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 tran scription of many eucaryotic genes by catalyzing the phosphorylation o f 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) p hosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcrip tion from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was e nhanced over the level of PEPCK gene transcription observed in cells t reated 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 stim ulate transcription from the PEPCK promoter in cell-free reactions. Th e ability of okadaic acid to enhance PKA-stimulated transcription in v itro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extr acts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromato graphically 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 PPI. Nuclear PP2A dephosphorylated P-CREB 3 0-fold more efficiently than did nuclear PP1. Finally, when PKA-phosph orylated CREB was treated with immunopurified PP2A and PPI, the PP2A-t reated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PPI-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase tha t dephosphorylates PKA-phosphorylated CREB.