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PMID: 8386317 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

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

Molecular and cellular biology ·Vol. 13 ·No. 5 ·1993-05-00 ·Pages 2822-34

Wadzinski BE, Wheat WH, Jaspers S, Peruski LF, Lickteig RL, Johnson GL, Klemm DJ

Abstract

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
Amino Acid Sequence Base Sequence Bucladesine/pharmacology Carcinoma, Hepatocellular Cell Nucleus/enzymology Cloning, Molecular Cyclic AMP Response Element-Binding Protein/genetics,metabolism Ethers, Cyclic/pharmacology Female Gene Expression Regulation, Neoplastic/drug effects Humans Kinetics Leukemia, Promyelocytic, Acute Liver Neoplasms Macromolecular Substances Molecular Sequence Data Okadaic Acid Oligodeoxyribonucleotides Phosphoenolpyruvate Carboxykinase (GTP)/genetics Phosphoprotein Phosphatases/antagonists & inhibitors,genetics,metabolism Phosphorylation Placenta/metabolism Polymerase Chain Reaction Pregnancy Promoter Regions, Genetic/drug effects Protein Kinases/metabolism Protein Phosphatase 2 Recombinant Proteins/metabolism Transcription, Genetic/drug effects Tumor Cells, Cultured
Chemicals
Cyclic AMP Response Element-Binding Protein Ethers, Cyclic Macromolecular Substances Oligodeoxyribonucleotides Recombinant Proteins Okadaic Acid Bucladesine Protein Kinases Phosphoprotein Phosphatases Protein Phosphatase 2 Phosphoenolpyruvate Carboxykinase (GTP)
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Wadzinski B E
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
Wheat W H
Jaspers S
Peruski L F
Lickteig R L
Johnson G L
Klemm D J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.13.5.2822-2834.1993
Published
1993-05-00
Pages
2822-34
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC359667
Subset
IM
Grants
NIDDK NIH HHS · DK37871 · United States
External Links
PubMed source DOI: 10.1128/mcb.13.5.2822-2834.1993 PMC Full Text View journal details
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