Figure 7
PKAII is less efficient compared to PKAI in regulating CRE activity in vivo. 293T cells were co-transfected with or without (empty vector, M), pDeCα1 (300 ng) together with either pExRIIα (320 ng) or pDeRIα (640 ng) for 24 h (A- C). (A) R subunit-specific activity (pmol cAMP/mg protein) was monitored in the presence of 25 μM [3H]-cAMP and showed comparable levels of RIα and RIIα. (B) Catalytic activity of Cα1 was monitored in the absence (0) and presence of low (5 nM) and high (5000 nM) concentrations of cAMP. In the presence of 5000 nM cAMP levels of transfected Cα1 released from RIα and RIIα were equal. (C) The stability of RIα and RIIα was monitored in the presence (+) and absence (-) of Cα1 and the absence (0) and presence of incremental doses (1-320 μM) of 8-CPT-cAMP. R subunit stability was determined according to immunoreactive R subunits after separation of cell extracts (25 μg total protein) on SDS-PAGE (12.5 % gels) and anti-RIα (1 : 300 dilution, upper panel) and anti-RIIα (1 : 400 dilution, lower panel). (D) 293T cells were left untreated (0) or stimulated for 1 hour with 8-CPT-cAMP (320 μM) before harvesting. Cell extracts were adjusted to 1 mg total protein/mL and assayed for PKA-specific phosphorylation of CREB monitored as relative luciferase activity at 560 nm. Data points represent relative luciferase activity +/- SD (n = 2-6).