Ethanol-induced phosphorylation and potentiation of the activity of type 7 adenylyl cyclase: Involvement of protein kinase C δ

Abstract

Ethanol can enhance Gsα-stimulated adenylyl cyclase (AC) activity. Of the nine isoforms of AC, type 7 (AC7) is the most sensitive to ethanol. The potentiation of AC7 by ethanol is dependent on protein kinase C (PKC). We designed studies to determine which PKC isotype(s) are involved in the potentiation of Gαs-activated AC7 activity by ethanol and to investigate the direct phosphorylation of AC7 by PKC. AC7 was phosphorylated in vitro by the catalytic subunits of PKCs. The addition of ethanol to AC7-transfected HEK 293 cells increased the endogenous phosphorylation of AC7, as indicated by a decreased "back-phosphorylation" of AC7 by PKC in vitro. The potentiation of Gαs-stimulated AC7 activity by either phorbol 12,13-dibutyrate or ethanol, in HEL cells endogenously expressing AC7, was not through the Ca2+-sensitive conventional PKCs. However, the potentiation of AC7 activity by ethanol or phorbol 12,13-dibutyrate was found to be reduced by the selective inhibitor of PKCδ (rottlerin), a PKCδ-specific inhibitory peptide (δV1-1), and the expression of the dominant negative form of PKCδ. Immunoprecipitation data indicated that PKCδ could bind and directly phosphorylate AC7. The results indicate that the potentiation of AC7 activity by ethanol involves phosphorylation of AC7 that is mediated by PKCδ.