B56 regulatory subunit of protein phosphatase 2A mediates valproic acid-induced p300 degradation

Abstract

Transcriptional coactivator p300 is required for embryonic development and cell proliferation. Valproic acid, a histone deacetylase inhibitor, is widely used in the therapy of epilepsy and bipolar disorder. However, it has intrinsic teratogenic activity through unidentified mechanisms. We report that valproic acid stimulates proteasome-dependent p300 degradation through augmentation of gene expression of the B56gamma regulatory subunits of protein phosphatase 2A. The B56gamma3 regulatory and catalytic subunits of protein phosphatase 2A interact with p300. Overexpression of the B56gamma3 subunit leads to proteasome-mediated p300 degradation and represses p300-dependent transcriptional activation, which requires the B56gamma3 interaction domain of p300. Conversely, silencing of the B56gamma subunit expression by RNA interference increases the stability and transcriptional activity of the coactivator. Our study establishes the functional interaction between protein phosphatase 2A and p300 activity and provides direct evidence for signal-dependent control of p300 function.

FIG. 1.

Valproic acid induces p300 degradation through the 26S proteasome. (A) Western blot analysis of endogenous p300 in extracts (50 μg) from HeLa cells treated with sodium butyrate (NaB) (5 mM) and/or MG132 (MG) (5 μM). The blot was then stripped and reprobed for protein loading with an anti-SRC antibody. The treatment time (in hours) is indicated above the gel. (B) The experimental procedure was as described for panel A except that valproic acid (VPA) (2 mM) was used. (C and D) Quantitative analysis of Western blot results is expressed as fold variation compared to the values for untreated controls after the values were normalized to those of the loading controls. Values are means ± standard deviations (error bars) from a minimum of three independent experiments with each experiment performed in duplicate. (E) Cells were pretreated with valproic acid or sodium butyrate for 6 h, then pulsed for 3 h, and chased for 7 h in the different treatment conditions. The endogenous p300 protein was immunoprecipitated with a p300 antibody, separated by SDS-PAGE, and analyzed with a PhosphorImager. (F) The relative stability of p300 in cells treated with valproic acid or sodium butyrate, upon 7 h of chase (lanes 5 and 6), is presented as a percentage of the labeled p300 in untreated cells (lane 4) after being normalized to nonchased cells in the different treatment conditions (as in lanes 1 to 3 of panel E). Values are means ± standard deviations (error bars) from a minimum of three independent experiments with each experiment performed in duplicate.

FIG. 2.

Protein phosphatases play an important role in the control of the steady-state level of p300. (A) Equal amounts (50 μg) of whole-cell extracts from HeLa cells were used for Western blot analysis of endogenous p300 after treatment with okadaic acid (OA) (0.4 μM [+] or 0.8 μM [++]). The treatment time (in hours) is indicated above the lanes. The blot was then stripped and reprobed with a p53 antibody. (B) The same experimental setup as in panel A, except the cells were treated with okadaic acid (OA) (0.2 μM) in the presence or absence of valproic acid (VPA) (2 mM) or sodium butyrate (NaB) (5 mM) for 16 h. (C) The same procedure as in panel B, except the blot was probed with p300 antibody, stripped, and reprobed with a RAR antibody. A protein band cross-reacting with the RAR antibody is shown as a control (Ctl).

FIG. 3.

Valproic acid and sodium butyrate augment the expression of the B56 regulatory subunits of PP2A. (A) Cells were treated with valproic acid (VPA) (2 mM) or sodium butyrate (NaB) (5 mM) for 4, 8, and 16 h. Total RNA was isolated, and the mRNA levels of B56γ3 (γ3) were assessed by quantitative RT-PCR. Results show fold variations of the treated cells compared to those of untreated control (Ctl). Values are means ± standard deviations (error bars) from a minimum of three independent experiments with each experiment performed in duplicate. (B) The experimental setup was as in panel A, except the mRNA level of B56γ2 was examined. (C) Cells were treated as in panel A. Equal amounts of whole-cell extract (50 μg) were used for Western blotting. The blot was probed with an antibody specific to endogenous B56γ2 and B56γ3 and with an anti-RAR antibody for loading control.

FIG. 4.

The B56γ3 regulatory subunit of PP2A inhibits p300 activity. (A) HeLa cells were transfected with increasing amounts of B56γ3 (γ3) expression plasmid (1, 1.5, and 2 μg in lanes 2, 3, and 4, respectively; 2 μg in lanes 6 to 8) and treated with MG132 (MG) (5 μM) for 16 h or left alone. The abundance of endogenous p300 protein was analyzed on a Western blot. The blot was then stripped and reprobed for the B56γ3 subunit. (B) The experimental procedure was as in panel A, except the cells were transfected with increasing amounts of expression plasmid for the B56γ2 subunit (1, 2, and 4 μg). (C) Cells were transfected with RAR luciferase reporter (0.3 μg), respiratory syncytial virus β-galactosidase (RSV-β-Gal) (0.3 μg), and increasing amounts of B56γ3 expression plasmid (0.5 and 2.0 μg). The cells were then induced with arotinoid acid (AA) (1 μM) for 24 h and assayed for luciferase (Luc) assay. Values shown were normalized to β-galactosidase activity and are expressed as the fold induction relative to the values for untreated controls. Values are means ± standard deviations (error bars) of triplicates within the representative experiments. (D) Western blot analysis of RARβ from cells transfected with 2 μg of B56γ3 expression plasmid. (E) siRNA (100, 150, and 200 nM [lanes 2, 3, and 4, respectively]) specific for the B56 subunits was transfected twice at 20-h intervals. Cells were then harvested and subjected to Western blot analysis for the abundance of p300 and B56γ3 and B56γ2 subunits. A protein band that cross-reacts with the p300 antibody is used for a loading control (Ctl). (F) Cells were first transfected with RAR luciferase reporter (0.3 μg), RSV-β-Gal (0.3 μg), and B56 siRNA (100 and 150 nM), followed by a second transfection of the siRNA alone 24 h later. The cells were then induced with arotinoid acid (AA) (1 μM) for 20 h and subjected to luciferase (Luc) assay. β-Galactosidase activity was used as an internal control. Values are means ± standard deviations (error bars) of three experiments.

FIG. 5.

The B56γ3 regulatory subunit of PP2A associates with p300. (A) Equal amounts of whole-cell extracts were used for immunoprecipitation (IP) with a p300 antibody (lanes 2 and 3) or protein A agarose alone (lane 1) as a negative control. The precipitations were then analyzed sequentially with the antibody specific to the catalytic subunit (C) of PP2A and p300 on Western blots. Portions (10%) of the extracts used in the immunoprecipitation were subjected to Western blot analysis (lanes 4 to 6) as input control. (B) GST pull-down assays were performed with 500 μg of cell lysate from HeLa cells expressing the B56γ3 subunit and the indicated GST-p300 fusion proteins spanning the full length of p300 (p300-1, amino acids 1 to 672; p300-2, amino acids 672 to 1193; p300-3, amino acids 1069 to 1459; p300-4, amino acids 1459 to 1892; p300-5, amino acids 1893 to 2414). The blot was probed sequentially for the B56γ3 (γ3) subunit and GST tag. (C) Cells were transfected with expression plasmid for the Flag-tagged N terminus of p300 (p300N, amino acids 1 to 670; 10 μg) or the C terminus of p300 (p300C, amino acids 1135 to 2414; 10 μg). The truncated forms of p300 were purified with anti-Flag affinity gel and subjected to Western blotting with anti-Flag antibody. The blot was then stripped and reprobed for the B56γ subunits of PP2A. (D) Cells were transfected with plasmids encoding either wild-type p300 (p-WT) (0.2 μg) or p300 in which the C terminus had been deleted (p-ΔC) (2 μg) and allowed to express for 36 h. The cells were then stained with specific anti-Flag antibody and fluorophore-labeled secondary antibody for immunofluorescence microscopy. (E) Cells were transfected with RAR luciferase reporter (0.3 μg), RSV-β-Gal (0.3 μg), B56γ3 (0.3 μg), and the wild-type p300 or p300ΔC (0.2 μg) for luciferase (Luc) assay. Values shown were normalized to β-galactosidase activity and expressed as the fold induction relative to the values for untreated controls (P < 0.001 as measured by repeated analysis of variance tests). Values are means ± standard deviations (error bars) of triplicates within the representative experiments.