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

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.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 ...

G1/S transcriptional networks modulated by the HOX11/TLX1 oncogene of T-cell acute lymphoblastic leukemia

“…Considered together with our observation of prolonged AKT phosphorylation following serum stimulation of HOX11-expressing NIH3T3 cells, these results point to an expanded role of this phosphatase in T-cell oncogenesis beyond disruption of the G 2 /M cell-cycle checkpoint (Kawabe et al, 1997). Along these lines, CREB is another transcription factor whose activity has been reported to be negatively modulated by PP1 and PP2A (Wadzinski et al, 1993;Alberts et al, 1994;Canettieri et al, 2003). We found that a number of CREB target genes were more highly expressed in HOX11 þ human and murine T-cell tumors.…”

“…Given that PP1 and PP2A were reported to be the primary phosphatases involved in dephosphorylating activated CREB (Wadzinski et al, 1993;Alberts et al, 1994;Canettieri et al, 2003), and the implied role of CREB in regulating a number of the genes whose expression patterns were modulated by HOX11 (Table 1), we next performed luciferase reporter assays in NIH3T3 cells using the pCRE-Luc plasmid which contains multiple copies of a CRE-binding site. Compared with the empty vector pcDNA3, cotransfection with pcDNA3-HOX11 resulted in a two-fold increase in expression from pCRE-Luc (Figure 6a).…”

Failure of stress-induced downregulation of Bcl-2 contributes to apoptosis resistance in senescent human diploid fibroblasts

“…14 To investigate whether PP2A is involved in dephosphorylation of p-CREB in stress-induced apoptosis of young and senescent HDFs, we determined PP2A activities in young and senescent HDFs before and after agent treatments with H 2 O 2 , staurosporine and thapsigargin. As shown in Figure 6, apoptotic stimuli significantly increased PP2A activities only in young HDFs but not in senescent HDFs.…”

Purification and Identification of a Novel Subunit of Protein Serine/Threonine Phosphatase 4

“…Preparation of Phosphorylated Substrates and Phosphatase AssayPhosphorylated substrates were prepared as described previously for casein (25) and phosphorylase a and histone H1 (26). Purified proteins (approximately 0.14 ng of PP2A C and approximately 11.3 ng of PP4 C contained in PP4) were assayed for phosphatase activity in a 100-l reaction volume containing 25 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, 0.5 mg/ml bovine serum albumin, and 32 P-labeled substrate.…”