Epac1 translocates toward the PM upon elevation of cAMP levels. (A) In HEK293 cells, stimulation with forskolin (25 μM) induces the translocation of GFP-Epac1 toward the cell periphery. The inset shows fluorescence intensity along the red line, showing a sharp demarcation of the plasma membrane (width at half maximum, ∼300 nm). On the right, fluorescence intensity at the PM (red) and in the cytosol (Cyt) (blue) as well as the PM/Cyt ratio (green) during the response to forskolin is shown; fluorescence levels in the nucleus (Nucl) (gray) were constant. Traces are representative for n > 15. (B) Representative TIRF experiment showing the forskolin-induced accumulation of GFP-Epac1 at the basal membrane of HEK293 cells. The mean increase relative to prestimulus levels was 60% ± 4% (means ± SEM; n = 17). (C) A431 cells expressing GFP-Epac1 were imaged during isoproterenol (1 nM) stimulation. The translocation of GFP-Epac1 was observed within 1 min (n = 8). (D) Immunofluorescence of OVCAR-3 cells stained for endogenous Epac1. In resting cells, little PM localization of Epac1 is observed. After forskolin stimulation (25 μM, 10 min), the amount of PM-localized Epac1 is markedly increased. Note that the nuclear immunofluorescence is background staining rather than that of Epac1, as it is insensitive to the small interfering RNA-mediated silencing of Epac1 (data not shown). (E) Measurement of FRET between the PM marker CFP-CAAX (see Materials and Methods) and translocating YFP-tagged Epac1 (traces are representative for n > 5). FRET, expressed as a YFP/CFP ratio, increases upon the addition of forskolin (25 μM) (black). FRET increases also were induced by the Epac-specific cAMP analogue 007 (100 μM) (blue) or the more membrane-permeable analogue 007-AM (1 μM) (red). (F) Forskolin (25 μM) treatment of HEK293 cells transfected with the FRET sensor CFP-Epac1-YFP. The sensor reports the cAMP-induced conformational change as a loss of intramolecular FRET. In contrast to the wild-type sensor (red), the mutant FRET construct CFP-Epac1(R279L)-YFP (blue) lacks the ability to change conformation upon changing cAMP concentrations. (G) Mutagenesis of arginine 279 to leucine eliminated the ability of YFP-Epac1(R279L) to translocate upon forskolin stimulation (25 μM). Scale bars, 10 μm.

Rap activity is not involved in Epac1 translocation. (A) Western blotting of the Rap activation assay (see Materials and Methods) confirming that RapGAP1 overexpression effectively inhibits the 007-AM-mediated activation of Rap1 and Rap2. Images are HEK293 cells expressing GFP-Epac1 plus overexpressed RapGAP1 (left) or constitutively active Rap1A(G12V) (right). Neither RapGAP1 nor Rap1A(G12V) overexpression had any effect on the distribution of unstimulated GFP-Epac1 (upper) or on the 007-AM-induced translocation (lower). Scale bars, 10 μm. (B) Kinetic analyses were performed on cells transfected as described for panel A. Kinetics of the 007-AM-induced translocation of GFP-Epac1 were not affected by the coexpression of RapGAP1 or Rap1A(G12V). Translocation was quantified from the depletion of cytosolic fluorescence. Traces of >10 experiments per condition were averaged after normalization to the basal level (set to 100%) and the end level (set to 0%). (C) A431 cells expressing CFP-Epac1-YFP were imaged by the simultaneous detection of CFP and YFP emission (see Materials and Methods), allowing the analysis of both the Epac1 activation state (FRET) (YFP/CFP ratio) and its translocation to the PM (PM/Cyt ratio). Cyt, cytosol. The submaximal stimulation of A431 cells with 0.5 nM isoproterenol evokes slow cAMP accumulation and thereby induces the gradual activation of Epac1; subsequently, forskolin (25 μM) was added to saturate CFP-Epac1-YFP. The experiment illustrates that the kinetics of the construct's PM translocation strongly resemble its gradual activation (representative for n = 6).

Translocation of Epac1 enhances Rap activation and Rap-mediated adhesion of Jurkat T cells. (A) The trace on the left shows the in vivo TIRF imaging of Rap1 activation in HEK293 cells using YFP-RalGDS(RBD). When coexpressed with HA-Epac1, YFP-RalGDS(RBD) translocates to the basal membrane within seconds after 007-AM stimulation (1 μM). The 007-AM-induced increase was 46% ± 6% (means ± SEM) relative to prestimulus levels (n = 9). The images on the right are confocal pictures of YFP-RalGDS(RBD) translocation, showing accumulation at the PM as well as the simultaneous depletion of cytosol (Cyt); nuclear fluorescence was constant (Nucl) (gray). The accumulation of YFP-RalGDS(RBD) was not observed on intracellular membranes. Scale bar, 10 μm. (B) HEK293 cells were transfected with CFP-Epac1 or CFP-Epac1(ΔDEP), and the recruitment of cotransfected YFP-RBD(RalGDS) to the basal membrane was measured by TIRF microscopy. Cells expressing low levels of CFP- and YFP-tagged proteins were selected. Traces are from representative experiments. The bar graph shows the relative occurrence of the 007-AM-induced membrane accumulation of YFP-RBD(RalGDS): for CFP-Epac1, 12 out of 14 cells; for CFP-Epac1(ΔDEP), 1 out of 9 cells. (C) Jurkat T cells were transfected with either wild-type Epac1 or the nontranslocating Epac1 mutants [Epac1(ΔDEP) and Epac1(R82A)] together with a luciferase reporter. Transfected cells were allowed to adhere to a fibronectin-coated surface for 45 min, and adhesion subsequently was detected as luciferase emission. Wild-type Epac1, when activated by 007 (100 μM) (black bars), greatly enhanced the adhesion compared to that of unstimulated conditions (white bars). This effect was impaired when the translocation-deficient mutant Epac1(ΔDEP) or Epac1(R82A) was transfected, and it was absent in empty vector-transfected cells (EV). Shown are data from a representative experiment performed in triplicate (n = 4). Total luciferase levels were comparable in all transfections. On the right is a Western blot labeled with the Epac1 antibody (5D3) showing the expression levels of the transfected wild-type and mutant Epac1 used in the adhesion assay. (D) Jurkat T cells were transfected with similar amounts of either wild-type Epac1 or the translocation-deficient mutants Epac1(ΔDEP) and Epac1(R82A), and GTP-bound Rap1 was pulled down from lysates of cells after stimulation with 007 (100 μM, 10 min).

Epac1 translocation is a highly dynamic and reversible event. (A) cAMP-uncaging experiments in HEK293 cells. The upper trace shows that the release of NPE-caged cAMP (arrows; see Materials and Methods for details) saturates the FRET-based cAMP sensor CFP-Epac(ΔDEP,C.D.)-Venus, in that a subsequent UV flash did not induce further FRET changes. The lower trace shows the ratio between PM and cytosolic fluorescence of GFP-Epac1 [expressed at levels comparable to those of CFP-Epac1(ΔDEP,C.D.)-Venus] showing the immediate translocation upon the identical photolysis of caged-cAMP (τ_1/2 < 5 s). (B) cAMP uncaging in GE11 cells. The upper trace shows that due to the high speed of cAMP clearing in these cells, the dosed release of NPE-caged cAMP evokes transient cAMP rises. The amounts of released cAMP are approximately proportional to the duration of UV flashes. The lower trace shows that the release of identical amounts of caged cAMP in GE11 cells induces transient translocations of GFP-Epac1. The PM/cytosol ratio shows that the degree of translocation correlates with the dose of released cAMP.

DEP domain is essential but not sufficient for Epac1 translocation. (A and B) Overview of Epac1 mutants and their abilities to translocate (TL) upon the addition of 007-AM (1 μM). Full-length Epac1 consists of a DEP domain (amino acids 50 to 148), the cyclic nucleotide-binding domain (CNB), the Ras exchange motive (REM), a putative RA domain, and the catalytic CDC25 homology GEF domain. The removal of the DEP domain (amino acids 50 to 148) or the disruption of the interaction surface within the DEP domain (R82A) abolishes the translocation of Epac1 in response to 007-AM. The separate regulatory region (YFP-Epac1-Reg; amino acids 1 to 328), containing both the DEP domain and the cAMP-binding domain (CNB), did not translocate after 007-AM stimulation, indicating that the DEP domain cannot mediate the localization of Epac1 at the PM in the absence of the catalytic region. In accordance with the crucial role of the DEP domain, the complete catalytic region (CFP-Epac1-Cat; amino acids 330 to 881) was not present at the PM either. (C) In contrast to the separately expressed regulatory and catalytic region, the coexpression of YFP-Epac-Reg (upper images) and CFP-Epac-Cat (lower images) restored the 007-AM-induced translocation, resulting in the colocalization of both constructs at the PM. Scale bars, 10 μm.