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. 2013 Apr;25(4):796-804.
doi: 10.1016/j.cellsig.2012.12.014. Epub 2012 Dec 28.

Protein phosphatase 1 inhibits p53 signaling by dephosphorylating and stabilizing Mdmx

Zengxin Lu  1 , Guohui Wan, Huarong Guo, Xinna Zhang, Xiongbin Lu
Affiliations

Protein phosphatase 1 inhibits p53 signaling by dephosphorylating and stabilizing Mdmx

Zengxin Lu et al. Cell Signal. 2013 Apr.

Abstract

The activation and stabilization of the p53 protein play a major role in the DNA damage response. Protein levels of p53 are tightly controlled by transcriptional regulation and a number of positive and negative posttranslational modifiers, including kinases, phosphatases, E3 ubiquitin ligases, deubiquitinases, acetylases and deacetylases. One of the primary p53 regulators is Mdmx. Despite its RING domain and structural similarity with Mdm2, Mdmx does not have an intrinsic ligase activity, but inhibits the transcriptional activity of p53. Previous studies reported that Mdmx is phosphorylated and destabilized in response to DNA damage stress. Three phosphorylation sites identified are Ser342, Ser367, and Ser403. In the present study, we identify protein phosphatase 1 (PP1) as a negative regulator in the p53 signaling pathway. PP1 directly interacts with Mdmx and specifically dephosphorylates Mdmx at Ser367. The dephosphorylation of Mdmx increases its stability and thereby inhibits p53 activity. Our results suggest that PP1 is a crucial component in the ATM-Chk2-p53 signaling pathway.

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Figures

Figure 1
Figure 1
PP1 inhibits the p53 signaling pathway. (A) The effects of overexpressed Ser/Thr protein phosphatases on the transcriptional activity of p53. U2OS cells were transfected with empty vector or phosphatase expression vector. 24 hr after transfection, cells were treated by NCS (500 ng/ml) and then harvested 12 hr after treatment. The relative luciferase activity was measured and normalized to the Renilla activity. Error bars indicate ± standard deviation in triplicate experiments. (B) PP1 inhibits the activity of p53. U2OS cells were transfected with p53RE-Luciferase or Mdm2-Luciferase vector, and expression vector encoding PP1 catalytic subunits or PP1 shRNAs. Cell lysates were harvested 12 h after NCS (500 ng/ml) treatment. Relative luciferase activity was determined as above. (C) Inhibition of p53 by PP1α is independent of Ser15 phosphorylation. p53 S18/S18 and p53 A18/A18 MEFs were transfected with control vector or vector expressing PP1α or PP1 shRNAs. 24 h after transfection, the cells were treated with NCS (500 ng/ml) and harvested 12h after treatment. Relative luciferase activity was determined as above.
Figure 2
Figure 2
PP1 inhibits the transcriptional activity of p53 by stabilizing Mdmx. (A) PP1 stabilizes Mdmx in the DNA damage response. U2OS cells stably overexpressing PP1α, PP1β, PP1γ or common PP1 shRNAs were treated with NCS (500 ng/ml). Protein levels were detected by immunoblotting at the indicated time points. (B) PP1 upregulates the half-life of Mdmx. U2OS cells stably overexpressing PP1α, PP1β, or PP1γ were treated with NCS (500 ng/ml) and cyclohexamide (CHX, 50 µg/ml) to inhibit nascent protein synthesis. Protein levels of Mdmx, PP1 and β-actin were measured by immunoblotting. (C) Levels of Mdmx at each time point in B were quantitated and half-life of Mdmx was calculated (from two separate experiments).
Figure 3
Figure 3
PP1 interacts with and dephosphorylates Mdmx at Ser367. (A) Three PP1isoforms interact with Mdmx. U2OS cells were transfected with vector expressing HA-Mdmx and FLAG-tagged PP1α, PP1β or PP1γ. Immunoprecipitates suing control or anti-FLAG antibody were analyzed by immunoblotting using anti-HA antibody (left panel). A reciprocal experiment using HA-PP1-containing immunoprecipitates confirmed the PP1-Mdmx interaction (right panel). WB: Western blot; WCL: whole cell lysate. (B) PP1α directly dephosphorylates Mdmx at Ser367, but not at Ser403 and Ser342. Phosphopeptides from Chk1 (pS345, positive control) and Mdmx containing phosphorylated Ser342, Ser367 or Ser403 were incubated with immunopurified PP1α enzymes in in vitro phosphatase assays. Reactions were also performed in the absence of manganese or peptide, or in the presence of okadaic acid. (C) All of three PP1 isoforms have similar phosphatase activity on Mdmx. Control peptides (negative control and positive Chk1 pS345 control) and Mdmx phosphopeptides were incubated with immunopurified PP1α, PP1β or PP1γ in the in vitro phosphatase assays. Phosphatase activity was calculated according to a phosphate standard curve.
Figure 4
Figure 4
PP1 directly interacts with Mdmx in vitro. (A) Schematic illustration for wildtype and mutant Mdmx. (B) Wildtype and mutant Mdmx with GST tag were expressed and purified from bacterial cell extracts. (C) In vitro Mdmx-PP1 interaction assays were performed by mixing PP1α-bound beads with wildtype or mutant Mdmx. The Mdmx proteins that bound PP1 were separated by SDS-PAGE gel and shown with Coomassie Blue staining.
Figure 5
Figure 5
PP1 dephosphorylates Mdmx at Ser367 in vivo. U2OS cells stably overexpressing PP1α, PP1β, PP1γ or common PP1 shRNAs were treated with or without NCS (500 ng/ml). The cells were harvested 4 h after treatment and cell lysates were subject to immunoblotting analyses using antibodies as indicated.
Figure 6
Figure 6
PP1 stabilizes wildtype Mdmx, but not the S367A mutant Mdmx. (A) PP1 inhibits destabilization of wildtype Mdmx, but not the S367A mutant. U2OS cells stably expressing FLAG-tagged wildtype or S367A mutant Mdmx were transfected with control or PP1α expression vector. 24 h after transfection, cells were treated with NCS (500 ng/ml) and harvested at 0 and 2 h after treatment. Protein levels were detected by immunoblotting using antibodies as indicated. (B) Altered PP1 levels have no impact on the half-life of the S367A mutant Mdmx. U2OS cells stably overexpressing wildtype or S367A mutant Mdmx were transfected with control vector or vector expressing PP1α or PP1 shRNAs. 24 h after transfection, cells were treated with NCS (500 ng/ml) and cyclohexamide (CHX, 50 µg/ml) to inhibit nascent protein synthesis. Protein levels of Mdmx were measured by immunoblotting and quantitated as in Fig. 2C. Half-lives of Mdmx were calculated from two separate experiments. (C) PP1 inhibits the ubiquitination of Mdmx. U2OS cells stably expressing HA-Mdmx and HA-Mdmx (S367A) were transfected with PP1α, PP1 shRNA, or control expression vector. Transfected cells were treated with NCS (200 ng/ml) and protease inhibitors MG132 and MG101, and harvested 4 h after NCS treatment. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted by anti-ubiquitin antibody. (D) Overexpression of PP1α inhibits Mdm2-Mdmx interaction in the DNA damage response. 293 HEK cells were transfected with HA-Mdmx and PP1α or control expression vector, treated with NCS (200 ng/ml), and harvested 2 h after treatment. Cell lysates were immunoprecipitated with anti-Mdm2 antibody. Mdmx in the immunoprecipitates and various proteins in whole cell lysate (WCL) were assessed by immunoblotting as indicated.
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