FIGURE 7.

PKA-dependent activation of Rap1 requires C3G. A, Hek293 cells were treated with F/I for the times indicated and pretreated with H89 or vehicle, as indicated. Rap1 activity (Rap1-GTP) was measured and shown in the 1st panel. Levels of total Rap1 are shown in the 2nd panel. B, cells transfected with either NS shRNA or shRNA against C3G were treated with F/I for the times indicated. The levels of pERK and total ERK2 are shown in the 1st and 2nd panels, respectively. The efficacy of the C3G knockdown is shown in the 3rd panel. C, cells were transfected with FLAG-ERK2 and either NS shRNA or shRNA against C3G and either vector or GFP-tagged shRNA-resistant C3G (GFP-C3G*), as indicated. Cells were treated with F/I for 20 min, lysed, and subjected to FLAG IP. The levels of FLAG-ERK2 activation (pFLAG-ERK2) and total FLAG-ERK2 within the IP are shown in the top two panels. The levels of total ERK2, GFP-C3G*, and C3G within the TCL are shown in the lower three panels, respectively. D, cells were transfected with FLAG-Src and treated with increasing doses of PP2 as indicated. The levels of phosphorylation of Y416 Src were determined using the phospho-Y416 antibody (top panel). The levels of FLAG-Src were determined using the FLAG antibody (bottom panel). E, cells were treated with F/I for the times indicated and pretreated with 2.5 μm PP2 or vehicle, as indicated. Rap1-GTP was measured in the 1st panel. Total levels of Rap1 are shown in the 2nd panel. The levels of pERK and total ERK2 are shown in the 3rd and 4th panels, respectively.