Fig. 3. Phosphorylation of endogenous Src by PKA is required for Rap1 and ERK activation in PC12 cells. (A) cAMP induction of Src phosphorylation at S17 and Y416 via PKA by cAMP. PC12 cells were stimulated with forskolin/IBMX (F/I) at various time points in the presence or absence of H89 or left untreated (Un) as indicated. Src was immunoprecipitated and analyzed for phosphorylation at S17 and Y416 by using PSAb (pS17) and phospho-Src (pY416) antibody, respectively. Total Src levels are shown as a loading control. (B) Involvement of S17 phosphorylation in Rap1 activation by cAMP. PC12 cells were cotransfected with Flag-Rap1 and pcDNA3 (vector), Flag-Src wild type, Flag-SrcS17A or Flag-SrcS17D, then stimulated with forskolin/IBMX (F/I) for 20 minutes or left untreated (Un). Lysates were analyzed for activation of Rap1 (Flag-Rap1-GTP). Total Flag-Rap1 and Flag-Src are shown as transfection and loading controls. (C) Involvement of S17 phosphorylation in ERK activation by cAMP. PC12 cells were cotransfected with myc-ERK2 and vector, Flag-Src wild type or Flag-SrcS17A (left panels). Cells were then stimulated with forskolin/IBMX (F/I) for 20 minutes or left untreated (Un) and mycERK2 was immunoprecipitated. Cells were also transfected with vector or Flag-SrcS17D as indicated in the right panels. Activation of mycERK2 was determined by western blot (p-myc-ERK2) and total myc-ERK2 levels are shown as a loading control. Total Flag-Src is shown as a transfection control (lower panels).