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

Abstract

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. 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. The exchange protein directly activated by cAMP (Epac) 1The 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.1The 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. is a cAMP-responsive guanine nucleotide exchange factor (GEF) for the small GTP-binding proteins Rap1 and Rap2 (1de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1594) Google Scholar, 2Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1158) Google Scholar). 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 (3Coccetti P. Mauri I. Alberghina L. Martegani E. Parmeggiani A. Biochem. Biophys. Res. Commun. 1995; 206: 253-259Crossref PubMed Scopus (34) Google Scholar, 4Lenzen C. Cool R.H. Prinz H. Kuhlmann J. Wittinghofer A. Biochemistry. 1998; 37: 7420-7430Crossref PubMed Scopus (196) Google Scholar), 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 (5Boriack-Sjodin P.A. Margarit S.M. Bar-Sagi D. Kuriyan J. Nature. 1998; 394: 337-343Crossref PubMed Scopus (603) Google Scholar). More recently, an activity-modulating function of the Ras exchange motif domain was suggested (6Margarit S.M. Sondermann H. Hall B.E. Nagar B. Hoelz A. Pirruccello M. Bar-Sagi D. Kuriyan J. Cell. 2003; 112: 685-695Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). 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 (7de Rooij J. Rehmann H. van Triest M. Cool R.H. Wittinghofer A. Bos J.L. J. Biol. Chem. 2000; 275: 20829-20836Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). 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 (8Rehmann H. Prakash B. Wolf E. Rueppel A. de Rooij J. Bos J.L. Wittinghofer A. Nat. Struct. Biol. 2003; 10: 26-32Crossref PubMed Scopus (165) Google Scholar). Based on a structural comparison with cAMP-bound regulatory subunits of PKA (9Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (341) Google Scholar, 10Diller T.C. Madhusudan Xuong N.H. Taylor S.S. Structure (Camb.). 2001; 9: 73-82Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) it was proposed that cAMP binding induces a conformational change in the phosphate binding cassette (8Rehmann H. Prakash B. Wolf E. Rueppel A. de Rooij J. Bos J.L. Wittinghofer A. Nat. Struct. Biol. 2003; 10: 26-32Crossref PubMed Scopus (165) Google Scholar). 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 (8Rehmann H. Prakash B. Wolf E. Rueppel A. de Rooij J. Bos J.L. Wittinghofer A. Nat. Struct. Biol. 2003; 10: 26-32Crossref PubMed Scopus (165) Google Scholar, 11Rehmann H. Rueppel A. Bos J.L. Wittinghofer A. J. Biol. Chem. 2003; 278: 23508-23514Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 (11Rehmann H. Rueppel A. Bos J.L. Wittinghofer A. J. Biol. Chem. 2003; 278: 23508-23514Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 (12Enserink J.M. Christensen A.E. de Rooij J. van Triest M. Schwede F. Genieser H.G. Doskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Crossref PubMed Scopus (610) Google Scholar). 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. 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 (13Kraemer A. Rehmann H.R. Cool R.H. Theiss C. de Rooij J. Bos J.L. Wittinghofer A. J. Mol. Biol. 2001; 306: 1167-1177Crossref PubMed Scopus (58) Google Scholar). All constructs are human Epac1 and correspond to the following amino acids: Epac, 149–881; Epac149–328, 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 (14van den Berghe N. Cool R.H. Horn G. Wittinghofer A. Oncogene. 1997; 15: 845-850Crossref PubMed Scopus (86) Google Scholar). 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. (11Rehmann H. Rueppel A. Bos J.L. Wittinghofer A. J. Biol. Chem. 2003; 278: 23508-23514Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In Vitro Activation of Rap1—The experiments were performed as described earlier (11Rehmann H. Rueppel A. Bos J.L. Wittinghofer A. J. Biol. Chem. 2003; 278: 23508-23514Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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). Interactions of Epac with Various cNMPs—We have used the isolated cAMP-binding domain of Epac 1, Epac-RD149–328, to determine the equilibrium dissociation constant Kd 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. 14van den Berghe N. Cool R.H. Horn G. Wittinghofer A. Oncogene. 1997; 15: 845-850Crossref PubMed Scopus (86) Google Scholar). 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. 8Rehmann H. Prakash B. Wolf E. Rueppel A. de Rooij J. Bos J.L. Wittinghofer A. Nat. Struct. Biol. 2003; 10: 26-32Crossref PubMed Scopus (165) Google Scholar), 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 Epac149–881, 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 affinity of cIMP with cAMP. were obtained for cGMP and cXMP (data not The motif is conserved in the regulatory domains of cAMP and is by in was shown that mutations in PKA change the from cAMP to cGMP and L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. S.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In the of the mutation indeed the affinity for cGMP, the affinity for cAMP is not (Fig. in the of the Epac, the shows not an activation after with cGMP but a of k max after activation by cAMP (Fig. From these we that although a of cyclic nucleotides bind to Epac, only cAMP is able to activate GEF activity for Rap1. cAMP from the other cNMPs by the structure of the nucleotide and cGMP, cIMP, and cXMP have in that the of the in cAMP is by To investigate whether indeed the is for the activation of Epac, we tested the activity of the analogue in vitro. As this compound not activate Epac (data not shown) but inhibits cAMP-induced activation in vitro efficiently (Fig. of the Cyclic of the phosphate which are not involved in the by in a and (Fig. 1). is an of PKA, Sp-cAMPS, which has a is able to activate PKA B. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, B. PubMed Scopus Google Scholar). to the for PKA, not active Epac but inhibits cAMP-induced activation of Epac (Fig. A and In to PKA, is able to activate Epac (Fig. with a of both AC50 and k max. the and of the the membrane of cAMP, the of the is We have tested a of these analogues for on the activation of GEF activity of Epac. All analogues tested are but the maximal GEF activity saturating of is (Fig. In these modifications the AC50, of activating more efficiently than cAMP. Most is the in AC50 of with cAMP (Fig. Whereas activate Epac and both the k max and the AC50, has k max and AC50 for Epac similar to of cAMP (data not of the of the of the between the cAMP-binding domains of Epac and of other proteins is the presence of a of Epac, which to a that is conserved in other or in PKA this was shown to a with the 2′-OH of the (9Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (341) Google Scholar) and was shown to for high affinity binding Biochemistry. PubMed Scopus Google Scholar), we whether Epac modifications of the 2′-OH in in a of the AC50, and k max not even μm nucleotide (Fig. This that the 2′-OH is for binding and activation. To maintain the we and found a more of the AC50, which was to in the of 100 Surprisingly, guanine nucleotide exchange (k obs) much higher then the k max of cAMP, even was used the was found to the AC50 (Fig. we whether this on affinity with the on k max of the Indeed, 8-pCPT-2′-O-Me-cAMP a AC50 and an k max (Fig. A and The AC50 of 8-pCPT-2′-O-Me-cAMP μm) is only than the AC50 of μm) and than the AC50 found for cAMP. In the k max of 8-pCPT-2′-O-Me-cAMP is higher than and higher than cAMP. To that this of Epac by 8-pCPT-2′-O-Me-cAMP is caused by a change in equilibrium between an active an inactive of Epac, not by of the assay, we incubated Epac by 8-pCPT-2′-O-Me-cAMP with increasing of cAMP. We found that with increasing of cAMP, the GEF activity to a observed for cAMP inhibits GEF activity to a by this analogue, which is that of cAMP. We that the of the and the modifications on GEF activity to a cAMP analogue that activates Epac and used as a to the role of Rap1 in (12Enserink J.M. Christensen A.E. de Rooij J. van Triest M. Schwede F. Genieser H.G. Doskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Crossref PubMed Scopus (610) Google Scholar, G. M. Bos J.L. Schwede F. Genieser H.G. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). of the in and analyze the role of in the interaction with the of cAMP we the and The mutation the conserved in the affinity of cAMP for the isolated cAMP-binding domains of both was not that PKA, this is not involved in binding of cAMP (Fig. The were then further in the of the Epac, for activation properties in to by cAMP, and 8-pCPT-2′-O-Me-cAMP (Fig. For cAMP, a higher k max was observed for both the AC50 that is not for the activation mechanism of cAMP has an inhibitory role on activation. Activation by is by the the mutation had on the k max but the AC50, the mutation k max and the AC50 The AC50 of is high to determine k max either for and proteins (Fig. From these we that the presence of a or in the of to normal activation by cAMP and by 8-pCPT-2′-O-Me-cAMP in a that is not in the absence of a cAMP-bound The lid in we conserved residues and in the lid of Epac1 (11Rehmann H. Rueppel A. Bos J.L. Wittinghofer A. J. Biol. Chem. 2003; 278: 23508-23514Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As isolated cAMP-binding these proteins have a affinity for cAMP for which the affinity for cAMP was In these mutations cAMP-induced activation of Epac 11Rehmann H. Rueppel A. Bos J.L. Wittinghofer A. J. Biol. Chem. 2003; 278: 23508-23514Abstract Full Text Full Text PDF PubMed Scopus (54) Google and Fig. By the of cAMP, and 8-pCPT-2′-O-Me-cAMP, we that and have a similar on the activation of Epac by both cAMP and an on the activation by 8-pCPT-2′-O-Me-cAMP (Fig. not the k max of and cAMP but the k max of 8-pCPT-2′-O-Me-cAMP almost the k max of cAMP and 8-pCPT-2′-O-Me-cAMP but has on the k max of For the k max with was with wild for the k max was even by about In the AC50 of the analogues was by the Most although the affinity for cAMP for the isolated cAMP-binding domain of and was the AC50 was From these we that the lid region is involved in the of Epac by In the of 8-pCPT-2′-O-Me-cAMP with cAMP and on and that the communicates with the lid to super-activation. We have the of cAMP analogues on on Epac activation in vitro. We first noted that although cGMP, cIMP, and cXMP bind to Epac with an affinity similar to cAMP, are to activate Epac. This that the of the the is for the activation of Epac. In cGMP, cIMP, and cXMP are the IC50 is the it is that this inhibition is In this Epac from cGMP L. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and cIMP D. Doskeland S.O. J. Biol. Chem. Full Text PDF PubMed Google Scholar) are able to activate PKA, although only high inhibits Epac but activates PKA D. Doskeland S.O. J. Biol. Chem. Full Text PDF PubMed Google Scholar). All these nucleotides have an the interaction of the with the lid, the in the of cAMP the ability of cyclic nucleotides to activate Epac but not This that able to between PKA and Epac. to cyclic protein the and the motif were identified to determine the cAMP and cGMP From an bound between the of the guanine and the first in was Scopus Google Scholar). were by site-directed mutagenesis for both PKA L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Taylor S.S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and M. S.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which an cGMP affinity of the presence of the an affinity for cGMP as activation by cGMP in of k max. the the affinity of cGMP for Epac but had on the activation of Epac by Interestingly, as from with PKA L. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and M. S.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the had or on the affinity for cAMP. it the maximal activation of Epac by cAMP, that the sequence is involved not only in in binding but also in the cAMP binding to the activation of Epac. From the structural analysis of Epac and PKA, it was that the cyclic phosphate is for the activation of both Indeed, Rp-cAMPS, in which of the of the phosphate is by a is an of PKA B. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, B. PubMed Scopus Google Scholar), inhibits E. van M. L. B. PubMed Scopus Google Scholar). Indeed, also inhibits cAMP-induced Epac activation. This the activation mechanism of these proteins as we proposed previously (8Rehmann H. Prakash B. Wolf E. Rueppel A. de Rooij J. Bos J.L. Wittinghofer A. Nat. Struct. Biol. 2003; 10: 26-32Crossref PubMed Scopus (165) Google the of the the between the phosphate and an in the phosphate binding cassette and the of the phosphate binding the first in the activation of both the of the are used as analogues for PKA and PKG, these modifications both the for and the AC50 to activate these proteins D. Doskeland S.O. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D. R.H. B. Doskeland S.O. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In and are used for in vivo experiments. The was shown to stabilize the of cAMP, the of cAMP in the of and (9Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (341) Google Scholar, 10Diller T.C. Madhusudan Xuong N.H. Taylor S.S. Structure (Camb.). 2001; 9: 73-82Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), which a for the The analogues also have a AC50 for Epac with cAMP; in the resulted in a in the maximal activation of Epac by these analogues is with cAMP, that modifications the also the k max of the protein. not modifications of the the of cAMP on Epac. The highly conserved in the sequence of the phosphate binding cassette of PKA, PKG, cyclic ion and even the is by a in Epac1 and a in Epac2. was found to a with the 2′-OH of the cyclic nucleotide and was shown to for the binding of cAMP to PKA Biochemistry. PubMed Scopus Google Scholar). the Epac structure analysis (8Rehmann H. Prakash B. Wolf E. Rueppel A. de Rooij J. Bos J.L. Wittinghofer A. Nat. Struct. Biol. 2003; 10: 26-32Crossref PubMed Scopus (165) Google Scholar) that the to the 2′-OH a potential interaction between not to the binding affinity for cAMP. the AC50 of or nor the Kd of or was which that the in Epac is not involved in the direct binding of cAMP and the of the 2′-OH for activation of Epac. on activation of GEF activity with to both AC50 and k max were obtained by modifications of the 2′-OH of the of the 2′-OH by or by to an of the AC50, that interactions of the to the binding of Epac but not Indeed, the affinity of for the cAMP binding domain was this was much but the was by the for an A.E. F. de Rooij J. S. Schwede F. A. C. Bos J.L. Genieser H.G. Doskeland S.O. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). This indicated that the binding for cAMP to Epac, interaction with the conserved in the Interestingly, concentration induces k that are higher then the k max of activation. the the conformational equilibrium of the Epac more efficiently to the active with the affinity of the both modifications resulted in a highly of Epac (12Enserink J.M. Christensen A.E. de Rooij J. van Triest M. Schwede F. Genieser H.G. Doskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Crossref PubMed Scopus (610) Google Scholar). The of the k max is in part by The k max of activation of not is significantly. the other the k max of activation for both is with wild This that the cAMP-bound active state but the active of the lid a role of this region in the of 8-pCPT-2′-O-Me-cAMP, although the role of individual residues in this is A potential between the lid and the is In PKA a between the in the sequence to in and a in the lid is This correspond to Indeed, and activation are in responsive to the the k max of cAMP is not the k max of 8-pCPT-2′-O-Me-cAMP is In responsive to the structural and mutational analysis of both the lid and the catalytic domain are required to the mechanism of Epac activation by cAMP and cAMP analogues. We Genieser for and for Rueppel for and for