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Fig. 2. cAMP activates Rap1 and ERKs in a PKA and Src tyrosine kinase-dependent manner. (A) Time course of ERK activation by forskolin/IBMX. PC12 cells were stimulated with forskolin/IBMX (F/I) for the indicated times in the presence or absence of H89 or PP2. Activation of endogenous ERKs was determined by western blot (pERK1/2). Total ERK2 levels are shown as a loading control. (B) Time course of ERK activation by 8-CPT. PC12 cells were stimulated with 8-CPT for the indicated periods in the presence or absence of H89 or PP2. Activation of endogenous ERK1/2 was determined by western blot (pERK1/2) as above. Total ERK2 levels are shown as a loading control. (C) cAMP activation of Rap1 via PKA and Src. PC12 cells were stimulated with forskolin/IBMX (F/I) for 20 minutes in the presence or absence of H89 or PP2, and lysates were analyzed for activation of endogenous Rap1 (Rap1-GTP). Total Rap1 levels are shown as a loading control. (D) cAMP does not activate Ras. PC12 cells were stimulated with forskolin/IBMX (F/I) for 10 minutes and lysates were analyzed for activation of endogenous Ras (Ras-GTP) or ERKs (pERK1/2). Total Ras and ERK2 levels are shown as loading controls. (E) 8-CPT does not activate Ras. PC12 cells were stimulated with 8-CPT (15 minutes) or EGF (5 minutes) and lysates were analyzed for activation of endogenous Ras (Ras-GTP). Total Ras levels are also shown.
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