Ethanol induces translocation of the catalytic subunit (C␣) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108 -15 cells. Ethanol also induces translocation of the RII␤ regulatory subunit of PKA to the nucleus; RI and C␤ are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of C␣ with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of C␣ by the PKA inhibitor PKI. Pre-incubation of C␣ with ethanol decreases phosphorylation of Leu-Arg-Arg-AlaSer-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response elementbinding protein (CREB) phosphorylation by C␣ is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R p )-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of C␣ and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.

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We have shown that ethanol induces translocation of cAMP-dependent protein kinase (PKA) to the nucleus, cAMP response element-binding protein (CREB) phosphorylation, and cAMP response element-mediated gene transcription in NG108-15 cells. However, little is known about which PKA types regulate this process. We show here that under basal conditions NG108-15 cells contain type I PKA (C␤RI␤) primarily in cytosol and type II PKA (C␣RII␤) in the particulate and nuclear fractions. Antagonists of both type I and type II PKA inhibit forskolin-and ethanol-induced cAMP response element-mediated gene transcription. However, only the type II PKA antagonist inhibits forskolin-induced C␣ and ethanol-induced C␣ and RII␤ translocation to the nucleus and CREB phosphorylation; the type I antagonist is without effect. Our data suggest that forskolin-and ethanol-induced CREB phosphorylation and gene activation are differentially mediated by the two types of PKA. We propose that type II PKA is translocated and activated in the nucleus and induces CREB phosphorylation that is necessary but not sufficient for gene transcription. By contrast, type I PKA is activated in the cytoplasm, turning on a downstream pathway that activates other transcription cofactors that interact with phosphorylated CREB to induce gene transcription.cAMP signaling is a multiple component system that requires production of cAMP and induces activation of PKA, 1 phosphorylation of CREB, and cAMP-dependent gene expression. This pathway has been implicated in learning and memory (1, 2) as well as addictive behaviors (3) and responses to ethanol (4). Ethanol activates the cAMP pathway to its full extent by stimulating adenylyl cyclase activity (4), C␣ translocation to the nucleus (5), CREB phosphorylation (6), and CREmediated gene transcription (7).The primary intracellular receptors for cAMP in mammalian cells are various isoforms of PKA. In the absence of cAMP, PKA is a tetrameric holoenzyme consisting of two catalytic subunits (C) bound to a regulatory subunit (R) dimer. After binding of two molecules of cAMP to each R monomer, the two C subunits are released and activated to phosphorylate intracellular substrates. The PKA family consists of four regulatory subunits (RI␣, RI␤, RII␣, RII␤) and three catalytic subunits (C␣, C␤, and C␥), each encoded by a unique gene (8, 9). Type I or type II PKA are classically defined by their specific regulatory subunits. The mechanisms by which the cAMP-signaling pathway achieves specificity include 1) compartmentalization of PKA via binding to scaffolding proteins such as A kinase-anchoring protein (AKAP) near target substrates; 2) regulated expression of distinct R and C subunit isoforms in cells and tissues; and 3) differential combinations of R and C subunit isoforms. All these mechanisms can affect PKA interaction with cAMP, substrates, and inhibitors. Studies with knockout and transgenic mouse models have shed some light on the physiological functions of specific isoforms of PKA in vivo (8). Models used in ethanol studies...

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