doi:10.1006/jmbi.2001.4444 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 306, 1167±1177

Dynamic Interaction of cAMP with the RapGuanine-nucleotide Exchange Factor Epac1

Astrid Kraemer1, Holger R. Rehmann2, Robbert H. Cool1

Christiane Theiss1, Johan de Rooij2, Johannes L. Bos2

and Alfred Wittinghofer1*

1Max-Planck-Institut fuerMolekulare Physiologie, Otto-Hahn-Strasse 1144227 Dortmund, Germany2Department of PhysiologicalChemistry and Centre forBiomedical Genetics, UMCUtrecht, Universiteitsweg 1003584 CG Utrecht, TheNetherlands

Present address: R. H. Cool, RijksNN Haren, The Netherlands.

Abbreviations used: GEF, guanin8-(4-(N-methylanthraniloyl)aminobu(6-(N-methylanthraniloyl)aminohexyethenoadenosine-30,50-cyclic monoph20-O-(N-methylanthraniloyl)adenosibenzofurazanyl)amino]ethyl]thio]admethylanthraniloyl)guanosine diphkinase A; REM, Ras exchanger motsaline; EDTA, [ethylenedinitrilo]tetrcNMP, cyclic nucleotide monophos

E-mail address of the correspond

0022-2836/01/051167±11 $35.00/0

Epac1 is a Rap-speci®c guanine-nucleotide exchange factor (GEF) whichis activated by the binding of cAMP to a cyclic nucleotide monophos-phate (cNMP)-binding domain. We investigated the equilibrium anddynamics of the interaction of cAMP and Epac1 using a newly designed¯uorescence analogue of cAMP, 8-MABA-cAMP. We observed that theinteraction of cAMP, measured by competition with 8-MABA-cAMP,with an isolated cNMP binding domain of Epac1 has an overall equili-brium constant (Kd) of 4 mM and that the kinetics of the interaction arehighly dynamic. The binding properties of cAMP are apparently notaffected when the catalytic domain is present, despite the fact that bind-ing of cAMP results in activation of Epac1. This indicates that for theactivation process, no appreciable binding energy is required. However,when bound to Rap1b, the apparent Kd of Epac to cAMP was about ®ve-fold lower, suggesting that substrate interaction stabilizes cAMP binding.Since the ¯uorescent analogues used here were either less able or unableto induce activation of Epac1, we concluded that the binding of nucleo-tide to Epac and the activation of GEF activity are uncoupled processesand that thus appropriate cAMP analogues can be used as inhibitors ofthe Epac1-mediated signal transduction pathway of Rap.

# 2001 Academic Press

Keywords: cAMP; Epac; Rap; guanine nucleotide exchange factor;¯uorescent cAMP analogues

*Corresponding author

Introduction

Ras-related GTP-binding proteins are molecularswitch proteins which cycle between an inactiveGDP-bound state and an active GTP-bound state.Activation is achieved by the exchange of tightlybound GDP for GTP, whereas return to the inac-tive state is through the GTPase reaction.1,2 Both of

universiteit Groningen, D

e-nucleotide exchange factyl)aminoadenosine-30,50-l)adenosine-30,50- cyclic mosphate; e-cAMP, 1,N6-et

ne-30,50-cyclic monophospenosine-30,50-cyclic monop

osphate; mant, N-methylaif; DEP domain, Dishevellaacetic acid; DTE, dithioephate; ¯, full-length.ing author: Alfred.Witting

these reactions are very slow and are activated byguanine-nucleotide exchange factors (GEFs) andGTPase-activating proteins (GAPs), respectively.The former stimulate the intrinsic GDP-dissociationreaction by several orders of magnitude, as wasshown for Ras,3 Rap,4 and Ran-speci®c GEFs.5

Rap is a close homologue of Ras,6,7 (for a reviewsee ref. 8) which was isolated as an antagonist of

epartment of Molecular Microbiology, Kerklaan 30, 9751

tor; GAP, GTP-ase activating proteins; 8-MABA-cAMP,cyclic monophosphate; 6-MAH-cAMP, N6-onophosphate; 2-aza-(-cAMP, 2-aza-1,N6-henoadenosine-30,50-cyclic monophosphate; 20-m-cAMP,hate; 8-NBD-cAMP, 8-[[2-[(7-nitro-4-hosphate; DAG, diacylglycerol; mGDP, 30-O-(N-

nthraniloyl; GST, glutathione S-transferase; PKA, proteined, Egl-10, Pleckstrin domain; PBS, phosphate bufferedrythritol; Epac, exchange protein activated by cAMP;

hofer@mpi-dortmund.mpg.de

# 2001 Academic Press

1168 Dynamic Interaction of cAMP with Epac1

Ras whose transfection could revert K-Ras-inducedtransformation.9 It was also shown to inhibitgrowth-factor-induced MAP kinase activation,10

suggesting that Rap functions as an inhibitor ofRas signaling. However, no inverse correlationbetween the GTP loading of Ras and Rap inducedby a number of growth factors could bedemonstrated,11 except for insulin stimulation ofCHO cells.12 Rap has also been implicated to act asa mitogenic stimulus in certain types of cells13,14

and as an inducer of neurite outgrowth in PC12cells.15,16 More recently, Rap1 was found to beinvolved in the inside-out regulation of integrinactivation.17 ± 19

The importance of the Rap signaling pathway(s)is best demonstrated by the fact that a large varietyof stimuli induce the activation (GTP-loading) ofRap1. This activation is among others mediated bya number of second messenger molecules such asCa2�, DAG and cAMP.20 ± 22 The action of thesemessengers is mediated by several different Rap-speci®c GEFs23 ± 26 which are highly conservedbetween nematode and man. The most fascinatingof these are two different proteins called Epac(exchange protein activated by cAMP) 1 and 2.Epac1 contains one and Epac2 two cyclic nucleo-tide monophosphate (cNMP)-binding domains andboth GEFs are tightly controlled by direct bindingof cAMP.23,26 In addition to the cNMP-bindingdomains, both Epac proteins contain the REM andCDC 25 homology domains which together formthe structural unit for the exchange reaction27

and a DEP domain involved in membranelocalization.28

We have investigated the equilibrium and kin-etics of the interaction of cAMP and Epac1 utilizing¯uorescent cAMP analogues, and different domainconstructs from Epac1. We observe that the af®nityof cAMP to the cNMP binding domain is in themicromolar range, compatible with the level ofintracellular cAMP after stimulation of cells. Theinteraction, however, is highly dynamic, with highassociation and dissociation rate constants. Thepresence of the DEP domain or the GEF domaindid not affect these kinetics. However, binding toRap increases the af®nity of cAMP.

Results

The cNMP binding domain (cNMP-Epac) andthe cNMP binding domain together with thecatalytic region (���DEP-Epac) are stableprotein domains

Epac 1 consists of a DEP domain, a cNMP-bind-ing domain, a REM and a catalytic domain(Figure 1). The REM and the catalytic domaintogether constitute the GEF domain with the REMmotif29 being required for the stabilization of thecatalytic domain.27 Figure 1 shows the constructsmade to express various fragments and domains ofEpac1. Whereas full-length Epac1 is not expressedvery well and is dif®cult to purify, the �DEP-EpacL (149-881; L for large) can be produced andpuri®ed in large amounts and is stable during bio-physical measurements. The ®rst 36 residues ofthis construct are necessary for stability, since asomewhat shorter construct, �DEP-EpacS (S forshort), which starts at the N terminus of the mini-mal cNMP domain, as predicted from the structureof the regulatory domain of PKA,30 is unstable. Forthe same reason, the cNMP construct, cNMP-EpacL

(149-318) is much more stable and can be puri®edin larger amounts than cNMP-EpacS, residues 183-318. The isolated regulatory (2-329) and catalytic(324-881) domain can also be expressed, but onlyin small amounts and cannot be completely puri-®ed, similar to the full-length protein. From theexpression pattern and puri®cation protocol,�DEP-EpacL and the cNMP-EpacL are the mol-ecules of choice for the main biochemical andbiophysical investigations designed to understandcAMP regulation.

8-MABA-cAMP is a suitable fluorescentanalogue for kinetic studies

We wanted to probe the binding of cAMP to thecNMP binding domain in real time in homogenoussolution and to be able to develop a ¯uorescence-based assay to measure the af®nity of cAMP ana-logues. Since we could not observe changes in the¯uorescence signal of the intrinsic tryptophanegroups upon the addition of cAMP, we looked for

Figure 1. Constructs of Epacemployed here, indicating thelocation and boundaries of the pro-teins with single or multipledomains. On the right the proper-ties of the fragments with respectto expression and puri®cation areindicated, �� means excellent,� good, ÿmeans low, ÿÿ meansinsuf®cient.

Table 1. Characteristic properties of the ¯uorescent analogues

Fluorescent cAMPanalogue lex (nm) lem (nm)

Fluorescence intensitychange (%) Kd (mM)

Kd analogue/Kd

cAMP

e-cAMP 300 415 not measurable not measurable -2-aza-e-cAMP 358 483 280 2.4 � 0.1 0.4 � 0.1NBD-cAMP 471 540 600 0.2 � 0.0 0.1 � 0.020-m-cAMP 350 440 - - -MABA-cAMP 340 430 35 1.6 � 0.0 0.3 � 0.1MAH-cAMP 340 430 11 1.2 � 0.3 n.d.

Listed are the excitation and emission maxima, the ¯uorescence intensity change on binding to cNMP-EpacL, the dissociationconstant (Kd) for binding to that domain, and the ratio of Kd analogue/Kd cAMP.

Dynamic Interaction of cAMP with Epac1 1169

appropriate ¯uorescent derivatives. The spectralproperties of these compounds and their structuresare summarized in Table 1 and Figure 2. The ana-logues were tested for whether the interactioninduces a ¯uorescent change large enough to serveas a basis for measurement of equilibrium and kin-etic data and if so, whether the af®nity is in thesame order of magnitude as the af®nity of cAMP.The interaction of the protein with e-cAMP31 istechnically dif®cult to measure due to the low exci-tation maximum of e-cAMP, which is within therange of the ¯uorescence absorbance of the protein,

although it has been used in the study of the regu-latory subunits of PKA.32,33

8-NBD-cAMP is commercially available but toour knowledge only the cGMP derivative has sofar been used.34 This derivative appears suitabledue to the large ¯uorescence change induced bybinding (Figure 3(a)). The Kd of the interaction wasdetermined by an equilibrium titration experimentand was found to be 0.2 mM (Figure 3(a) andTable 1). This af®nity is approximately 10-20-foldhigher as compared to cAMP, as shown by thecompetition binding assay (Figure 3(b)).

Figure 2. Structures of ¯uor-escent cAMP derivatives employed.e-cAMP, 2-aza-e-cAMP, 8-NBD-cAMP and 20-m-cAMP have beensynthesized before and arecommercially available (BIOLOG).6-MAH-cAMP and 8-MABA-cAMPhave been synthesized on order asdescribed in Materials andMethods.

Figure 3. Use of 8-NBD-cAMPand 8-MABA-cAMP to probe theequilibrium interaction with cNMP-EpacL. In (a), 8-NBD-cAMP istitrated with the cNMP-bindingdomain and gives a very large(600 %) ¯uorescent change, whichis much smaller for 8-MABA-cAMP(35 %). (c) The ¯uorescence changeseen with NBD-cAMP and MABA-cAMP can be reversed by theaddition of excess unlabelledcAMP, (b) and (d), and is used todetermine the af®nity of the latter(Tables 1 and 2).

1170 Dynamic Interaction of cAMP with Epac1

cNMP derivatives in which the ribose group ismodi®ed with the commonly used mant ¯uoro-phore (N-methylanthraniloyl (m-)) have beenintroduced by Hiratsuka,35 and ribose-modi®edm-derivatives of guanine nucleotides have beenwidely used, but the 20-m-cAMP interaction withPKA has never been reported. Furthermorethe structure of the regulatory subunit of PKA incomplex with cAMP suggests that this derivativewould not be tolerated by the cNMP binding sitedue to the contact of the ribose with the protein.30

20-m-cAMP indeed appears to be unable to bind tothe cNMP-binding domain of Epac (not shown),suggesting a similar location of the ribose moietyin the structure.

2-aza-e-cAMP has been introduced as a ¯uor-escent derivative by Tsou et al.36 and its 8-azidoderivative was used for ¯uorescent af®nity labelingof the regulatory subunit of protein kinase A.37 Ithas an absorption maximum of 358 nm, well separ-ated from the tryptophane ¯uorescence, and givesa large ¯uorescence change upon interaction withthe protein. Competition experiments with cAMPshow that the ¯uorescence increase can be comple-tely reversed, and that the relative af®nity com-pared to cAMP is 2.7-fold.

8-MABA-ATP, where the mant ¯uorophore isconnected to the base via a butyl linker, has beenshown to be a valuable tool for the study of themechanism of chaperone function of DnaK.38,39

Accordingly, we had custom synthesized 8-MABA-cAMP where the mant group is bound to the 8position of the adenine base via a butyl linker, aswell as a related 6-MAH-cAMP, where the mantgroup is bound to the 6 position of the adeninebase via a hexyl linker. The ¯uorescent increasewith 6-MAH-cAMP is only 11 % and thus appearsto be less suitable for further analysis. 8-MABA-cAMP induces a 35 % ¯uorescent increase upon

binding, and binds with an af®nity of 1.6 mM(Figure 3(c)). Thus, in principle, since a 35 % ¯uor-escence intensity change is suf®cient for themeasurements, both 8-MABA-cAMP and 2-aza-e-cAMP are suitable analogues. An argument infavor of the mant group of 8-MABA-cAMP are thenumerous examples of it being a ¯uorescent repor-ter group sensitive to a variety of protein-proteinand protein-nucleotide interactions in the ®eld ofGTP-binding proteins. Also, the linker analoguemight be more generally applicable for in vivocell-based assays as the 8-(4-aminobutyl) amino-adenosine synthesis intermediate would allow thecoupling of many different and potentially morestable ¯uorescent dyes. Therefore 8-MABA-cAMPwill be exclusively used in the following studies.The competition experiment between 8-MABA-cAMP and cAMP (Figure 3(d)) gives an af®nity ofcNMP-EpacL for cAMP of 4.5 mM, which is furthercon®rmed by isothermal calorimetry (not shown,see also reference 28) whereby a dissociationconstant of 4.2 mM is obtained. The same methodshows a large favorable enthalpy change �Hof ÿ8.4 kcal/mol on binding of cAMP tocNMP-EpacL.

The interaction of 8-MABA-cAMP withcNMP-Epac is highly dynamic

Next we investigated the kinetics of the inter-action between 8-MABA-cAMP and cNMP-Epac(Figure 4). In a stopped-¯ow experiment the associ-ation rate constant can be measured under pseudo-®rst-order conditions, i.e. cNMP-Epac is present inexcess with varying concentrations of the protein(Figure 4(a)). The rates obtained from mono-exponential ®tting of the ¯uorescence transients(kobs) are plotted against the protein concentration(Figure 4(b)). This gives a linear dependence up to

Figure 4. Kinetics of interaction of cAMP derivativeswith cNMP-EpacL. (a) Epac (10 mM) was reacted with1 mM 8-MABA-cAMP in a stopped-¯ow apparatus. The¯uorescence change is ®tted to a single exponential togive kobs. (b) From the concentration dependence of the®tted ®rst-order association rates, kon can be obtained asthe slope of the linear ®t (14 mMÿ1 sÿ1). In (c) a pre-formed complex between 8-MABA-cAMP and cNMP-EpacL was reacted with excess (400 mM) unlabelledcAMP in a stopped-¯ow apparatus to obtain a dis-sociation rate constant of 22.3 secondÿ1.

Dynamic Interaction of cAMP with Epac1 1171

40 mM. The slope of this relationship determinesthe association rate constant (kass), which is1.4 � 107 Mÿ1 sÿ1 and thus much slower thanexpected for a diffusion-limited interaction (esti-mated 109 Mÿ1 sÿ1), but in the range of normalenzyme-ligand interactions (106 to 108 Mÿ1sÿ1).40

The dissociation rate constant (kdiss), as measuredby the decrease of ¯uorescence of cNMP-Epac pre-bound with 8-MABA-cAMP in a stopped-¯ow

experiment is 22 secondÿ1 (Figure 4(c)). Both rateconstants can be used to calculate the overall equi-librium dissociation constant (as kdiss/kass) betweencNMP-Epac and 8-MABA-cAMP as 1.5 mM. Thisvalue very nicely agrees with the equilibriumtitration value (1.6 mM) as determined inFigure 3(a). From these results we conclude that8-MABA-cAMP binds to cNMP-Epac with anaf®nity in the low micromolar range, but that thisbinding interaction is highly dynamic, with veryfast dissociation and association kinetics.

Although the complete regulatory domain ofEpac1, Epac1-RD, could not be puri®ed to a singleband we were nevertheless able to measure itsaf®nity to cyclic nucleotide, using as a probe8-MABA-cAMP. The af®nity was estimated byequilibrium titration, as in Figure 3, to be approxi-mately 2 mM, very similar to that of the isolatedcNMP domain (Table 2). This indicates that theDEP domain does not modify the binding propertyof the cNMP domain.

The catalytic domain of Epac does not affectthe binding kinetics of cAMP

Since full-length Epac1 (¯ Epac1) is onlyexpressed in small amounts and cannot be puri®edto homogeneity, we have investigated whether thewell-expressing �DEP-Epac1 can be used as aproper model for cAMP regulation of the GEFactivity. GEF activity of the constructs is measuredin an assay where Rap1-bound ¯uorescent mant-GDP (mGDP) is exchanged against an excessunlabelled free GDP. Released mGDP has adecreased ¯uorescence compared to bound mGDP.As reported,23 ¯ Epac is not capable of stimulatingthe mGDP-dissociation in the absence of cAMP(Figure 5(a)). Adding saturating amounts of cAMP(500 mM) induced the exchange activity, asevidenced by the increase in the rate of mGDPdissociation monitored in real time. Just as thefull-length protein, �DEP-EpacL is tightly regu-lated by the second messenger cAMP. In fact, itappears to be more active than the ¯ protein whichcould however be due to the full-length proteinbeing impure, and/or partly degraded-denaturedand/or labile during the course of the experiment.Interestingly, the short version of �DEP-Epac,�DEP-EpacS, also has a high GEF activity in thepresence of cAMP, but appears to be less tightlyregulated, seen by its activity in the absence ofcAMP (Figure 5(a)). The experiments together withthe similar binding properties of cNMP-Epac andEpac-RD however unequivocally show that theDEP domain of Epac is not necessary for the GEFactivity or its regulation by cAMP. Therefore,�DEP-EpacL can be used as a model to study thekinetics of cAMP binding to the intact protein.

In order to show if and how the catalytic domainof Epac1 affects the reaction with cAMP and/or itsanalogues, we intended to measure the af®nitybetween �DEP-Epac and 8-MABA-cAMP underequilibrium conditions. This turned out to be

Table 2. Af®nities of various protein domains from Epac to cAMP or the ¯uorescent analogue 8-MABA-cAMP

Kd MABA-cAMP(mM)

Kd MABAcAMP/Kd

cAMPKd cAMP (mM) comp.

titration exp. Kd cAMP (mM)

cNMP-EpacL 1.6 � 0.0 0.3 � 0.1 5.0 � 1.4 4.2 � 0.2 (ITC)Epac-RD 2.0 � 0.2 n.d. n.d. n.d.

�DEP-EpacL 3.3(kinet.) n.d. n.d.4.0 � 0.9 (initial rates of

exchange)�DEP-EpacL

a 0.8 � 0.2 1.4 � 0.3 0.6 � 0.3 -

a In the presence of Rap1B and EDTA.

1172 Dynamic Interaction of cAMP with Epac1

experimentally dif®cult, since upon addition ofcAMP, part of the protein precipitates out of sol-ution. We interpret this observation as indicativeof a conformational change upon the addition ofcAMP which exposes hydrophobic residues to thesolvent.

Although a stable complex between 8-MABA-cAMP and �DEP-EpacL could not be obtained forequilibrium-titration measurements, the stopped-¯ow measurements of rate constants are suf®-ciently fast to not allow the protein �nucleotidecomplex to precipitate out of solution. Further-more, addition of 300 mM salt was found to stabil-ize the cAMP �protein complex to a certain extent.Figure 6(a) shows an association reaction underpseudo-®rst-order conditions. Although the signal-to-noise ratio is lower, precipitation does notappear to be a major problem such that kobs can bemeasured and plotted against the concentration(Figure 6(b)), and give an association rate constantof 6 � 106 Mÿ1 sÿ1 (Table 3). To relate this tocNMP-Epac, the association rate for the latter wasalso measured at 300 mM salt, and found to besimilar (Table 3). These data further show that theassociation is weakly dependent on ionic strength,as it is decreased from 14.4 to 8.8 � 106 Mÿ1sÿ1.The dissociation of 8-MABA-cAMP from the com-plex with �DEP-EpacL is dif®cult to interpret. Itfollows a double-exponential function with a veryfast rate of 144 secondÿ1 which we interpret to bedue to scattering artefacts. The slower rate of 20secondÿ1, however, corresponds to the dissociationrate constants found for cNMP-Epac. Therefore,with a rate constant for dissociation of 20 secondÿ1,an equilibrium dissociation constant of 3.2 mM canbe calculated, very similar (twofold difference) tothat for the isolated cNMP domain.

Since the dissociation of 8-MABA-cAMP fromthe complex with �DEP-EpacL is very complex

Table 3. Association and dissociation rate constants and eqconstants for the interaction of MABA-cAMP with cNMP-Ep

Construct [NaCl] (mM) kon (mMÿ

cNMPL-Epac 50 14.4cNMPL-Epac 300 8.8�DEP-EpacL 300 6.0

and titration experiments cannot be evaluated dueto precipitation of the protein, we tried to con®rmthe data from the kinetic experiments betweencAMP and �DEP-EpacL by analyzing the velocityof the exchange reaction in dependence on the con-centration of cAMP. We performed the guaninenucleotide exchange reaction in the presence of100 nM �DEP-EpacL and varying concentrationsof cAMP. The initial rates of the exchange reactionwere plotted against the concentration, resulting ina half-maximal response at 4.0 mM cAMP(Figure 5(b)). This indicates that the af®nity is verysimilar between the isolated regulatory domainand the complete protein, again under the likelyassumption (see above) that the DEP domain hasno effect on cAMP binding. It also demonstratessimilar af®nities of �DEP-EpacL for cAMP and 8-MABA-cAMP.

The binding of Rap1 to Epac stabilizescAMP binding

�DEP-EpacL precipitates after binding to cAMPor its ¯uorescent analogues, but, interestingly, thisdoes not occur in the presence of Rap1 and EDTA,which presumably form a tight nucleotide-free ter-tiary complex via the GEF domain under these con-ditions. Titrating �DEP-Epac in the presence ofRap1b �GDP and EDTA with 8-MABA-cAMP givesan apparent dissociation constant of 0.8 mM, morethan fourfold lower that in its absence. From thisresult we conclude that binding of Rap to the cata-lytic domain of Epac results in a stabilization ofthe cAMP binding.

The binding of cAMP analogues to Epac doesnot correlate with activation

To investigate whether binding of cAMP ana-logues correlates with GEF activity of Epac1, we

uilibrium dissociation constant derived from the kineticacL and �DEP-EpacL

1 sÿ1) koff (sÿ1) Kd (mM)

22 1.521 2.420 3.2

Figure 6. Kinetics of binding of MABA-cAMP with�DEP-EpacL measured by stopped-¯ow. (a) Fluorescenttransient observed upon association of 50 mM �DEP-Epac with 1 mM 8-MABA-cAMP in a stopped-¯owapparatus. The ¯uorescence change is ®tted to a singleexponential to give kobs. (b) Plot of the ®tted ®rst-orderassociation rates against the concentration of �DEP-Epac. kon can be obtained as the slope of the linear ®t(6 mMÿ1 sÿ1).

Figure 5. Activation of GEF activity of �DEP-Epac bycAMP. (a) Rap1 (100 nM) loaded with ¯uorescentmGDP was incubated at 20 �C with excess unlabelledGDP, in the presence of various concentrations of either¯ Epac or �DEP-Epac as indicated. cAMP was added tothe reaction at the time indicated by the arrow. In (b),the GEF activity of �DEP-EpacL was measured, undersimilar conditions as in (a), with increasing concen-trations of cAMP. The initial rate of mGDP release isplotted against the cAMP concentration as indicated.Half maximal stimulation of GEF activity was obtainedat 4 mM cAMP.

Dynamic Interaction of cAMP with Epac1 1173

tested the effect of various ¯uorescent analoguesfor their ability to induce GEF activation on Epac.However, due to the overlapping ¯uorescence ofmGDP and the cAMP analogues, we had to switchto a more conventional radioactive GEF assay. Inthis assay, radioactive [H3]GDP bound to Rap1 isincubated with excess unlabelled GDP and �DEP-EpacL, in the presence of cAMP and analogues,and the radioactivity remaining bound to the pro-tein is determined by nitrocellulose ®lter binding(Figure 7). As expected, the GEF activity of �DEP-EpacL is induced by cAMP in this assay. Sur-prisingly, the ¯uorescent analogues, even at aconcentration 100� the calculated Kd for the iso-lated cNMP domain (Table 2), are much less ableto induce the GEF activity. Whereas 2-aza-e-cAMPis almost completely inactive, the other analoguesare less active, the activity decreasing in the order8-MABA-cAMP > 6-MAH-cAMP > NBD-cAMP.From this result we conclude that the binding ofcAMP derivatives to the cNMP-binding domain

and activation of the catalytic activity are indepen-dent processes that can be uncoupled.

Discussion

Epac is multi-domain protein whose GEFactivity towards Rap1, located in the C-terminalhalf of the molecule, is tightly regulated by theN-terminal half, consisting of a cNMP-binding siteand a DEP domain.23 Here we have shown thatfragments of the protein can be expressed asrecombinant proteins in Escherichia coli, the moststable of which are the cNMP-binding domain(cNMP-EpacL) and the cNMP-binding domaintogether with the catalytic domain (�DEP-EpacL).cNMP-EpacL is a monomeric cyclic nucleotide-binding domain whose properties can convenientlybe studied by ¯uorescence spectroscopy. �DEP-EpacL retains the properties of full-length proteinin that its GEF activity is tightly regulated by cyclicAMP. We have con®rmed and extended an earlier

Figure 7. Radioactivity assay of �DEP-EpacL usingcAMP and ¯uorescent analogues thereof at a concen-tration 100� the Kd calculated for the isolated cNMPdomain. Measured is the decrease of incorporated radio-active GDP with time in the presence of excessunlabeled GDP and �DEP-EpacL by the nitrocellulose®lter-binding assay.

Figure 8. Schematic view of the equilibrium constantsof Epac in the presence and absence of cAMP. Epac isthus switching between an active (a) and inactive (i)conformation which is on the inactive side in theabsence and on the active side in the presence of cAMP.Analogues of cAMP, such as those presented here,although binding, do not switch Epac to the active con-formation.

1174 Dynamic Interaction of cAMP with Epac1

observation that the DEP domain, originally foundin the proteins dishevelled, Egl-10 and pleckstrin,41

is dispensable both for the GEF activity of Epacand its regulation, and is likely to be required onlyfor the localization of the protein,28 as was shownfor the DEP domain of dishevelled.42

We have screened a number of ¯uorescent ana-logues, two of which were newly designed, to ®ndthat all except the ribose-modi®ed 20-m-cAMP bindwith suf®ciently high af®nity and give reasonable¯uorescence changes on binding. The most appro-priate for further ¯uorescence studies turned out tobe 2-aza-e-cAMP and/or 8-MABA-cAMP. Bothanalogues were shown to bind with an af®nitysimilar to that of cAMP and to be environmentallysensitive probes for thermodynamic and kineticstudies. Using 8-MABA-cAMP we could show thatboth the ¯uorescent analogue and cAMP itselfbind with micromolar af®nity in a highly dynamicfashion, as both ON and OFF rates are fast.

Since the discovery of Epac27,29 it has becomeobvious that treating cells with cAMP, and mostlikely with some of its analogues as well, inducesnot only the activation of the cAMP-dependentprotein kinase and cyclic-nucleotide-dependent ionchannels, but also the loading of Rap1 with GTPand thus the activation of the Rap1 signaling path-way. It will thus be necessary to compare the af®-nities of the proteins to cAMP and eventually to allthe analogues that are commonly used in cell bio-logical studies. The af®nity of cAMP for both theisolated cNMP domain and �DEP-Epac is around4 mM. The induction of a voltage-gated and cyclic-nucleotide-dependent channel by cAMP has beendescribed by a single binding isotherm with a K1/2

(half-maximal saturation value) of 0.74 mM43 indi-cating a somewhat higher af®nity of cAMP for thisprotein. The regulatory subunits of PKA have twocNMP-binding sites per polypeptide chain with

different binding af®nities and speci®cities,44

which show co-operative binding in the holo-enzyme but are apparently independent in the iso-lated regulatory subunit. The kinetic and thermo-dynamic analysis of the protein-nucleotideinteraction is thus very complicated and cannot bedescribed by a simple binding isotherm. The dis-sociation constant of the regulatory subunits ofPKA has been determined as around 18-22 nM45,46

and is expected to be higher in the presence of thecatalytic subunit and even higher in the presenceof the catalytic subunit and ATP.44 Since there arecon¯icting data and the af®nities are also depen-dent on the different types of PKA used, these dataare dif®cult to compare, although in general theaf®nity to PKA seems to be higher than for Epac.Whether this means that Rap activation needsmuch higher concentration in cells remains to bedetermined. In principle this difference in cAMPaf®nity could re¯ect differences in the cellularregulation of the two proteins by cAMP concen-trations. Alternatively, the lower binding of cAMPby Epac could be compensated by a higher localconcentration of the second messenger at the siteof Rap activation.

The addition of cAMP to �DEP-EpacL leads topartial precipitation of the protein. We had thusexpected that this would be accompanied by anextensive conformational change. Concomitantlywe had expected the af®nity of the �DEP-EpacL

protein to be signi®cantly different from that of theisolated cNMP domain, the difference in energybeing used for the conformational change. Surpris-ingly, we found that the af®nity and kinetics ofbinding, taking cAMP itself and the MABA-cAMPas examples, were very similar between these twoproteins, with fast dissociation and association kin-etics in both cases. We interpret this to mean thatthe structural change between the active and inac-tive form of Epac, although apparently exposinghydrophobic surfaces on the protein, appears to berather subtle and that the energetic difference

Dynamic Interaction of cAMP with Epac1 1175

between these two states is rather minor. This issupported by CD spectroscopic measurements ofthe proteins which also show no signi®cant changeon cAMP addition (data not shown). From theor-etical considerations we propose that there is apre-existing equilibrium between an inactive andactive conformation, which in the presence ofcAMP is shifted towards the latter, as shown inFigure 8. The ¯uorescent analogues bind with af®-nities that are sometimes much higher than that ofcAMP, but are either unable or much less able toactivate the GEF activity of Epac, even when testedat 100 times above their Kd towards the isolatedcNMP domain. Thus the binding activity and theability to induce the conformational change areapparently uncoupled from each other. Using themodel we would thus anticipate that the analoguesbind to the inactive conformation of Epac and beunable to ef®ciently drive it into the catalyticallyactive conformation. The analogues presented here,in particular those with a higher af®nity comparedto cAMP, can thus actually be used as lead com-pounds for inhibitors of the Epac activation bycAMP, 2-aza-e-cAMP being the most promising. Inaddition, the ¯uorescent analogues can be used ina ¯uorescence-based assay to screen for cAMPderivatives and other small molecules that interactwith cAMP binding and which can therefore serveas inhibitors or perhaps activators of the Epac reac-tion. Using derivatives thus obtained and using theinformation from the structure of the regulatorydomain of PKA,30 it might also be possible to ren-der these compounds able to speci®cally interactwith Epac without acting on the cyclic AMP-dependent protein kinase.

Materials and Methods

Preparation of proteins

All constructs of Epac were expressed as GST-fusionproteins (pGEX4T2) in the E. coli strain CK600K. Thecells were grown in Standard I media (Merck) containing100 mg/l ampicillin and 50 mg/l kanamycin to a A600 of0.8, induced with 100 mM IPTG and incubated overnightat 25 �C. Cells were resuspended in PBS buffer with 10 %(v/v) glycerol, 10 mM EDTA, 5 mM DTE and 500 mMPefabloc and lysed in a ¯uidizer. After centrifugation thesupernatant was loaded onto a GSH column (Pharma-cia). The column was washed three times with PBS in360 mM NaCl, with 50 mM Tris-HCl (pH 7.6), 100 mMKCl, 10 mM MgCl2, 10 % glycerol, 5 mM DTE, 2 mMATP and with 50 mM Tris-HCl (pH 7.6), 50 mM NaCl,

10 % glycerol, 5 mM DTE. Thrombin cleavage (thrombinfrom bovine plasma, Serva, Heidelberg) was performedwith 150 units for three hours at 4 �C (1 unit clots a stan-dard ®brinogen solution in 15 seconds at 37 �C) and theprotein was eluted with standard buffer: 50 mM Tris-HCl (pH 7.6), 50 mM NaCl, 5 % glycerol, 5 mM DTE, fol-lowed by gel ®ltration on Superdex 200 or Superdex 75in the same buffer.

Rap1 proteins were expressed from the ptac vector inthe E. coli strain CK600K as described.4

Fluorescent analogues

8-MABA-cAMP and 6-MAH-cAMP were custom syn-thesized (BIOLOG, Bremen, Germany) by coupling succi-nyl-N-methylanthranilate (Molecular Probes, Eugene,USA) to 8-(4-aminobutyl)aminoadenosine-30,50-cyclicmonophosphate and N6-(6-aminohexyl)adenosine-30,50-cyclic monophosphate, respectively, and subsequentchromatographic isolation and puri®cation on reversedphase silica. e-cAMP, 2-aza-e-cAMP, 20-m-cAMP and 8-NBD-cAMP (Figure 2) were purchased from BIOLOG,Bremen, Germany.

Fluorescence assay

Equilibrium-titration measurements were usuallydone in standard buffer. Titrations of the ¯uorescentcAMP analogues with proteins were carried out on aFluoromax2 ¯uorimeter (S.A. Instruments, USA) by add-ing increasing amounts of protein to 100 nM of the ¯uor-escent analogue. For competitive-titration experiments ofthe cNMP-EpacL �¯uorescent analogue complex withcAMP equimolar concentrations of cNMP-EpacL and the¯uorescent analogue (at a concentration 10 � Kd) weretitrated with increasing amounts of cAMP. The ¯uor-escent signal was monitored for typically ®ve to ten min-utes to ascertain a stable value (for the excitation andemission wavelengths see Table 1). Data were ®ttedaccording to Equation (1) for titration or Equation (2) forcompetitive titration experiments:

Fminÿ�E�L�K� ÿ�����������������������������������������������E�L�K�2ÿ4�E�L

q��FmaxÿFmin�

2� E�1�

where Fmin, minimal ¯uorescence; Fmax, maximal ¯uor-escence; E, concentration of ¯uorophore, L, concentrationof ligand (Epac); K, Kd:

Fmax ÿ �Fmax ÿ Fmin�

�Kfluo

KcAMP� �cAMP� � Kfluo

KcAMP� �E� ÿ

�������������������������������������������������������������������������������������������������������������������������������������������������Kfluo

KcAMP� �cAMP� � Kfluo

KcAMP� �E�

� �2

ÿ4� Kfluo

KcAMPÿ 1

� �� �cAMP� � �E�

s2� � Kfluo

KcAMPÿ 1� � �E�

�2�

1176 Dynamic Interaction of cAMP with Epac1

where Fmin, minimal ¯uorescence; Fmax, maximal ¯uor-escence; [E], concentration of protein; [cAMP], concen-tration of competitor (cAMP); K¯uo , KD for ¯uorescentanalogue; KcAMP, KD for cAMP. The equation has beenderived under the assumption that no free protein isavailable.

Measurements of the nucleotide exchange onRap1 �mGDP, using mostly Rap1B, and the correspond-ing domains from Epac1, were performed in standardbuffer with 5 mM MgCl2 as described elsewhere4 withthe concentration of cAMP as indicated.

Fast kinetic measurements using ¯uorescence werecarried out on a stopped-¯ow apparatus (Applied Photo-physics SX16MV) in standard buffer at 20 �C, usingeither standard buffer or standard buffer with 300 mMNaCl instead of 50 mM. An excitation wavelength of340 nm was used for MABA-cAMP and the emissionwas followed using a KV408 cut-off ®lter. Data collectionand primary analysis of rates were performed with thepackages from Applied Photophysics, with monoexpo-nential ®tting to the observed ¯uorescence transients,while secondary analysis was performed with theprogram Gra®t 3.0 (Erithacus software).

Radioactivity measurements

Rap1b (185 nM) was radiolabeled by incubation witha tenfold excess of [3H]GDP (37MBeq/ml) and EDTA(5 mM) in standard buffer for 1.5 hours on ice. The reac-tion was stopped by the addition of 20 mM MgCl2 andanother 30 minutes on ice. For measurements of theexchange activity of �DEP-EpacL on Rap1b at roomtemperature, 400 ml of the Rap[3H]-GDP were mixedwith 100 nM �DEP-EpacL and the ¯uorescent cAMP-analogues at a concentration 100 � Kd (400 mM cAMP,330mM 8-MABA-cAMP, 20 mM 8-NBD-cAMP, 150 mM 6-MAH-cAMP, 240 mM 2-aza-e-cAMP). The reaction wasstarted by the addition of a 250-fold excess of GDP. A50 ml sample was taken at different time points (0, 5, 10,20, 30, 40 and 60 minutes) and applied to a nitrocellulose®lter presoaked with standard buffer containing 10 mMMgCl2. After washing with 3 ml of buffer, the ®lter wasdried and the bound radioactivity was measured byscintillation counting. The results of three independentexperiments were averaged, the error of the mean wasapproximately 20 %.

Acknowledgements

We thank Rita Schebaum for very ef®cient secretarialassistance and Margaret Spechtel for help with proteinpuri®cation, Stein Ove Dùskeland and Hans-G. Genieserfor expert advice on cAMP and its analogues and KlausScheffzek for a very helpful observation concerning theprecipitation of Epac.

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Edited by A. R. Fersht

(Received 27 September 2000; received in revised form 4 January 2001; accepted 4 January 2001)