Differential Rap1 activation properties of cAMP analogs. A,
Western blot analysis demonstrating the weak effect of
8-pCPT-2′-O-Me-cAMP (ESCA, left panel) and the strong
effect of 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM, right
panel) to activate Rap1 in lysates prepared from monolayers of INS-1
cells transfected with FLAG-Rap1 and Eapc1. Active Rap1 is labeled as Rap1-GTP
and the quantity of active Rap1 was assessed by densitometry. For the left
panel, treatment with 3.0 μm
8-pCPT-2′-O-Me-cAMP increased the amount of active Rap1 by
1.41-fold relative to the untreated control value. For the right
panel, treatment with 3.0 μm
8-pCPT-2′-O-Me-cAMP-AM increased the amount of active Rap1 by
4.79-fold relative to the untreated control value. B, time course of
Rap1 activation by 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM).
The exposure time corresponds to the time in which INS-1 cells were exposed to
the test solution. C, contrasting actions of PKA-selective
Bt2cAMP-AM (db-cAMP-AM) and Epac-selective
8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM) to activate Rap1
under conditions in which INS-1 cells were or were not pretreated with the PKA
inhibitor H-89 (10 μm). For panels A-C, each experiment
was repeated 3 times with similar results.