Phosphorylation Cascades Control the Actions of Ethanol on Cell cAMP Signalling

Abstract

Our studies indicate that, in the presence of particular isoforms of adenylyl cyclase (i.e., type 7 AC), moderately intoxicating concentrations of ethanol will significantly potentiate transmitter-mediated activation of the cAMP signaling cascade. Activation of this signaling cascade may have important implications for the mechanisms by which ethanol produces intoxication, and/or for the mechanisms of neuroadaptation leading to tolerance to, and physical dependence on, ethanol. We initiated a series of studies to investigate the phosphorylation of AC7 by PKC, the role of this phosphorylation in modulating the sensitivity of AC7 to activation by Gsα, and the PKC isotype(s) involved in the phosphorylation of AC7. The T7 epitope-tagged AC7 expressed in Sf9 and HEK293 cells was found to be phosphorylated in vitro by the catalytic subunit of PKC. Treatment of AC7-transfected HEK293 cells with phorbol dibutyrate (PDBu) or ethanol increased the phosphorylation of AC7 and its responsiveness to Gsα. In human erythroleukemia (HEL) cells, which endogeneously express AC7, ethanol and PDBu increased AC activity stimulated by PGE₁. The potentiation by both PDBu and ethanol was found to be sensitive to the PKC δ-selective inhibitor, rottlerin. The potentiation of AC activity by ethanol in HEL cells was also selectively attenuated by the RACK inhibitory peptide specific for PKC δ, and by expression of the dominant negative, catalytically inactive, form of PKC δ. These data demonstrate that AC7 can be phosphorylated by PKC, leading to an increase in functional activity, and ethanol can potentiate AC7 activity through a PKC δ-mediated phosphorylation of AC7.