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. 2008 Sep 29;28(23):7109–7125. doi: 10.1128/MCB.01060-08

FIG. 4.

FIG. 4.

Ras-GTP potentiates Rap1 activation by the catalytic region of Epac2 in vivo but not in vitro. (A) Comparison of Rap1 activation by Epac2Δ430 and Epac2Δ430-684E. Increasing amounts of Epac2Δ430 or Epac2Δ430-684E were expressed in COS cells, and lysates were subjected to a Rap1 activation assay. The left panel shows linear regression of data from one representative experiment. Levels of Rap1 activation were plotted against the expression levels of constructs as indicated. The right panel shows representative gels from one experiment. Three independent experiments were performed. AU, artificial units; Ctrl, control. (B) RasV12 enhancement of Rap1 activation by Epac2Δ430 requires an intact RA domain. GFP-Epac2Δ430 and Epac2Δ430-684E were transfected alone or cotransfected with mCherry (mc)-RasV12, and lysates were subjected to a Rap1 activation assay. The left panel shows the quantification of data from three experiments (mean ± standard error); the right panel displays gels from one representative experiment. (C) Coomassie staining of purified Epac2Δ430, Epac2Δ430-684E, GST-Rap1, and RasV12. Protein molecular size markers are shown on the left. (D) Both Epac2Δ430 and Epac2Δ430-684E catalyzed nucleotide exchange reactions on Rap1 at identical rates in vitro. The upper panel shows a comparison of the intrinsic (red) exchange reaction and that catalyzed by Epac2Δ430 (cyan) or Epac2Δ430-684E (pink). Rap1-mant-dGDP (100 nM) was incubated in buffer containing 100 μM unlabeled GTP in the absence or presence of 1 μM Epac2Δ430 or Epac2Δ430-684E. Dissociation of mant-dGDP was monitored by the decrease in fluorescence emission at 435 nm over time. The bottom panel shows reaction rates fitted to single exponentials, and three to six independent measurements for each condition were pooled in the bar graph (mean ± standard error). (E) RasV12 does not enhance the exchange activity of Epac2Δ430 in vitro. The upper panel shows a comparison of the intrinsic (red) exchange reaction and that catalyzed by Epac2Δ430 in the presence (orange) or absence (cyan) of Ras-GTP. Rap1-mant-dGDP (100 nM) was incubated in buffer containing 100 μM unlabeled GTP in the absence or presence of 1 μM Epac2Δ430 alone or in addition to GTPγS-loaded RasV12. Dissociation of mant-dGDP was monitored. The bottom panel shows reaction rates fitted to single exponentials, and three independent measurements are summarized in the bar graph (mean ± standard error).