EPAC proteins transduce diverse cellular actions of cAMP

Abstract

It has now been over 10 years since efforts to completely understand the signalling actions of cAMP (3'-5'-cyclic adenosine monophosphate) led to the discovery of exchange protein directly activated by cAMP (EPAC) proteins. In the current review we will highlight important advances in the understanding of EPAC structure and function and demonstrate that EPAC proteins mediate multiple actions of cAMP in cells, revealing future targets for pharmaceutical intervention. It has been known for some time that drugs that elevate intracellular cAMP levels have proven therapeutic benefit for diseases ranging from depression to inflammation. The challenge now is to determine which of these positive actions of cAMP involve activation of EPAC-regulated signal transduction pathways. EPACs are specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, which they activate independently of the classical routes for cAMP signalling, cyclic nucleotide-gated ion channels and protein kinase A. Rather, EPAC activation is triggered by internal conformational changes induced by direct interaction with cAMP. Leading from this has been the development of EPAC-specific agonists, which has helped to delineate numerous cellular actions of cAMP that rely on subsequent activation of EPAC. These include regulation of exocytosis and the control of cell adhesion, growth, division and differentiation. Recent work also implicates EPAC in the regulation of anti-inflammatory signalling in the vascular endothelium, namely negative regulation of pro-inflammatory cytokine signalling and positive support of barrier function. Further elucidation of these important signalling mechanisms will no doubt support the development of the next generation of anti-inflammatory drugs.

Figure 1

Structural basis of EPAC regulation. (A) Schematic diagram of EPAC domain organization. (B) Conformational changes upon cAMP binding. Cartoon representations of the regulatory CNB domain and catalytic regions of EPAC2 are shown in the inactive (PDB 2byv) and active (PDB 3cf6) conformations with the molecular surfaces of the CNB domain and bound RAP1B depicted. The yellow ovals indicate the two parts of the cAMP-binding site (the lid and the pocket) in the inactive conformation. (C) Close-up view of the cAMP-binding site with Sp-CAMPS bound & with 8-pCPT-2′OMe–cAMP docked in its place showing how the pCPT and OMe groups might be accommodated and contribute to tighter binding between the core and lid of the CNB domain. Images generated by using PyMOL [DeLano, W.L.The PyMOL Molecular Graphics System (2002) DeLano Scientific, San Carlos, CA, USA]. 8-pCPT-2′OMe–cAMP, 8- (4- chlorophenylthio)- 2′- O- methyladenosine- 3′, 5′-cyclic monophosphate; cAMP, 3′-5′-cyclic adenosine monophosphate; CDC25-HD, CDC25 homology domain; CNB, cyclic nucleotides binding; DEP, Dishevelled-Egl-10-Pleckstrin; EPAC, exchange protein activated by cAMP; HH, helix hairpin; IL, ionic latch; RA, Ras association; REM, Ras exchange motif; Sp-CAMPS, adenosine- 3′, 5′-cyclic monophosphorothioate, Sp-isomer; SW, switchboard.

Figure 2

Control of cell fate by EPAC-activated intracellular signalling. The involvement of EPAC in intracellular signalling controlling cell division, cell differentiation and cell hypertrophy is shown. Generally activation of EPAC by cAMP leads to the activation of Rap1 GTPase, which, in turn, controls downstream signalling as indicated by black arrows. EPAC, exchange protein activated by cAMP; cAMP, 3′-5′-cyclic adenosine monophosphate; GPCR, G-protein-coupled receptor; PKA, protein kinase A; NFAT, nuclear factor of activated T-cell.

Figure 3

Anti-inflammatory effects of EPAC in VECs. EPAC mediates at least three anti-inflammatory signalling pathways in VECs. IL-6 signalling via STAT3 is inhibited by EPAC-dependent SOCS3 induction; this occurs via Rap1 and C/EBP transcription factors (black pathway). EPAC mediates activation of integrins involved in adhesion of VECs to the basement membrane; the signalling pathway between EPAC-activated Rap1 and integrin activation is not known, but may involve RIAM and talin, as reported for the αIIbβ3 platelet integrin (purple pathway). Agents such as thrombin and TNFα reduce adhesion of VECs to adjacent cells at adherens junctions via Rho, actin reorganization to reduce cortical ring and increase contractile stress fibres and destabilization of microtubules, resulting in reduced cadherin-mediated adhesion, increasing inter-endothelial gaps and thus increasing endothelial permeability (red pathway). In the presence of permeability-increasing agents, EPAC reduces endothelial permeability by inhibiting Rho activation and actin cytoskeleton reorganization, by promoting microtubule lengthening and by unidentified signalling pathways not requiring Rho or the cytoskeleton, all of which result in increased cadherin-mediated adhesion (blue pathway). C/EBP, CCAAT/enhancer-binding protein; EPAC, exchange protein activated by cAMP; GPCR, G-protein-coupled receptor; MT, microtubule; RIAM, Rap1-GTP-interacting adaptor molecule; SOCS3, suppressor of cytokine signalling 3; VECs, vascular endothelial cells.