanine nucle-otide exchange factors directly activated by cAMP (Epac1 andEpac2) raised the possibility that effects hitherto attributed toactivation of cAPK were in fact mediated by Epac. Epac1 hasan N-terminal DEP (dishevelled, Egl-10, pleckstrin) domain,involved in membrane docking (9) and cell adhesion (10), acAMP binding domain (CNBD), a Ras exchange motif, and aguanine nucleotide exchange factor homology domain (see Fig.1A). Epac2 has been crystallized recently (11). It has an addi-tional CNBD (8), which is dispensable for cAMP-induced Rapactivation (9, 12).

There is increasing but still modest knowledge about thebiological consequences of cAMP activation of endogenous Epacin intact cells. Epac appears not to mediate modulation ofextracellular signal-regulated kinase activity by cAMP (13) butmay mediate the stimulation by cAMP of exocytosis (14, 15)and the calcitonin-induced H,K-ATPase activation in kidneycells (16) and may modulate integrin-mediated cell adhesion(17). Less is known about how cAMP signaling through cAPKand Epac might be integrated, but there is evidence that over-expressed Epac1 can activate Akt/protein kinase B, whereasstimulation of cAPK inhibits Akt/protein kinase B (18), sug-gesting that Epac and cAPK serve opposite functions. The aimof the present study was to develop and identify cyclic nucleo-tide analogs that could help distinguish the roles of cAPK andEpac1 in intact cells. We obtained first a detailed map of thecAMP binding site of Epac1 using more than 50 analogs, manyof which are novel. We studied next the ability of selectedanalogs to activate Epac and cAPK in vitro. We found that2�-O-alkyl-modified cAMP analogs were only partial agonists ofcAPK activation, while being stronger than cAMP itself asactivators of Epac. On the other hand, several 6-modified cAMPanalogs were full cAPK agonists and poor agonists of Epacactivation, even if they bound well to Epac.

* A preliminary account of this work was presented by Christensen etal. (42). This work was supported in part by The Norwegian ResearchCouncil (NFR), The Novo Nordic Insulin Foundation, and The Norwe-gian Cancer Society (DNK). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

� Researcher of the Fonds National de la Recherche Scientifique.§§ To whom correspondence should be addressed: Dept. of Anatomy

and Cell Biology, University of Bergen, Jonas Lies vei 91, N-5009Bergen, Norway. Tel.: 47-55-58-63-76; Fax: 47-55-58-63-60; E-mail:stein.doskeland@iac.uib.no.

1 The abbreviations used are: cAPK, cAMP-dependent protein kinase;

RI and RII, regulatory subunit of cAPK isozyme I and II, respectively;Epac, exchange protein directly activated by cAMP; Epacfl, Epac full-length protein; CNBD, cyclic nucleotide binding domain; NGF, nervegrowth factor; GST, glutathione S-transferase; CREB, cAMP-responseelement-binding protein; cPuMP, cyclic purine monophosphate; Sp- andRp-cAMPS, axial and equatorial diastereoisomer, respectively, ofcAMPS; DCl-cBIMP, dichloro-cyclic benzimidazole monophosphate;6-Bnz-cAMP, N6-benzoyl-cAMP; 6-MB-cAMP, N6-monobutyryl-cAMP.The following abbreviations were used to designate substituents: Pip,piperidino; pCPT, para-chloro-phenyl-thio; AHA, aminohexylamino;Phe, phenyl; Me, methyl; PT, phenylthio; HPT, hydroxyphenylthio;MABA, 4-[N�-methylanthraniloyl]aminobutylamino.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 37, Issue of September 12, pp. 35394–35402, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Intact cell studies confirmed the cAPK specificity of 6-modi-fied analogs and the Epac specificity of 2�-O-Me-cAMP analogslike 8-pCPT-2�-O-Me-cAMP. Using these analogs as tools weshowed that Epac acted synergistically with NGF to promoteneurite extension in PC-12 rat pheochromocytoma cells, amodel for neuronal differentiation (19, 20). Activation of Epacsensitized the cells toward cAPK. This is the first demonstra-tion that Epac and cAPK act in synergy to mediate a cAMPeffect.

EXPERIMENTAL PROCEDURES

Cyclic Nucleotide Analogs—The cAMP and cGMP analogs modifiedonly in the purine ring or the cyclic phosphate ring and all 2�-deoxycAMP analogs were provided by BIOLOG Life Science Institute, Bre-men, Germany. 2�-O-Ethyl-, 2�-O-propyl-, 2�-O-butyl-, and 2�-O-isobu-tyl-cAMP were kindly provided by Drs. B. Jastorff and J. Kruppa,Bio-Organic Unit, University of Bremen, Bremen, Germany. 8-Br-2�-O-

Methyladenosine was phosphorylated and cyclized in a one pot reactionto 8-Br-2�-O-Me-cAMP as described earlier (21). 8-pCPT-2�-O-Me-cAMPwas synthesized by nucleophilic substitution (22) of 8-Br-2�-O-Me-cAMP. Other 8-substituted 2�-O-Me-cAMP analogs were synthesizedessentially as described for 8-pCPT-2�-O-Me-cAMP. 8-pCPT-2�-O-Me-cAMP benzyl ester was prepared from 8-pCPT-2�-O-Me-cAMP by alkyl-ation with benzyl bromide (23). All analogs, including the axial andequatorial stereoisomers of 8-pCPT-2�-O-Me-cAMP benzyl ester, werepurified by reversed phase high pressure liquid chromatography (YMCODS-120A; 10 �m) and isolated to purity �98%. The structure of eachanalog was confirmed by UV-visible and electrospray ionization-massspectrometry analysis (8-Br-2�-O-Me-cAMP and 8-pCPT-2�-O-Me-cAMP also by 1H NMR and 31P NMR).

Epac and cAPK Purification—Recombinant GST-hEpac1fl, GST-hEpac12–329, GST-hEpac1149–318, GST-hEpac1149–881, GST-hRI�,and His-hRII� were expressed in E. coli BL21 cells. The GST fusionproteins were adsorbed to GST-agarose and either eluted with gluta-thione, or the GST cleaved off in situ with thrombin (7, 24). Epac1149–

FIG. 1. Determination of the affinity of full-length and truncated Epac1 for cAMP. Panel A shows a schematic representation of thedomains of the Epac1 protein. DEP, disheveled; Egl-10, pleckstrin homology domain; CNBD, cAMP binding domain; REM, Ras exchange motif;GEF, guanine nucleotide exchange factor homology domain. Numbers indicate start and stop residues for the truncated versions of hEpac1 used.Panel B shows typical gel filtration experiments to determine binding of [3H]cAMP to Epac under equilibrium conditions. Size exclusion gelfiltration columns were equilibrated at 37 °C with 2.8 or 80 �M [3H]cAMP, loaded with Epac1149–318 preincubated with the same concentrationof [3H]cAMP, and the amount of [3H]cAMP was determined in the collected fractions. Note the peak of Epac-associated [3H]cAMP in the excluded(macromolecular) fractions and the depression of [3H]cAMP in the later fractions. Panel C shows the saturation of Epac149–318 with [3H]cAMPas a function of free [3H]cAMP, based on data from experiments like those shown in panel B. The columns were equilibrated with variousconcentrations of [3H]cAMP (abscissa). Panel D shows the saturation of GFP-Epacfl (�) and GFP-Epac149–881 (‚) as a function of free [3H]cAMP,as assayed by scintillation proximity assay. The proteins were immobilized to GSH-coated wells of a microplate with solid scintillant (flashplate),and the radioactivity close to the wall (bound to immobilized Epac) was determined. The data points represent the mean � S.E. of at least fourseparate experiments for Epac149–318 and for Epac149–881 and the mean of two experiments for Epacfl. The concentration of [3H]cAMP at whichhalf-maximal saturation was observed (apparent KD) is indicated in panels C and D.

FIG. 2. Determination of the efficiency of various cAMP analogs to compete with [3H]cAMP for binding to Epac. Panel A showsdisplacement of [3H]cAMP from its complex with Epac149–318 by increasing concentrations (Conc.) of unlabeled cAMP analog. The data are givenas the ratio (C0/Cx) of Epac-associated [3H]cAMP in the absence (C0) and presence (Cx) of competing nucleotide. Open symbols refer to dataobtained using the ammonium sulfate precipitation method. Filled symbols refer to results obtained using the gel filtration method illustrated inpanel B, which shows the effect of a low concentration of unlabeled 8-pCPT-cAMP on the elution of the [3H]cAMP-Epac complex.

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318 was a gift from Drs. A. Kramer and A. Wittinghofer (25). Theproteins were further purified by size exclusion fast protein liquidchromatography as described (24). The catalytic subunit of cAPK wasprepared as described (24).

Determination of [3H]cAMP Binding to the R Subunits of cAPK andto Epac1—An extensively validated ammonium sulfate precipitationmethod was used to assay [3H]cAMP bound to the RI and RII subunitsof cAPK (26). To determine [3H]cAMP bound to the more rapidly ex-changing binding site of Epac we used cold (�10 °C) 98% saturatedammonium sulfate. Unbound isotope was removed by immediate filter-ing and rinsing with 1 ml of the ammonium sulfate solution.

A size-exclusion chromatography method (27) was used to assay thebinding of [3H]cAMP to Epac under strict equilibrium conditions. Thebuffer (Buffer A; 15 mM Hepes, pH 7.2, with 1 mM Na2PO4, 130 mM KCl,2 mM Mg(CH3COO)2, 2 mM glutathione, 0.1 mM �-mercaptoethanol, 0.3mM EGTA, 1 mM EDTA, 0.2 mM AMP, and 0.2 mg/ml soybean trypsininhibitor) had near physiological pH and ionic strength. To assay for[3H]cAMP without interference from cAMP-induced Epac precipitationGST-Epac was attached to GSH-coated Flashplates (PerkinElmer LifeSciences). To determine [3H]cAMP binding, the plates were incubatedwith Buffer A (0.1 ml) and various concentrations of [3H]cAMP andanalyzed (at 25 °C) in a TopCount NXT scintillation counter (Packard,Meriden, CT).

Determination of the cAMP Analog Binding Affinity for Sites A and Bof RI and RII and for Epac1—The equilibrium inhibition constant ofbinding (Ki) of cAMP analog was determined by competitive displace-ment of [3H]cAMP binding to R or Epac. The analog affinity relative tocAMP (K�ianalog) is KicAMP/Kianalog. The determination of K�ianalogfor Epac was, in principle, as described previously (28) and validated forRI and RII.

In Vitro Assay of Epac-induced Rap Activation and cAMP-dependentProtein Kinase Activity—Cyclic AMP analogs were tested for in vitroRap1 or Rap2 activation by determination of their effect on the rate ofEpac-induced fluorescent GDP analog (3�-O-(N-methylantraniloyl)-GDP) exchange from Rap (9). The determination of cAMP analog effectson protein kinase activity was routinely by incubation for 40 min at37 °C in Buffer A with 1 mM [�-32P]ATP, using kemptide as substrate(24).

Assay of cAMP Analog Actions in Intact Cells—The activation of Rapwas determined by GST pull-down assay as described (29), except thatNaF was omitted, and 5 mM Mg(CH3COO)2 added in lysis and washbuffers. The amount of immobilized Ral-GDS-RBD-GST protein (whereGDS is GDP dissociation stimulator and RBD is Ras binding domain)used to capture Rap1-GTP was in excess to ensure that variation inpipetting error of the slurry would not affect the pull down of Rap-GTP.Phospho-CREB was determined by Western blotting using the Ab5322(a kind gift from Dr. M. Montminy, Salk Institute, La Jolla, CA) andcompared with the amount of total CREB using a non-discriminatingCREB antibody (number 9192; www.cellsignal.com). Primary dog thy-roid cells were cultured and assessed for rounding as described earlier(5, 30). Rat pheochromocytoma PC-12 cells were cultured in RPMI with10% horse serum and 5% fetal calf serum. The cells were kept for 20 hin intact medium after seeding and 1 h with low (5% horse and 2.5%fetal calf) serum, before being stimulated with cAMP analog or NGF for3 to 72 h, and fixed for evaluation by phase and differential interferencecontrast microscopy (31).

Molecular Modeling—The structures of the CNBDs A and B of RI�(Protein Data Bank accession code 1RGS) (4) were used as templates toconstruct a structural model of the CNBD of Epac1. The model obtainedusing the Homology module of InsightII 2000 (MSI found at www.msi.

TABLE IAffinity of purine base-modified cAMP analogs the for Epac1 and site A and B of RI� and RII�

The table shows the affinity of cAMP analogs relative to cAMP (i.e. K�i, which is Ki cAMP/Ki analog) for Epac1 and site A and B of RI� (AI andBI) and RII� (AII and BII). The data were obtained by competitive isotope displacement, as detailed under “Experimental Procedures.” The resultsrepresent the mean of three determinations, ranging � 15% from the average.

Compound K�i Epac1K�i RI� K�i RII�

AI BI AII BII

cAMP 1.0 1.0 1.0 1.0 1.02-NH2-cAMP 0.13 0.043 0.35 0.036 0.0298-Pip-cAMP 0.21 2.1 0.060 0.047 2.78-Br-cAMP 8.1 1.3 1.0 0.11 6.88-pCPT-cAMP 65 3.9 1.7 0.054 198-AHA-cAMP 1.3 0.055 4.1 0.010 0.396-Phe-cAMP 2.8 11 0.38 19 0.346-Bnz-cAMP 1.3 4.0 0.26 3.8 0.0376-MB-cAMP 0.77 3.6 0.093 0.74 0.041cIMP (6 � O) 0.28 0.12 0.025 0.094 0.0051cGMP (2-NH2, 6 � O) 0.078 0.0051 0.012 0.0041 0.000936-Phe-8-pCPT-cAMP 110 8.1 0.90 0.040 9.6

TABLE IIMapping of Epac1, RI�, and RII� with cAMP analogs modified in their cyclic phosphate or ribose moiety

The table shows the affinities for Epac1, RI� (sites AI and BI), and RII� (AII and BII) of cAMP analogs with sulphur (Sp- or Rp- diastereomi-somer) substitution in the cyclic phosphate ring or with modification in the 2 position of the ribose ring. Several analogs had additionalsubstitutions in the purine ring. Other details are described in the legend to Table I.

Compound K�iEpac1

K�i RI� K�i RII�

AI BI AII BII

Sp-cAMPS 0.085 0.18 0.034 0.038 0.43Sp-8-pCPT-cAMPS 5.2 1.6 0.087 0.0059 2.3Sp-5,6-DC1-cBIMPS 0.69 0.022 0.13 0.034 14Rp-cAMPS 0.0083 0.0013 0.0028 0.0021 0.022Rp-8-Br-cAMPS 0.098 0.0008 0.00071 0.00048 0.094Rp-8-pCPT-cAMPS 0.36 0.015 0.0068 0.0012 0.172�-dcAMP 0.0025 0.000065 0.00028 0.000070 0.000118-pCPT-2�-dcAMP 0.17 0.002 0.00069 0.00014 0.00916-Phe-8-pCPT-2�-dcAMP 0.46 0.031 0.011 0.20 0.0462�-O-Me-cAMP 0.12 0.0090 0.0026 0.0019 0.0142�-O-Et-cAMP 0.050 0.0049 0.00382�-O-Pr-cAMP 0.025 0.0011 0.000512�-O-Bu-cAMP 0.025 0.0010 0.000638-Br-2�-O-Me-cAMP 0.90 0.0037 0.00047 0.0028 0.00418-PT-2�-O-Me-cAMP 3.4 0.018 0.000528-pCPT-2�-O-Me-cAMP 4.6 0.043 0.0016 0.00059 0.0248-pHPT-2�-O-Me-cAMP 6.98-pMeOPT-2�-O-Me-cAMP 7.1 0.025 0.00060

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com) was similar to that obtained by the Swiss-Pdb Viewer in conjunc-tion with SWISS-MODEL (32). Final optimization of the structure ofthe complexes was performed using Discover (MSI). The programsInsightII (MSI) and WebLab Viewer (MSI) were used to prepare thefigures.

RESULTS

Binding of [3H]cAMP and cAMP Analogs to Full-lengthEpac1, the CNBD Fragment of Epac, and Sites AI, BI, AII, andBII of cAPK—The isolated CNBD (Epac149–318) of Epac iswell expressed and does not aggregate in the presence of cAMP,unlike Epacfl and Epac149–881 (25). To know whether thebinding properties of CNBD are relevant for full-length Epac,we compared their affinity for cAMP. The isolated CNBD(Epac149–318) of Epac1 bound [3H]cAMP with an apparent KD

of 2.9 �M (Fig. 1, B and C) at close to physiological pH (7.2) andionic strength, as determined by the time-honored size exclu-sion gel chromatography method (27). To prevent aggregation,we anchored GST-Epacfl and GST-Epac149–881 to GSH-coated plates with intrinsic scintillant and determined[3H]cAMP binding by scintillation proximity assay. GST-Epacfland GST-Epac149–881 bound [3H]cAMP with an apparent KD

of 2.8 �M (Fig. 1D).We conclude that the CNBD of Epac has the same affinity for

cAMP whether in a 170-residue peptide (Epac149–318) or in-cluded in the full-length Epac molecule. Cyclic AMP analogmapping of the binding sites of Epac1, RI and RII, was under-taken to probe for differences between the Epac and R subunitbinding sites. We showed first that the estimated K�i of cAMPanalogs for binding to Epac149–318 was similar whether as-sayed by the routine ammonium sulfate precipitation methodor the equilibrium binding chromatography assay (Fig. 2).

Mapping data for analogs modified only in the adenine moi-ety are shown in Table I. We noted that the commonly usedcAPK activators 8-Br-cAMP, 8-AHA-cAMP, and particularly

8-pCPT-cAMP had higher affinity than cAMP itself for Epac.No analog was severely restricted from binding to Epac,whereas several had very low K�i for binding to one or more of

FIG. 3. The activation of cAPKI and cAPKII by 2�-modifiedcAMP analogs. The activity of the catalytic subunit of cAPK (0.25 nM),repressed by 10 nM hRI� (panel A) or hRIIa subunit (panel B), wasde-repressed by cAMP (E), 2�-O-methyl-cAMP (●), 2�-O-ethyl-cAMP(‚), 2�-O-propyl-cAMP (�), 2�-O-butyl-cAMP (f), or 2�-dcAMP (Œ). Theactivity is the percentage of that observed in the absence of added Rsubunit. The incubation was for 40 min at 37 °C with 1 mM ATP and 100�M kemptide. Most of the data represent the average of two separateexperiments, whereas some are given as mean � S.E. and represent theaverage of four experiments.

FIG. 4. The activation of cAPKI by cAMP and by 8-pCPT- and8-Br-substituted 2�-O-Me-cAMP at low and near physiologicalconcentration of RI�. The assay was as described for Fig. 3, exceptthat the concentration of hRI� was 3 nM (�), 30 nM (Œ), or 300 nM (E).De-repression of kinase activity was by increasing concentrations ofcAMP (A), 8-pCPT-2�-O-Me-cAMP (B), or 8-Br-2�-O-Me-cAMP (C). Thedata represent mean � S.E. (n � 3–4), except for panel C, whichpresents the average from two separate experiments.

FIG. 5. The activation of cAPKII by cAMP and by 8-pCPT-2�-O-Me-cAMP at low and near physiological concentration of RII�.The experimental details were as for the experiments shown in Fig. 4,except that hRII� at 1 nM (�), 15 nM (Œ), 100 nM (E), or 400 nM (�) waspresent rather than RI�.

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the binding sites of RI� (AI, BI) or RII� (AII, BII). All the Rsubunit binding sites discriminated better than Epac againstcGMP (Table I). The modestly (12-fold) decreased K�i of cGMPfor Epac was because of a combined effect of the introduction of2-NH2 and 6�O into the cAMP molecule (Table I). The Sp- andRp-diastereoisomers of cAMPS bound to Epac with a relativeaffinity similar to that for the cAPK sites (Table II). The pre-sumed cAPK-specific agonists Sp-8-pCPT-cAMPS and Sp-5,6-DCl-cBIMPs (33, 34) bound to Epac with an affinity similar tothat of cAMP itself (Table II).

The loss of the 2�-OH of cAMP was more detrimental forbinding to cAPK than to Epac, because both 2�-deoxy- and2�-O-alkyl-cAMP had higher K�i for Epac than for RI or RII(Table II). A more detailed survey of 8-substituted analogs of2�-O-Me-cAMP indicated that the highest affinity was achievedwhen the S-phenyl ring was substituted in the para-positionwith a polar group, like Cl�, HO�, O-methyl (Table II), or F�

(not shown).Comparison of cAMP Analogs as Modulators of cAPK and

Epac Activity in Vitro and in Intact Cells—It is not knownwhether cAMP analogs modified in the 2�-position are full orpartial agonists for cAPK. We found that 2�-O-Me-cAMP, 8-Br-2�-O-Me-cAMP, and 8-pCPT-2�-O-Me-cAMP failed to activatecAPK completely in vitro (Figs. 3-5). This indicates that 2�-O-Me analogs of cAMP are only partial agonists with respect toactivation of cAPK. At near physiological concentration (300–600 nM) of R subunit (24) 8-Br-2�-O-Me-cAMP (1 mM) achievedless than 15% activation of cAPKI, and 8-pCPT-2�-O-Me-cAMP(1 mM) achieved less than 25% activation of either isozyme.

The 2�-O-Me-cAMP analogs were next tested for ability tostimulate the Epac1-catalyzed dissociation of the GDP-Rapcomplex. The compounds 8-pCPT-2�-O-Me-cAMP, 8-pHPT-2�-O-Me-cAMP, and 8-pMeOPT-2�-O-Me-cAMP all stimulated thedissociation of GDP more strongly than cAMP or 8-Br-cAMP(Fig. 6A and data not shown). We conclude that 2�-O-Me-cAMPanalogs super-activate Epac.

All cAMP analogs modified in the 6-position of the adeninering, including cPuMP, 6-Cl-cPuMP, cIMP, cGMP, 6-MB-cAMP, 6-Bnz-cAMP, and to a lesser extent 6-Phe-cAMP, wereonly partial Epac agonists (data for 6-Bnz-cAMP are shown inFig. 6A). Furthermore, the partial stimulation was observed ata higher concentration than expected from the analog affinityfor free Epac (not shown).

In fibroblasts with enforced expression of Rap1 and Epac1,the analogs 6-MB-cAMP and 6-Bnz-cAMP stimulated stronglythe phosphorylation of the cAPK substrate CREB but failed toactivate Rap1 (not shown). Preferential stimulation by 6-mod-ified cAMP analog of CREB phosphorylation was observed alsoin primary dog thyrocytes. In these cells rounding is a specificreaction to cAPK (5). Cell rounding was induced by 6-MB-cAMP (Fig. 7A) and 6-Bnz-cAMP (not shown) without Rap1activation (Fig. 7A). In contrast, 8-pCPT-cAMP and the adeny-late cyclase activator forskolin induced Rap activation, CREBphosphorylation, and cell rounding, whereas 8-pCPT-2�-O-Me-cAMP only induced Rap activation (Fig. 7A). We conclude that6-modified cAMP analogs, notably 6-Bnz-cAMP and 6-MB-cAMP, may be useful to preferentially activate cAPK in intactcells.

It was of interest to know whether Rp-cAMPS analogs couldbe used to inhibit Epac, and we used Rp-8-p-CPT-cAMPS totest this hypothesis, because this compound had a high affinityfor Epac (Table II). Rp-8-pCPT-cAMPS is an inhibitor of bothcAPKI and cAPKII in intact cells (33, 35). It was a very weakEpac agonist in vitro (Fig. 6B) and did not activate Rap inintact thyrocytes (Fig. 7A). Rp-8-pCPT-cAMPS had a weak

ability to counteract 8-Br-cAMP-induced Rap activation invitro (Fig. 6B) although it could inhibit 8-pCPT-cAMP-inducedRap activation in thyrocytes when present at high concentra-tion. The compound was a stronger inhibitor of CREB phospho-rylation and rounding than of Rap activation in thyrocytes (Fig.7, B and C). This suggests that Rp-8-pCPT-cAMPS would haveto be modified to act as a specific Epac antagonist in intactcells. Rp-8-Br-cAMPS, which inhibits cAPKI in intact cells (35),was a weak partial Epac agonist in vitro (Fig. 6B) and failed toinhibit either basal (Fig. 7A) or 8-pCPT-cAMP-induced (notshown) Rap activation in intact thyroid cells or fibroblasts. Weconclude that presently available Rp-cAMPS analogs are un-able to preferentially inhibit Epac and may be more potentantagonists of cAPK than Epac.

Use of cAMP Analogs to Elucidate the Roles of cAPK andEpac in Mediating cAMP-induced PC-12 Cell Neurite Exten-sions and Rap1 Activation—It is uncertain whether Epac orcAPK is responsible for the cAMP-induced Rap1 activation inPC-12 cells (20). The Epac activator 8-pCPT-2�-O-Me-cAMPactivated Rap1 without stimulating CREB phosphorylation.The cAPK activator 6-Bnz-cAMP stimulated CREB phospho-rylation but did not significantly activate Rap1 (Fig. 7D). Weconclude that Epac stimulation can activate Rap1 in the ab-sence of cAPK activation.

Neurite extensions were induced by the cAPK activator6-Bnz-cAMP, the Epac activator 8-pCPT-2�-O-Me-cAMP, orNGF (Fig. 8). Cells incubated with 8-pCPT-2�-O-Me-cAMP hadincreased sensitivity to 6-Bnz-cAMP. Cells incubated withNGF were also sensitized to 6-Bnz-cAMP. In the combinedpresence of NGF and 8-pCPT-2�-O-Me-cAMP the cells re-sponded strongly to 6-Bnz-cAMP at concentrations (0.02–0.05mM; see Fig. 9B) that hardly had any effect when given alone

FIG. 6. In Vitro stimulation of the guanine nucleotide ex-change factor activity of EPAC149–881 by cAMP analogs. Thedissociation of the Rap-mantGDP complex was monitored by decreasedfluorescence. In panel A, 20 nM EPAC149–881 was incubated with 300nM Rap2A-mantGDP at 20 °C in the absence of cyclic nucleotide (E) orin the presence of 10 �M 6-MB-cAMP (‚), 8-Br-cAMP (�), or 8-pCPT-2�-O-Me-cAMP (●). In panel B, 100 nM EPAC149–881 was incubatedwith 100 nM Rap1A-mantGDP in the absence of cyclic nucleotide (E) orthe presence of 10 �M 8-Br-cAMP (‚), Rp-8-Br-cAMPS (�), or Rp-8-pCPT-cAMPS (ƒ). After 5 min (arrow 1) the Rp-8-Br-cAMPS and Rp-8-pCPT-cAMPS incubations were supplied with more of the same ana-log to a final concentration of 100 �M. 10 min thereafter (arrow 2) thesame two incubations received 8-Br-cAMP to a final concentration of100 �M.

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(Fig. 9A). This suggested that Epac, and to some extent NGF,had a permissive and sensitizing effect on cAPK-stimulatedneurite extension.

To ensure that 6-Bnz-cAMP and not 8-pCPT-2�-O-Me-cAMPacted via cAPK also with respect to neurite extensions, wetested them in the presence of the ATP site-directed cAPKinhibitor H-89 (36, 37) and a combination of Rp-cAMPS andRp-8-Br-cAMPS. These Rp analogs have lower affinity for Epacthan Rp-8-pCPT-cAMPS (Table II) and should therefore bemore cAPK selective. Furthermore, when combined, they in-hibit both cAPKI and cAPKII, because Rp-8-Br-cAMPS is aninhibitor of cAPKI and Rp-cAMPS of cAPKII in intact cells (35).The cAPK antagonists inhibited the action of 6-Bnz-cAMPmuch more strongly than the action of 8-pCPT-2�-O-Me-cAMPand failed to affect the NGF action (Fig. 9, A and C). ThecAMP analog 8-pCPT-cAMP is expected to activate both cAPKand Epac. It had an effect comparable with that of 8-pCPT-2�-O-Me-cAMP and 6-Bnz-cAMP combined and was inhibited byRp-cAMPS analogs to a similar extent as 8-pCPT-2�-O-Me-cAMP and 6-Bnz-cAMP in combination (Fig. 9C). Similar re-sults were obtained with 8-Br-cAMP (data not shown).

We considered next whether 8-pCPT-2�-O-Me-cAMP couldact by binding to an as yet uncharacterized cAMP receptor. Wetested therefore whether cAMP analogs induced neurite exten-sion with potency expected from their affinity for Epac1. Theanalog 8-pCPT-2�-deoxy-cAMP had 27-fold lower affinity than8-pCPT-2�-O-Me-cAMP for Epac and failed to induce neuriteextensions. 8-Br-2�-O-Me-cAMP had 5-fold lower affinity andpromoted neurite extensions only weakly and at high (�0.5mM) concentrations (not shown). 8-pHPT-and 8-pMeOPT-2�-O-Me-cAMP bound to Epac1 with 50% higher affinity than8-pCPT-2�-O-Me-cAMP (Table II) and were slightly more po-tent than 8-pCPT-2�-O-Me-cAMP in inducing neurite exten-

FIG. 7. Effect of cAMP analogs on intact cell activation ofRap1, phosphorylation of CREB, and rounding. Panels A–C showtotal Rap1, active Rap1 (Rap1-GTP), phospho-CREB (P-CREB), andtotal CREB in primary dog thyrocytes. Rp-8-pCPT-cAMPS was presentfor 60 min before addition of 8-pCPT-cAMP (panels B and C). Other-wise, analogs were present for 10, 30, or 60 min for the determinationof Rap activation, rounding, and P-CREB, respectively. The concentra-tion of forskolin was always 10 �M. For panels A and D all cAMPanalogs were at 0.5 mM. Thyrocyte rounding was determined by phasecontrast microscopy. 3�, �90% of cells rounded; 2�, 30–60% cellrounding; 0, less than 4% rounded cells. For PC-12 cells (panel D)analogs were present for 30 min before cell lysis.

FIG. 8. The effect of cAMP analogs on PC-12 cell neurite exten-sions in the absence and presence of NGF. The upper panels showdifferential interference contrast micrographs of PC-12 cells treated for5 h (panels A–F) or 24 h (panels H–M) in the absence (panels A, C, E, H,J, and L) or presence (B, D, F, I, K, and M) of 0.6 mM of the Epacactivator 8-pCPT-2�-O-Me-cAMP. The cells received vehicle (control),0.1 mM 6-Bnz-cAMP, or NGF (50 ng/ml). The lower panels show thepercent of PC-12 cells with neurite extensions between 10 and 20 �m(open bars) or longer than 20 �m (solid bars) after incubation for 5 h(panel G) or 24 h (panel N) with the agents indicated below the abscissa.

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sions (Fig. 9D). We conclude that 8-pCPT-2�-O-Me-cAMP actedvia Epac1 or a cAMP receptor with binding properties nearlyidentical to Epac1.

It could be argued that 8-pCPT-2�-O-Me-cAMP had an extra-cellular action, e.g. by binding to a surface receptor. To test thispossibility we prepared the benzyl ester pro-drug of 8-pCPT-2�-O-Me-cAMP. This inactive compound penetrates cells betterthan the parent compound and is converted to the parentcompound within the cell (38). It was more potent than theparent compound as inducer of neurite extensions (Fig. 9E). Weconclude that Epac agonists like 8-pCPT-2�-O-Me-cAMP mostlikely acted intracellularly.

It was finally considered whether 8-pCPT-2�-O-Me-cAMPacted through inhibition of cyclic nucleotide phosphodiesteraseleading to increased cGMP and activation of cGMP targets. Wefound no inhibition of the action of 8-pCPT-2�-O-Me-cAMP bythe cGMP-dependent protein kinase inhibitor Rp-8-pCPT-cGMPS, and we failed to promote neurite outgrowths with thecGMP-dependent protein kinase activators 8-Br-cGMP and Sp-8-Br-PET-cGMPS (not shown). We conclude that cAMP analogscan be used to dissect separately the roles of Epac and cAPK asmediators of cAMP action and that Epac and cAPK act insynergy to promote PC-12 cell neurite extensions.

DISCUSSION

The cAMP analog mapping revealed significant differencesbetween the binding sites of Epac and the R subunits of cAPK.In general, Epac had less stringent requirements for binding ofeither adenine- or 2�-ribose-modified analogs than any of thecAPK binding sites (AI, BI, AII, BII). Molecular docking sug-gests that Epac could accommodate bulky adenine substituentsbecause of little steric hindrance in the region facing the ade-nine. In addition, perfect stacking between the adenine of an-alogs and an aromatic residue is not required for binding toEpac, which has no aromatic residue in a position correspond-ing to Tyr-371 in bRI� (Fig. 10A). Introduction of an 8-thio-phenyl substituent with a polar group like Cl-, HO- or MeO- inpara-position enhanced the affinity for Epac more than 50-fold.Docking of 8-pCPT-2�-O-Me-cAMP into the CNBD of Epac1(Fig. 10B) suggested that the high affinity of 8-thiophenylsubstituted cAMP related to the ability of His-317 to providestacking interactions with the phenyl ring. The further affinityenhancement from a polar group in the para-position of thethiophenyl substituent could be explained by the predictedsolvent exposure of the apical part of the phenyl ring (Fig. 10B).Modification of the 2�-position of the ribose was less detrimen-tal for binding to Epac than to cAPK. Comparative docking of8-pCPT-2�-O-Me-cAMP (Fig. 10B) and cAMP (Fig. 10A) into theCNBD of Epac1 suggested that the H-bonding of the oxygen of2�-OH of cAMP with the side chain amino of Gln-270 wasmaintained by 2�-O-Me. On the other hand, 2�-O-Me loses theparticularly strong (39) H-bond of the 2�-OH of cAMP to Glu-324 in site BI (Fig. 10C).

FIG. 9. Demonstration of specificity of and synergy in promo-tion of PC-12 cell extensions by cAPK-activating and Epac-acti-vating cAMP analogs. PC-12 cells were treated for 5 h (A–D) or 1.5 h(E) with various cyclic nucleotide analogs in the absence or presence of

NGF and analyzed for the presence of extensions �20 �m (panels A, B,D, and E) or 15 �m (panel C). Panel A, cells were incubated with variousconcentrations of 6-Bnz-cAMP alone (●), with 0.5 m M Rp-cAMPS � 0.5mM Rp-8-Br-cAMPS (�), or with 0.6 mM 8-pCPT-2�-O-Me-cAMP (Œ).Panel B, cells were treated with various concentrations of 6-Bnz-cAMPand either 50 ng/ml NGF (●) or NGF � 0.6 mM 8-pCPT-2�-O-Me-cAMP(Œ). Panel C shows the effect of the cAPK inhibitors H-89 (60 �M),Rp-cAMPS (0.5 mM), and Rp-8-Br-cAMPS (0.5 mM) on neurite out-growth in the presence of NGF (50 ng/ml), 8-pCPT-2�-O-Me-cAMP (0.6mM), 6-Bnz-cAMP (0.1 mM), or 8-pCPT-cAMP (0.2 mM). Panel D showsthe stimulation of cell extensions by various concentrations of 8-pHPT-2�-O-Me-cAMP, 8-pMeOPT-2�-O-Me-cAMP, and 8-pCPT-2�-O-Me-cAMP, and panel E shows stimulation by various concentrations of8-pCPT-2�-O-Me-cAMP (E) or the axial benzyl triester prodrug of8-pCPT-2�-O-Me-cAMP (‚).

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8-pCPT-2�-O-Me-cAMP and other 2�-O-alkyl analogs ofcAMP were unable to activate cAPK fully under physiologicallyrelevant conditions. This is a novel finding and indicates that

2�-O-Me-cAMP analogs are partial rather than full cAPK ago-nists. Even in the unlikely event that they should reach milli-molar concentration inside the cell, they would be unable to

FIG. 10. Modeling of the structure of the CNBD of Epac1 and docking of 8-pCPT-2�-O-Me-cAMP. A comparison with site BI of bRI� isshown. Panel A, composite of the structures of the CNBD of Epac1 (blue) and site BI of bRI� (orange). Residues at contact distance from cAMP (inscaled ball and stick representation) are shown as sticks. Panel B shows 8-pCPT-2�-O-Me-cAMP (ball and stick representation) docked in the CNBDof Epac1. Note the favorable stacking interaction between His-317 and the 8-pCPT group of the analog. Panel C shows 8-pCPT-2�-O-Me-cAMPdocked in the B site of RI�. Note the limited space around the cAMP analog molecule.

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activate cAPK above the normal resting level (20–30% of fullactivity).

The CNBD of free Epac showed only partial discrimination(about 10-fold) between cAMP and cGMP (Table I). This wouldnot be sufficient to prevent cGMP binding to Epac in cellswhere the cGMP level exceeds that of cAMP. We found that6-modified cAMP analogs, including cGMP, were only weakpartial agonists of Epac activation, indicating that Epac is onlyweakly activated by cGMP under physiological conditions. Ba-sically, guanine nucleotide exchange factors like Epac act bystabilizing the free, relative to the GDP-complexed form, ofG-proteins (40). An intact amino group in the 6-position ap-pears therefore to be crucial for cAMP analogs to induce theactive conformation of Epac with enhanced affinity for free Raprelative to Rap-GDP. The low efficiency of the 6-modified ana-logs was not because of introduction of bulk causing sterichindrance, because cPuMP and cIMP/cGMP have less bulkthan cAMP at the 6-position but still were only weak agonists.

The intact cell studies showed that 6-Bnz-cAMP and 6-MB-cAMP failed to activate Rap1. In contrast, 8-pCPT-2�-O-Me-cAMP, 8-pCPT-cAMP, 8-Br-cAMP, and the adenylate cyclasestimulator forskolin activated Rap1 in the same cells (fibro-blasts, dog thyrocytes, PC-12 cells) under comparable condi-tions. This suggests that 6-modified analogs are indeed poorEpac activators in the intact cell. It also suggests that Rap1activation by cAMP is more likely to be mediated by Epac thancAPK in the cells under the conditions of the present study.

Armed with discriminatory cAMP analogs it was possibleto dissect the relative role of Epac and cAPK in cAMP-induced neurite extension in rat pheochromocytoma PC-12cells. The Epac activator 8-pCPT-2�-O-Me-cAMP appearedable to induce neurite extensions on its own. Only analogswith a high affinity for Epac could substitute for 8-pCPT-2�-O-Me-cAMP, indicating that Epac and not another cAMPreceptor was the target. This suggests Epac to have an im-portant, hitherto unrecognized, role in cAMP-induced neuriteextension. Rp-cAMPS analogs and H-89 counteracted theaction of 6-Bnz-cAMP much more strongly than the action of8-pCPT-2�-O-Me-cAMP, further underscoring the Epac spec-ificity of 8-pCPT-2�-O-Me-cAMP.

Activation of Epac had a strongly enhancing effect on cAPK.One obvious consequence of the synergy between Epac andcAPK is to provide positive cooperativity of cAMP action, be-cause activation of Epac enhanced the action of cAPK activator,which in turn enhanced the action of Epac activator (Fig. 9).

NGF sensitized the cells to both cAPK and Epac activators. Achallenging observation was that the Epac stimulator 8-pCPT-2�-O-Me-cAMP acted more rapidly than NGF to induce neuriteextensions, although NGF gave rise to a higher early activationof Rap1.2 This suggests that activation of Epac does not merelyact to mimic and replace the Rap1 activation by NGF butprobably has a distinct action, possibly by activating Rap1 in adifferent compartment than NGF. The Epac activator can alsohave hitherto unrecognized effects not mediated via activationof Rap (41).

In conclusion, we have mapped fine structural differencesbetween the binding sites of Epac1 and cAPK and elucidateddifferences in the mechanism of activation of Epac and cAPKusing cAMP analogs. Selective analogs have been demon-strated to discriminately activate Epac or cAPK and therebypoint out a hitherto unknown synergism between Epac andcAPK in PC-12 cell differentiation.

Acknowledgments—We are grateful to Kirsten Brønstad, LarsHerfindal, Reidun Kopperud, Miranda van Triest, Holger Rehmann,and Jaques Dumont for expert help and discussions.

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Stein O. DøskelandKhanh K. Dao, Aurora Martinez, Carine Maenhaut, Johannes L. Bos, H.-G. Genieser and

Anne E. Christensen, Frode Selheim, Johan de Rooij, Sarah Dremier, Frank Schwede,SYNERGISTICALLY TO PROMOTE PC-12 CELL NEURITE EXTENSION

ANALOGS DEMONSTRATE THAT Epac AND cAMP KINASE ACT cAMP Analog Mapping of Epac1 and cAMP Kinase: DISCRIMINATING

doi: 10.1074/jbc.M302179200 originally published online June 20, 20032003, 278:35394-35402.J. Biol. Chem. 

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