Figure 4

Oligomerization of AKAP12 in mammalian cells: elevation of intracellular cyclic AMP as well as inhibition of protein kinase A, MEK1/2, cyclic AMP phosphodiesterase 4, or expression of either N- or C-terminal fragments do not block AKAP oligomerization. (A) HEK293 cells were transfected with an expression vector harboring either HA-tagged full-length AKAP12 (HA-AKAP12) or an HA-tagged AKAP12 in which alanine substitution of protein kinase A phosphorylation sites (S627A, S696-698A, S772A) has been performed (AKAP12M3). Cells were treated with the beta-adrenergic agonist isoproterenol (10 μM) for 0, 5, or 30 min prior to harvesting the cells for lysis. Whole-cell lysates of transfected cells expressing either AKAP12 or AKAP12M3 were subjected to SDS-PAGE in the absence of 8 M urea. (B) HEK293 cells were either untreated or incubated with a chemical inhibitor for protein kinase A (KT5720, 1 μM), MEK1/2 (PD98059, 20 μM), or cyclic AMP phosphodiesterase 4 (Rolipram, 10 μM) for 45 min prior to cell lysis and subsequent analysis by SDS-PAGE. (C) HEK293 cells were either untransfected or transfected with an expression vector harboring the AKAP12 N-terminal (1-938) fragment or the C-terminal (840-1782) fragment. Whole-cell lysates of cells expressing either AKAP12 or one of the fragments subjected to SDS-PAGE in the absence of 8 M urea. The resolved proteins were transferred to PVDF membrane, subjected to immunoblotting, and made visible by staining with anti-HA or anti-AKAP12 antibodies. Beta-catenin was employed as a loading control for each lane, also identified by immunoblotting, stained with anti-beta-catenin antibodies. The results displayed are representative of at least three separate experiments performed on as many separate cell cultures.