cAMP-dependent Protein Kinase Types I and II Differentially Regulate cAMP Response Element-mediated Gene Expression

Abstract

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...

The Two Regulatory Subunits ofAplysiacAMP-Dependent Protein Kinase Mediate Distinct Functions in Producing Synaptic Plasticity

“…Distinctive responses of the two types of PKA to ethanol treatment were seen in cultured neuroblastoma-Xglioma cells by Constantinescu et al (2002), who found that type II PKA is translocated to the nucleus to phosphorylate CREB, whereas type I remains in the cytoplasm to activate other transcription cofactors that then interact with phosphorylated CREB. In Aplysia sensory neurons, the functions of the two types of R differ primarily because of the polarity of neurons, type I PKA operating in the cell body and type II at nerve terminals.…”

Requirement for the RIIβ Isoform of PKA, But Not Calcium-Stimulated Adenylyl Cyclase, in Visual Cortical Plasticity

“…Translocation and activation of RII-, but not RI-, PKA induces CREB phosphorylation that is necessary but insufficient for gene transcription (Constantinescu et al, 2002), whereas downregulation of RII␤-PKA by antisense oligonucleotides inhibits cAMP-induced nuclear signaling and CREB phosphorylation (Paolillo et al, 1999). Furthermore, anchoring by AKAP150 increases the rate, magnitude, and sensitivity of PKA signaling to the nucleus, increasing by 10-fold both CREB phosphorylation and CRE-mediated gene transcription (Feliciello et al, 1997).…”

Ethanol–BDNF interactions: Still more questions than answers

“…The different exposure paradigms (IP injection vs. long-term self administration and withdrawal), rodent strain or cell-type selectivity may explain these differences. Acute ethanol can activate adenylate cyclase, which can activate CREB through PKA (Asher et al, 2002;Constantinescu et al, 2002). Chronic exposure leads to compensatory up-regulation of NMDA receptors, with withdrawal producing Ca 2+ -dependent hyper-excitibility (Hoffman and Tabakoff, 1994).…”