Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.

Wadzinski BE, Wheat WH, Jaspers S, Peruski LF, Lickteig RL, Johnson GL, Klemm DJ
Mol Cell Biol. 1993 13 (5): 2822-34

PMID: 8386317 · PMCID: PMC359667 · DOI:10.1128/mcb.13.5.2822

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.

MeSH Terms (30)

Amino Acid Sequence Base Sequence Bucladesine Carcinoma, Hepatocellular Cell Nucleus Cloning, Molecular Cyclic AMP Response Element-Binding Protein Ethers, Cyclic Female Gene Expression Regulation, Neoplastic Humans Kinetics Leukemia, Promyelocytic, Acute Liver Neoplasms Macromolecular Substances Molecular Sequence Data Okadaic Acid Oligodeoxyribonucleotides Phosphoenolpyruvate Carboxykinase (GTP) Phosphoprotein Phosphatases Phosphorylation Placenta Polymerase Chain Reaction Pregnancy Promoter Regions, Genetic Protein Kinases Protein Phosphatase 2 Recombinant Proteins Transcription, Genetic Tumor Cells, Cultured

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