Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.The activation of signal transduction pathways by hormonal and developmental stimuli ultimately leads to changes in the expression of specific genes. These changes often occur at the level of gene transcription and are mediated by rapid changes in the phosphorylation state of specific transcription factors. For example, the transcriptional transactivation function of the cyclic AMP (cAMP)-regulatory element binding protein (CREB) is stimulated by phosphorylation (15,28,45). CREB phosphorylation by the cAMP-dependent protein kinase A (PKA) is sufficient for the transcriptional transactivation in response to elevated intracellular cAMP levels (15,18,28,45).The stimulation of transcription of certain genes by cAMP is generally characterized by a rapid increase in the rate of transcription to a maximal level, followed by a slow decline in transcription to basal levels (29,38). The decrease in the rate of transcription from stimulated levels occurs even under conditions where PKA activity is elevated, and it is refractory to the addition of agents that ...

Long-term potentiation (LTP) is a form of synaptic plasticity that has been extensively studied as a putative mechanism underlying learning and memory. A late phase of LTP occurring 3-5 hours after stimulation and depending on transcription, protein synthesis and cyclic-AMP-dependent protein kinase (protein kinase A, or PKA) has been described, but it is not known whether transcription of presynaptic and/or postsynaptic genes is required to support late-phase LTP. Here we show that late-phase LTP can be obtained in rat hippocampal CA1 mini-slices in which the cell bodies of presynaptic Schaffer collateral/commissural fibres are removed. Thus, transcription of presynaptic genes is not necessary to support maintenance of late-phase LTP. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor is the predominant mediator of the ionotropic response to synaptically released glutamate in the hippocampus and it has been implicated in LTP maintenance. We find that synthesis of AMPA receptor subunits is increased three hours after LTP induction: this effect on the synthesis of the AMPA receptor is blocked by inhibitors of PKA and of transcription. Our results support the idea of a postsynaptic mechanism maintaining late-phase LTP, in which AMPA receptor synthesis is increased as a result of PKA-dependent gene transcription.

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