Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.
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.
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
B E
BE
Wadzinski
W H
WH
Wheat
S
S
Jaspers
L F
LF
Peruski
R L
RL
Lickteig
G L
GL
Johnson
D J
DJ
Klemm
D000595:Amino Acid Sequence; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002467:Cell Nucleus / Q000201:enzymology*; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D004988:Ethers, Cyclic / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D009838:Oligodeoxyribonucleotides; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D054648:Protein Phosphatase 2; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D017362:Cyclic AMP Response Element-Binding Protein; D004988:Ethers, Cyclic; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019319:Okadaic Acid; D003994:Bucladesine; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D010729:Phosphoenolpyruvate Carboxykinase (GTP)