HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 40, 38548-38556, October 3, 2003

This Article Abstract Full Text (PDF) All Versions of this Article: 278/40/38548    most recent M306292200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rehmann, H. Articles by Bos, J. L. Search for Related Content PubMed PubMed Citation Articles by Rehmann, H. Articles by Bos, J. L. Social Bookmarking                      What's this?

Ligand-mediated Activation of the cAMP-responsive Guanine Nucleotide Exchange Factor Epac^*

Holger Rehmann   ¶, Frank Schwede ||, Stein O. Døskeland **, Alfred Wittinghofer  and Johannes L. Bos  

From the Department of Physiological Chemistry and Centre of Biomedical Genetics, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands; Max-Planck-Institut für Molekulare Physiologie, D-44227, Dortmund, Germany; ^||BIOLOG Life Science Institute, 28071 Bremen, Germany, and ^**Department of Anatomy and Cell Biology, Medical Faculty, University of Bergen, 5009 Bergen, Norway

Received for publication, June 15, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The exchange protein directly activated by cAMP (Epac)¹ is a cAMP-responsive guanine nucleotide exchange factor (GEF) for the small GTP-binding proteins Rap1 and Rap2 (1, 2). Two isoforms of Epac, Epac1 and Epac2, were described in mammalian cells, both containing a regulatory and a catalytic region (Fig. 1). The catalytic region contains a CDC25 homology domain and a Ras exchange motif domain. Whereas the CDC25-homology domain alone is sufficient to mediate the catalytic activity (3, 4), the Ras exchange motif domain of SOS has been shown to stabilize the CDC25-homology domain structurally without being directly involved in the interaction with Ras (5). More recently, an activity-modulating function of the Ras exchange motif domain was suggested (6).

View larger version (26K): [in this window] [in a new window]   FIG. 1. Schematic representation of Epac1 and 2 and the structure of cyclic nucleotide analogues. A, indicated is the domain structure of Epac1 with a DEP domain, a cAMP binding domain (cAMP), a Ras exchange motif (REM), and a CDC25 homology domain (CDC25-HD). The deletion constructs used in this study are indicated. B, chemical structures of the cyclic nucleotides and nucleotide analogues used in the present study; numbering of the ring positions of the base and the ribose are indicated in the cAMP structure. All modifications are grouped according to the position of the substitutions.

The regulatory domain of Epac contains a cAMP-binding domain and a DEP domain. The DEP domain mediates membrane localization of Epac and is not involved in regulation of the exchange activity in vitro. The cAMP binding domain inhibits the catalytic activity in the absence of cAMP. The inhibition is released after binding of cAMP. An additional N-terminal cAMP binding domain is present in Epac2. This domain is not required to maintain the inactive state of Epac2 in the absence of cAMP (7).

Insight into the structural basis of the regulatory mechanism was obtained from the x-ray structure of the regulatory region of Epac2 containing both the cAMP binding domains and the DEP domain. The structure was solved in the absence of cAMP and was the first structure of a cAMP binding domain in the ligand-free state (8). Based on a structural comparison with cAMP-bound regulatory subunits of PKA (9, 10) it was proposed that cAMP binding induces a conformational change in the phosphate binding cassette (8). This conformational change allows a C-terminal helix containing a hinge region and a lid region to bend toward the cAMP binding site and to cover cAMP (8, 11). Considering the high conservation of residues involved in the conformational change, the proposed model seems to account for the activation of not only Epac but also PKAs and cAMP-regulated ion channels.

When analyzing the role of the lid region of Epac in more detail, we observed that a conserved VLVLE sequence in the C-terminal end of the lid region is mediating the inhibition of the catalytic region. Indeed, mutation of this region resulted in a constitutively active Epac. Surprisingly, single point mutations in the lid region either abolished cAMP-induced activity almost completely or resulted in an activity even higher then that of the wt protein. This indicated that after binding of cAMP, the lid region regulates the activation status of Epac (11).

Recently, we identified a novel cAMP analogue, 8-pCPT-2'-O-Me-cAMP, that binds and activates Epac efficiently both in vitro and in vivo. Most importantly, this compound was completely insensitive in activating PKA both in vitro and in vivo (12). In this study, we have characterized 8-pCPT-2'-O-Me-cAMP further and noted Epac that bound to this compound had a 3-fold higher v_max than Epac bound to cAMP. By testing other cAMP analogues and by mutational analysis, we found that the 2'-OH atom of cAMP is responsible for the super-activation. We propose a model of how it communicates with the lid region.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Preparation of Proteins—All Epac constructs were expressed as glutathione S-transferase fusion proteins from the pGEX-4T2 vector in the Escherichia coli strain CK600K as described previously (13). All constructs are human Epac1 and correspond to the following amino acids: Epac, 149–881; Epac_149–328, 149–328. Mutations were introduced according to the QuikChange site-directed mutagenesis system (Stratagene). The C-terminal truncated version of Rap1B was expressed and loaded with 2'(3')-O-(N-methylanthraniloyl)-GDP as described previously (14). Hereafter, we refer to this construct as Rap1B or simply Rap1.

Isothermal Titration Calorimetry—To analyze the binding of cAMP to the isolated cAMP-binding domain, a microcalorimeter (MicroCal, Inc.) was used. For details see Rehmann et al. (11).

In Vitro Activation of Rap1—The experiments were performed as described earlier (11). Briefly, 200 nM of Rap1B loaded with the fluorescent GDP analogue 2'(3')-O-(N-methylanthraniloyl)-GDP were incubated in the presence of 20 µM GDP (Sigma) and 100 nM Epac. cAMP (Sigma) and analogues thereof were added as indicated for the individual experiments. Cyclic nucleotides except cAMP itself were obtained from BIOLOG Life Science Institute (Bremen, Germany). The nucleotide exchange was measured in real time as decay in fluorescence using a spectrofluorometer "spex1" (Spex Inc.). The origin of the decay is the release of the protein bound mGDP, which shows a higher fluorescence intensity in the hydrophobic environment of the protein than in the buffer solution. The obtained data were fitted to a single exponential decay and obtained rate constants (k_obs) were plotted against the cAMP concentration. The concentration dependence of the rate constants were treated as a normal titration experiment.

All data analysis, fitting, and plotting were done with the Grafit 3.0 program (Erithacus Software).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Interactions of Epac with Various cNMPs—We have used the isolated cAMP-binding domain of Epac 1, Epac-RD_149–328, to determine the equilibrium dissociation constant K_d for the nucleotides cAMP, cGMP, cIMP, and cXMP (Fig. 1A) by isothermal titration calorimetry. Interestingly, although cAMP has the highest affinity (3.3 µM) of the cyclic nucleotides tested, the affinities of cIMP (6.4 µM), cGMP (14 µM), and cXMP (20 µM) are of similar magnitude. Next, we investigated whether these nucleotides are able to activate Epac using a fluorescence-based assay, where the ability of Epac to act as a GEF for Rap1 is tested with increasing concentration of nucleotide (see "Experimental Procedures" and Ref. 14). From this we derive the half-maximal concentration for activation, AC₅₀, and the maximal rate of exchange under saturating concentration of activator k_max. Whereas cAMP activated Epac with the expected AC₅₀ of about 45 µM (Fig. 2A and Ref. 8), neither cGMP (Fig. 2A) nor the other cyclic nucleotides (data not shown) were able to activate Epac significantly. To investigate whether the failure of cGMP, cIMP, and cXMP to activate Epac is caused by a failure to bind to full-length Epac_149–881, we measured whether these cNMPs were able to inhibit the cAMP-induced activation. As shown in Fig. 2B, cIMP indeed inhibited cAMP-induced activation of Epac. The IC₅₀ of cIMP to inhibit Epac in the presence of 60 µM cAMP is approximately 200 µM, compatible with a 2–3-fold lower affinity of cIMP compared with cAMP. Similar results were obtained for cGMP and cXMP (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 2. cIMP, cGMP, and cXMP are inhibitors of Epac. A, rate constants for Epac-mediated GEF reactions were measured under standard conditions, using 200 nM Rap1 · 2'(3')-O-(N-methylanthraniloyl)-GDP in the presence of 100 nM wild-type or mutant Epac with excess GDP, at different concentration of cyclic nucleotide, from the rate of the decrease in fluorescence intensity (kobs), as indicated under "Experimental Procedures." The kobs values were plotted against the indicated cyclic nucleotide concentration. B, inhibition of GEF activity was measured under standard conditions using a constant concentration of 60 µM cAMP and the indicated concentrations of 8-pCPT-cAMP, N6-phenyl-cAMP, and cIMP, respectively. kobs were determined from the single experiments and plotted against the concentration of cyclic nucleotide. C, the nucleotide affinity of the single cAMP binding domain (EpacRD149–328) was determined by isothermal titration calorimetry as described under "Experimental Procedures."

The RAAT motif is conserved in the regulatory domains of cAMP dependent kinases and is replaced by RTAT/N in cGMP-dependent kinases. It was shown that A-to-T mutations in PKA change the selectivity from cAMP to cGMP and vice versa (15–17). In the case of EpacRD_149–328 the A280T mutation does indeed increase the affinity for cGMP, whereas the affinity for cAMP is not altered (Fig. 2C). Nevertheless, in the context of the auto-inhibited Epac, the mutant shows not an improved activation behavior after stimulation with cGMP but rather a 2-fold reduction of k_max after activation by cAMP (Fig. 2A). From these results, we conclude that although a number of cyclic nucleotides can bind to Epac, only cAMP is able to activate its GEF activity for Rap1. cAMP differs from the other three cNMPs by the structure of the nucleotide base, and cGMP, cIMP, and cXMP have in common that the 6-amino group of the base in cAMP is replaced by oxygen. To investigate whether indeed the N6 group is crucial for the activation of Epac, we tested the activity of the commonly employed N6-phenyl-cAMP analogue in vitro. As expected, this compound did not activate Epac (data not shown) but inhibits cAMP-induced activation in vitro rather efficiently (Fig. 2C).

Modification of the Cyclic Phosphate—The exocyclic oxygens of the phosphate group, which are not involved in the ring formation, can be replaced by sulfur in a stereo-specific manner generating Rp- and Sp-cAMPS isomers, respectively (Fig. 1). Rp-cAMPS is an inhibitor of PKA, whereas Sp-cAMPS, which has a strongly reduced affinity, is able to activate PKA (18, 19). Similar to the findings for PKA, Rp-cAMPS does not active Epac but rather inhibits cAMP-induced activation of Epac (Fig. 3, A and B). In analogy to PKA, Sp-cAMPS is able to activate Epac (Fig. 3B), with a slight modification of both AC₅₀ and k_max.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Rp-cAMPS acts as an inhibitor of Epac. A, the inhibitory properties of Rp-cAMPS were analyzed as described in Fig. 2B. B, the dependence of the exchange activity on the sulfur-substituted analogues Rp- and Sp-cAMPS was analyzed as described in Fig. 2A.

Modifications at the 8- and 2-Positions of the Base—To optimize the membrane permeability of cAMP, the 8-position of the base is often modified. We have tested a number of these analogues for their effect on the activation of GEF activity of Epac. All 8-modified analogues tested are active, but the maximal GEF activity at saturating concentrations of is reduced (Fig. 4A). In addition, these modifications affect the AC₅₀, many of them activating more efficiently than cAMP. Most notable is the 50-fold decrease in AC₅₀ of 8-pCPT-cAMP compared with cAMP (Fig. 4). Whereas all 8-modifications activate Epac and affect both the k_max and the AC₅₀, 2-chloro-cAMP has k_max and AC₅₀ values for Epac similar to those of cAMP (data not shown).

View larger version (27K): [in this window] [in a new window]   FIG. 4. Epac is activated at lower concentrations by several 8-modified cAMP analogues than by cAMP itself. A, several analogues with different substitutions at the 8-position were tested for their ability to activate the GEF activity of Epac under standard conditions, with different concentrations of cyclic nucleotides as indicated. kobs values obtained were plotted against the concentrations of the analogues, and the AC50 was determined from these plots. B, table of the AC50 value of analogues determined from titration experiments as shown in A.

Modification of the 2'-Position of the Ribose—One of the most striking differences between the cAMP-binding domains of Epac and those of other proteins is the presence of a glutamine at position 270 of Epac, which corresponds to a glutamate that is totally conserved in all other cAMP- or cGMP-binding proteins. Because in PKA this glutamate was shown to form a hydrogen bond with the 2'-OH group of the ribose (9) and was shown to be crucial for high affinity binding (20), we wondered whether Epac would tolerate modifications of the 2'-OH group. Its "deletion" in 2'-deoxy-cAMP results in a drastic increase of the AC₅₀, and k_max could not be reached even at 1000 µM nucleotide (Fig. 5A). This argues that the 2'-OH group is important for binding and activation. To maintain the oxygen, we generate 2'-O-Me-cAMP and found a more moderate reduction of the AC₅₀, which was estimated to be in the rage of 100 µM. Surprisingly, 2'-O-Me-cAMP induced guanine nucleotide exchange rates (k_obs) much higher then the k_max of cAMP, even though 2'-O-Me-cAMP was used at non-saturating conditions.

View larger version (23K): [in this window] [in a new window]   FIG. 5. "Super-activation" of Epac1 by 8-pCPT-2'OMe-cAMP. A, activation of Epac with the indicated analogues is measured under standard conditions, as shown previously. Data for 8-pCPT-cAMP are taken from Fig. 4. B, left, concentration-dependent activity of cAMP, 8-chloro-cAMP, and 8-pCPT-2'-O-Me-cAMP on Epac1. The data for cAMP and 8-pCPT-2'-O-Me-cAMP are taken from A. Right, inhibition of 8-pCPT-2'-O-Me-cAMP mediated activity by cAMP and 2-chloro-cAMP, respectively. A fixed concentration of 80 µM 8-pCPT-2'-O-Me-cAMP was added to the reaction mixture as described in A and the concentration of the inhibitory nucleotides were as indicated. C, the table summarizes AC50 and kmax values of the analogues used in A and B. The kmax values are normalized to the cAMP induced activity.

Because the 8-pCPT modification was found to decrease the AC₅₀ (Fig. 4A), we wondered whether this positive effect on affinity could be combined with the positive effect on k_max of the 2'-O-Me substitution. Indeed, 8-pCPT-2'-O-Me-cAMP showed a decreased AC₅₀ and an increased k_max (Fig. 5, A and C). The AC₅₀ of 8-pCPT-2'-O-Me-cAMP (1.8 µM) is only slightly lower than the AC₅₀ of 8-pCPT-cAMP (0.9 µM) and still 25 times lower than the AC₅₀ found for cAMP. In addition, the k_max of 8-pCPT-2'-O-Me-cAMP is 7.2-fold higher than 8-pCPT-cAMP and still 3.6-fold higher than cAMP. To demonstrate that this superactivation of Epac by 8-pCPT-2'-O-Me-cAMP is caused by a change in equilibrium between an active an inactive conformation of Epac, not by any different conditions of the assay, we incubated Epac induced by 8-pCPT-2'-O-Me-cAMP with increasing concentrations of cAMP. We found that with increasing concentrations of cAMP, the GEF activity reduced to a level observed for cAMP alone. Similarly, 8-chloro-cAMP inhibits 8-pCPT-2'-O-Me-cAMP-induced GEF activity to a level normally achieved by this analogue, which is half that of cAMP. We can thus conclude that the diverse effects of the 8-pCPT and the 2'-O-Me modifications on inducing GEF activity can be combined to create a super-activating cAMP analogue that specifically activates Epac and can be used as a tool to decipher the role of Rap1 in signal transduction pathways (12, 21).

Role of the Gln-270 in Binding and Activation—To analyze the role of Q270 in the interaction with the 2'OH group of cAMP we generated the mutants EpacQ270A and EpacQ270E. The mutation Q270E mimics the residue conserved in PKA. Importantly, the affinity of cAMP for the isolated cAMP-binding domains of both mutants was not affected, demonstrating that unlike PKA, this residue is not involved in binding of cAMP (Fig. 6B). The mutants were then further analyzed, in the context of the auto-inhibited Epac, for their activation properties in response to stimulation by cAMP, 2'-deoxy-cAMP, and 8-pCPT-2'-O-Me-cAMP (Fig. 6A). For cAMP, a 2-fold higher k_max was observed for both mutants, whereas the AC₅₀ remained unaffected, indicating that Gln-270 is not crucial for the activation mechanism of cAMP and, if anything, has an inhibitory role on full activation. Activation by 8-pCPT-2'-OMe-cAMP is differently affected by the two mutations: whereas the Q270E mutation had no effect on the k_max but slightly decreased the AC₅₀, the Q270A mutation decreased k_max and left the AC₅₀ unaffected. The AC₅₀ of 2'deoxy-cAMP is too high to determine k_max either for wild-type and mutant proteins (Fig. 6). From these results, we conclude that the presence of a hydrogen bond donor or acceptor in the position of residue Gln-270 does contribute to normal activation by cAMP and super-activation by 8-pCPT-2'-O-Me-cAMP in a manner that is not immediately obvious in the absence of a cAMP-bound 3D structure.

View larger version (27K): [in this window] [in a new window]   FIG. 6. Gln-270 plays an important role in the activation mechanism of Epac. A, the mutants EpacQ270A and Q270E were generated and compared with wt with respect to their activation behavior after stimulation with cAMP, 2'-deoxy-cAMP, and 8-pCPT-2'-O-Me-cAMP. The cAMP and 8-pCPT-2'-O-Me-cAMP dependence of Epac wt is reproduced from Fig. 5A. B, the table summarizes the A50 and kmax values. The kmax are normalized to the wt protein for the respective analogue (normalization within the column). In addition, the kmax are normalized to cAMP for the respective mutant (normalization within the row), these values are given in brackets. The affinity of cAMP to EpacRD149–328, as determined by isothermal titration calorimetry, is given in the first column.

The lid Region Is Involved in Super-activation—Recently we mutated conserved polar residues Glu-308, Thr-311, Arg-313, and His-317 in the lid of Epac1 (11). As isolated cAMP-binding domains, these mutant proteins have a 2-fold lower affinity for cAMP (except H317A, for which the affinity for cAMP was unaffected). In addition, these mutations affected cAMP-induced activation of Epac (Ref. 11 and Fig. 7). By comparing the effects of cAMP, 8-pCPT-cAMP, and 8-pCPT-2'-O-Me-cAMP, we observe that R313A and T311A have a roughly similar effect on the activation of Epac by both cAMP and 8-pCPT-cAMP but, surprisingly, an opposite effect on the activation by 8-pCPT-2'-O-Me-cAMP (Fig. 7), R313A did not influence the k_max of 8-pCPT-cAMP and cAMP but reduced the k_max of 8-pCPT-2'-O-Me-cAMP almost 2-fold. T311A increased the k_max of cAMP and 8-pCPT-2'-O-Me-cAMP 2-fold, but has no effect on the k_max of 8-pCPT-2'-O-Me-cAMP. For H317A, the k_max achieved with 8-pCPT-2'-O-Me was greatly reduced compared with wild type, whereas for E308A, the k_max was even increased by about 25%. In addition, the AC₅₀ of the analogues was affected by the mutations. Most notably, although the affinity for cAMP for the isolated cAMP-binding domain of T311A and T311A was reduced, the AC₅₀ was considerably lower. From these results, we conclude that the lid region is involved in the super-activation of Epac by 8-pCPT-2'-O-Me-cAMP. In particular, the differential effect of 8-pCPT-2'-O-Me-cAMP compared with cAMP and 8-pCPT-cAMP on R313A and T311A indicates that the 2'-O-Megroup communicates with the lid to induce super-activation.

View larger version (35K): [in this window] [in a new window]   FIG. 7. The lid region is directly involved in the activation process. A, four conserved polar residues of the lid region were mutated to Ala and analyzed for the dependence of their activity on the cyclic nucleotide concentration. The analysis of Epac wt is shown in each individual graph for comparison; values for cAMP-induced activation are taken from Rehmann et al. (11). B, the table shows the AC50 and kmax values. The kmax are normalized to the wt protein for the respective analogue (normalization within the column). In addition, the kmax are normalized to cAMP for the respective mutant (normalization within the row); these values are given in brackets.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have analyzed the effect of various cAMP analogues on their effects on Epac activation in vitro. We first noted that although cGMP, cIMP, and cXMP do bind to Epac with an affinity similar to cAMP, they are unable to activate Epac. This implies that the composition of the base, particularly the N6 position, is absolutely critical for the activation of Epac. In contrasts, cGMP, cIMP, and cXMP are competitive inhibitors. Because the IC₅₀ is the upper micromolar range, it is unlikely that this inhibition is physiologically relevant. In this respect, Epac differs from PKA. Both cGMP (15) and cIMP (22) are able to fully activate PKA, although only at high concentrations. Similarly, N⁶-phenyl-cAMP inhibits Epac but activates PKA (22). All these nucleotides have an identical cyclic-phosphate ribose group; thus, the interaction of the base, presumably with the lid, determines the difference. Apparently, alterations in the N6 position of cAMP abolishes the ability of cyclic nucleotides to activate Epac but not PKA. This implies that suitably N6-altered cAMP-analogues may be able to differentiate between PKA and Epac.

With respect to cyclic nucleotide-dependent protein kinases, the RAAT and the RTAT/N motif were identified to determine the cAMP and cGMP specificity, respectively. From modeling studies, an extra hydrogen bound between the 2-NH₂ group of the guanine and the first threonine in RTAT/N was predicted (23). These theoretical considerations were confirmed by site-directed mutagenesis for both PKA (17, 24) and PKG (16), which demonstrated an increased cGMP affinity because of the presence of the threonine. Similarly, EpacRD_149–328A280T showed an increased affinity for cGMP as well. However, EpacA280T showed no improved activation by cGMP in terms of k_max. Thus, the A/T conversion increased the affinity of cGMP for Epac but had no effect on the activation of Epac by cGMP. Interestingly, as predicted from studies with PKA (15) and PKG (16), the A/T conversion had little or no affect on the affinity for cAMP. However it did reduce the maximal activation of Epac by cAMP, showing that the RAAT sequence is involved not only in conferring specificity in binding but also in transferring the cAMP binding information to the activation of Epac.

From the structural analysis of Epac and PKA, it was clear that the cyclic phosphate group is critical for the activation of both proteins. Indeed, Rp-cAMPS, in which one of the free oxygens of the phosphate group is replaced by a sulfur atom, is an inhibitor of PKA (18, 19), whereas Rp-cGMPS inhibits PKG (25). Indeed, Rp-cAMPS also inhibits cAMP-induced Epac activation. This supports strongly the common activation mechanism of these proteins as we proposed previously (8); i.e. the replacement of the equatorial oxygen perturbs the hydrogen bonding between the phosphate and an alanine in the phosphate binding cassette and prevents the reorientation of the phosphate binding cassette, the first step in the activation of both proteins.

Modifications at the 8-position of the base are commonly used as cNMP analogues for PKA and PKG, because these modifications both increase the permeability for cells and reduce the AC₅₀ to activate these proteins (22, 26). In particular, 8-pCPT-cAMP and 8-bromo-cAMP are widely used for in vivo experiments. The 8-bromo substitution was shown to stabilize the syn conformation of cAMP, the conformation of cAMP adopted in the crystal structures of PKAI and PKAII (9, 10), which provides a possible explanation for the decreased AC₅₀. The analogues also have a lower AC₅₀ for Epac compared with cAMP; in particular, the 8-pCPT modification resulted in a 50-fold reduction in AC₅₀. However, the maximal activation of Epac by these analogues is 2-fold lower compared with cAMP, indicating that modifications at the 8-position also affect the k_max of the protein. However, not all modifications of the 8-positions affect the behavior of cAMP on Epac. e.g. 8-ABA-cAMP.

The highly conserved glutamate in the FGELAL sequence of the phosphate binding cassette of PKA, PKG, cyclic nucleotidegated ion channels, and even the bacterial CAP protein, is replaced by a glutamine (Gln-270) in Epac1 and a lysine in Epac2. Glutamic acid was found to form a hydrogen bond with the 2'-OH group of the cyclic nucleotide and was shown to be absolutely essential for the binding of cAMP to PKA (20). Although the Epac structure analysis (8) showed that the corresponding residue would be close to the 2'-OH group, a potential interaction between Gln-270 would not significantly contribute to the binding affinity for cAMP. Neither the AC₅₀ of EpacQ270A or EpacQ270E nor the K_d of Epac_149–328Q270A or Epac_149–328Q270E was altered, which suggests that the glutamine in Epac is not involved in the direct binding of cAMP and questions the necessity of the 2'-OH group for activation of Epac. However, drastic effects on activation of GEF activity with respect to both AC₅₀ and k_max were obtained by modifications of the 2'-OH group of the ribose. Replacement of the 2'-OH group by H or by OCH₃ leads to an increase of the AC₅₀, indicating that interactions of the 2'-OH-group contribute to the binding of Epac but not via Gln-270. Indeed, the affinity of 2'-deoxy-cAMP for the cAMP binding domain was dramatically reduced. However, this reduction was much less, but still 10-fold, when the oxygen was preserved by replacing the OH group for an O-Me group (27). This indicated that the OH group still confers binding energy for cAMP to Epac, presumably through its interaction with the conserved glycine in the FGELAL sequence. Interestingly, 2'-O-Me-cAMP (and perhaps 2'-deoxy-cAMP) at non-saturated concentration induces k_obs values that are higher then the k_max of cAMP-mediated activation. Apparently, the 2'-O-Me-group shifts the conformational equilibrium of the nucleotide-bound Epac more efficiently to the active site. Combined with the increased affinity of the 8-pCPT-group, both modifications resulted in a highly specific "superactivator" of Epac (12). The effect of the increased k_max is in part mediated by Gln-270. The k_max of 8-pCPT-2'-O-Me-cAMP-mediated activation of EpacQ270A (but not EpacQ270E) is decreased significantly. On the other hand, the k_max of cAMP-mediated activation for both mutants is increased compared with wild type. This indicates that Gln-270 disfavors the cAMP-bound active state but favors the 8-pCPT-2-O-Me-cAMP-bound active state.

Mutagenesis of the lid demonstrate a role of this region in the super-activation behavior of 8-pCPT-2'-O-Me-cAMP, although the role of individual residues in this process is still unclear. A potential link between the lid and the OH/OCH₃-group and/or Gln-270 is His-317. In all known PKA structures, a hydrogen bond between the glutamate in the FGELAL sequence (corresponding to Gln-270 in Epac1) and a tyrosine/tryptophane in the lid is found. This tyrosine/tryptophane may correspond to His-317. Indeed, cAMP- and 8-pPCT-2'-O-Me-cAMP-mediated activation are drastically reduced in EpacH317A. Arg-313 may be specifically responsive to the 2'-O-Me-group, because the k_max of cAMP is not altered, whereas the k_max of 8-pCPT-2'-O-Me-cAMP is reduced 2-fold. In contrast, Thr-311 may be specifically responsive to the 2'-OH-group. Further structural and mutational analysis of both the lid and the catalytic domain are required to fully understand the molecular mechanism of Epac activation by cAMP and cAMP analogues.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by the Council of Earth and Life Science of the Netherlands Organization for Scientific Research (NWO-CW) and the Human Frontier Science Program (HFSP).

To whom correspondence should be addressed. Tel.: 31302538988; Fax: 31302539035; E-mail: j.l.bos{at}med.uu.nl.

¹ The abbreviations used are: Epac, exchange protein directly activated by cAMP; GEF, guanine nucleotide exchange factor; DEP, Dishevelled, Egal-10, Pleckstrin; PKA, protein kinase A; wt, wild type; 2'-O-Me, 2'-O-methyl; 8-pCPT, 8-(4-chlorophenylthio); Rp-cAMPS, adenosine-3',5'-cyclicmonophosphorothioate, Rp-isomer; Sp-cAMPS, adenosine-3',5'-cyclicmonophosphorothioate, Sp-isomer; PKG, protein kinase G.

	ACKNOWLEDGMENTS

 
We thank Hans-Gottfried Genieser for stimulating discussing and ongoing co-operation, Milica Vukmirovic for support, Alma Rueppel for technical assistance, and Rita Schebaum for secretarial assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M., Wittinghofer, A., and Bos, J. L. (1998) Nature 396, 474–477[CrossRef][Medline] [Order article via Infotrieve]
2. Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Science 282, 2275–2279[Abstract/Free Full Text]
3. Coccetti, P., Mauri, I., Alberghina, L., Martegani, E., and Parmeggiani, A. (1995) Biochem. Biophys. Res. Commun. 206, 253–259[CrossRef][Medline] [Order article via Infotrieve]
4. Lenzen, C., Cool, R. H., Prinz, H., Kuhlmann, J., and Wittinghofer, A. (1998) Biochemistry 37, 7420–7430[CrossRef][Medline] [Order article via Infotrieve]
5. Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337–343[CrossRef][Medline] [Order article via Infotrieve]
6. Margarit, S. M., Sondermann, H., Hall, B. E., Nagar, B., Hoelz, A., Pirruccello, M., Bar-Sagi, D., and Kuriyan, J. (2003) Cell 112, 685–695[CrossRef][Medline] [Order article via Infotrieve]
7. de Rooij, J., Rehmann, H., van Triest, M., Cool, R. H., Wittinghofer, A., and Bos, J. L. (2000) J. Biol. Chem. 275, 20829–20836[Abstract/Free Full Text]
8. Rehmann, H., Prakash, B., Wolf, E., Rueppel, A., de Rooij, J., Bos, J. L., and Wittinghofer, A. (2003) Nat. Struct. Biol. 10, 26–32[CrossRef][Medline] [Order article via Infotrieve]
9. Su, Y., Dostmann, W. R., Herberg, F. W., Durick, K., Xuong, N. H., Ten Eyck, L., Taylor, S. S., and Varughese, K. I. (1995) Science 269, 807–813[Abstract/Free Full Text]
10. Diller, T. C., Madhusudan, Xuong, N. H., and Taylor, S. S. (2001) Structure (Camb.) 9, 73–82
11. Rehmann, H., Rueppel, A., Bos, J. L., and Wittinghofer, A. (2003) J. Biol. Chem. 278, 23508–23514[Abstract/Free Full Text]
12. Enserink, J. M., Christensen, A. E., de Rooij, J., van Triest, M., Schwede, F., Genieser, H. G., Doskeland, S. O., Blank, J. L., and Bos, J. L. (2002) Nat. Cell Biol. 4, 901–906[CrossRef][Medline] [Order article via Infotrieve]
13. Kraemer, A., Rehmann, H. R., Cool, R. H., Theiss, C., de Rooij, J., Bos, J. L., and Wittinghofer, A. (2001) J. Mol. Biol. 306, 1167–1177[CrossRef][Medline] [Order article via Infotrieve]
14. van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Oncogene 15, 845–850[CrossRef][Medline] [Order article via Infotrieve]
15. Shabb, J. B., Buzzeo, B. D., Ng, L., and Corbin, J. D. (1991) J. Biol. Chem. 266, 24320–24326[Abstract/Free Full Text]
16. Reed, R. B., Sandberg, M., Jahnsen, T., Lohmann, S. M., Francis, S. H., and Corbin, J. D. (1996) J. Biol. Chem. 271, 17570–17575[Abstract/Free Full Text]
17. Shabb, J. B., Ng, L., and Corbin, J. D. (1990) J. Biol. Chem. 265, 16031–16034[Abstract/Free Full Text]
18. Van Haastert, P. J., Van Driel, R., Jastorff, B., Baraniak, J., Stec, W. J., and De Wit, R. J. (1984) J. Biol. Chem. 259, 10020–10024[Abstract/Free Full Text]
19. Botelho, L. H., Rothermel, J. D., Coombs, R. V., and Jastorff, B. (1988) Methods Enzymol. 159, 159–172[Medline] [Order article via Infotrieve]
20. Yagura, T. S., and Miller, J. P. (1981) Biochemistry 20, 879–887[CrossRef][Medline] [Order article via Infotrieve]
21. Kang, G., Joseph, J. W., Chepurny, O. G., Monaco, M., Wheeler, M. B., Bos, J. L., Schwede, F., Genieser, H. G., and Holz, G. G. (2003) J. Biol. Chem. 278, 8279–8285[Abstract/Free Full Text]
22. Ogreid, D., Doskeland, S. O., and Miller, J. P. (1983) J. Biol. Chem. 258, 1041–1049[Abstract/Free Full Text]
23. Weber, I. T., Shabb, J. B., and Corbin, J. D. (1989) Biochemistry (Mosc.) 28, 6122–6127
24. Huang, L. J., and Taylor, S. S. (1998) J. Biol. Chem. 273, 26739–26746[Abstract/Free Full Text]
25. Butt, E., van Bemmelen, M., Fischer, L., Walter, U., and Jastorff, B. (1990) FEBS Lett. 263, 47–50[CrossRef][Medline] [Order article via Infotrieve]
26. Corbin, J. D., Ogreid, D., Miller, J. P., Suva, R. H., Jastorff, B., and Doskeland, S. O. (1986) J. Biol. Chem. 261, 1208–1214[Abstract/Free Full Text]
27. Christensen, A. E., Selheim, F., de Rooij, J., Dremier, S., Schwede, F., Dao, K. K., Martinez, A., Maenhaut, C., Bos, J. L., Genieser, H. G., and Doskeland, S. O. (2003) J. Biol. Chem. 278, 35394–35402[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. J. Murray Pharmacological PKA Inhibition: All May Not Be What It Seems Sci. Signal., June 3, 2008; 1(22): re4 - re4. [Abstract] [Full Text] [PDF]

				S. M. Harper, H. Wienk, R. W. Wechselberger, J. L. Bos, R. Boelens, and H. Rehmann Structural Dynamics in the Activation of Epac J. Biol. Chem., March 7, 2008; 283(10): 6501 - 6508. [Abstract] [Full Text] [PDF]

				D. Hochbaum, K. Hong, G. Barila, F. Ribeiro-Neto, and D. L. Altschuler Epac, in Synergy with cAMP-dependent Protein Kinase (PKA), Is Required for cAMP-mediated Mitogenesis J. Biol. Chem., February 22, 2008; 283(8): 4464 - 4468. [Abstract] [Full Text] [PDF]

				J. Rein, M. Voss, W. Blenau, B. Walz, and O. Baumann Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A Am J Physiol Cell Physiol, January 1, 2008; 294(1): C56 - C65. [Abstract] [Full Text] [PDF]

				S. Dremier, M. Milenkovic, S. Blancquaert, J. E. Dumont, S. O. Doskeland, C. Maenhaut, and P. P. Roger Cyclic Adenosine 3',5'-Monophosphate (cAMP)-Dependent Protein Kinases, But Not Exchange Proteins Directly Activated by cAMP (Epac), Mediate Thyrotropin/cAMP-Dependent Regulation of Thyroid Cells Endocrinology, October 1, 2007; 148(10): 4612 - 4622. [Abstract] [Full Text] [PDF]

				J. Hong, R. C. Doebele, M. W. Lingen, L. A. Quilliam, W.-J. Tang, and M. R. Rosner Anthrax Edema Toxin Inhibits Endothelial Cell Chemotaxis via Epac and Rap1 J. Biol. Chem., July 6, 2007; 282(27): 19781 - 19787. [Abstract] [Full Text] [PDF]

				K. K. Dao, K. Teigen, R. Kopperud, E. Hodneland, F. Schwede, A. E. Christensen, A. Martinez, and S. O. Doskeland Epac1 and cAMP-dependent Protein Kinase Holoenzyme Have Similar cAMP Affinity, but Their cAMP Domains Have Distinct Structural Features and Cyclic Nucleotide Recognition J. Biol. Chem., July 28, 2006; 281(30): 21500 - 21511. [Abstract] [Full Text] [PDF]

				G. Kang, O. G. Chepurny, B. Malester, M. J. Rindler, H. Rehmann, J. L. Bos, F. Schwede, W. A. Coetzee, and G. G. Holz cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic {beta} cells and rat INS-1 cells J. Physiol., June 15, 2006; 573(3): 595 - 609. [Abstract] [Full Text] [PDF]

				M. T. Branham, L. S. Mayorga, and C. N. Tomes Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway J. Biol. Chem., March 31, 2006; 281(13): 8656 - 8666. [Abstract] [Full Text] [PDF]

				Y. Li, S. Asuri, J. F. Rebhun, A. F. Castro, N. C. Paranavitana, and L. A. Quilliam The RAP1 Guanine Nucleotide Exchange Factor Epac2 Couples Cyclic AMP and Ras Signals at the Plasma Membrane J. Biol. Chem., February 3, 2006; 281(5): 2506 - 2514. [Abstract] [Full Text] [PDF]

				E. Morel, A. Marcantoni, M. Gastineau, R. Birkedal, F. Rochais, A. Garnier, A.-M. Lompre, G. Vandecasteele, and F. Lezoualc'h cAMP-Binding Protein Epac Induces Cardiomyocyte Hypertrophy Circ. Res., December 9, 2005; 97(12): 1296 - 1304. [Abstract] [Full Text] [PDF]

				S. Seino and T. Shibasaki PKA-Dependent and PKA-Independent Pathways for cAMP-Regulated Exocytosis Physiol Rev, October 1, 2005; 85(4): 1303 - 1342. [Abstract] [Full Text] [PDF]

				S. Sedej, T. Rose, and M. Rupnik cAMP increases Ca2+-dependent exocytosis through both PKA and Epac2 in mouse melanotrophs from pituitary tissue slices J. Physiol., September 15, 2005; 567(3): 799 - 813. [Abstract] [Full Text] [PDF]

				T. B. Hucho, O. A. Dina, and J. D. Levine Epac Mediates a cAMP-to-PKC Signaling in Inflammatory Pain: An Isolectin B4(+) Neuron-Specific Mechanism J. Neurosci., June 29, 2005; 25(26): 6119 - 6126. [Abstract] [Full Text] [PDF]

				M. Keiper, M. B. Stope, D. Szatkowski, A. Bohm, K. Tysack, F. vom Dorp, O. Saur, P. A. Oude Weernink, S. Evellin, K. H. Jakobs, et al. Epac- and Ca2+-controlled Activation of Ras and Extracellular Signal-regulated Kinases by Gs-coupled Receptors J. Biol. Chem., November 5, 2004; 279(45): 46497 - 46508. [Abstract] [Full Text] [PDF]

				C. Krakstad, A. E. Christensen, and S. O. Doskeland cAMP protects neutrophils against TNF-{alpha}-induced apoptosis by activation of cAMP-dependent protein kinase, independently of exchange protein directly activated by cAMP (Epac) J. Leukoc. Biol., September 1, 2004; 76(3): 641 - 647. [Abstract] [Full Text] [PDF]

				K. A. Cullen, J. McCool, M. S. Anwer, and C. R. L. Webster Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis Am J Physiol Gastrointest Liver Physiol, August 1, 2004; 287(2): G334 - G343. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 278/40/38548    most recent M306292200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services