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. 2015 Aug;38(8):697-704.
doi: 10.14348/molcells.2015.0066. Epub 2015 Jul 21.

Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions

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Free PMC article

Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions

Jihye Lee et al. Mol Cells. .
Free PMC article

Abstract

Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the interaction between DBC1 and PP4C, a catalytic subunit. PP4C efficiently dephosphorylates pThr454 on DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells alters the kinetics of DBC1 phosphorylation and p53 activation, and increases apoptosis in response to DNA damage, which are compatible with the expression of the phosphomimetic DBC-1 mutant (T454E). These suggest that the PP4-mediated dephosphorylation of DBC1 is necessary for efficient damage responses in cells.

Keywords: DNA damage response; deleted in breast cancer-1; dephosphorylation; protein phosphatase 4.

Figures

Fig. 1.
Fig. 1.
DBC1 is a substrate of PP4C/PP4R2. (A) Silver staining of a PP4C- or PP4R2-containing complex isolated by Flag-HA tandem affinity purification from HeLa S3 cells expressing Flag-HA-PP4C, PP4R2 or empty construct. (B) PP4C/PP4R2 interacts with DBC1. HeLa S3 cells were subjected to reciprocal immuneprecipitation using antibodies against PP4C, PP4R2, PP4R3β, PP4R3α, PP4R1, DBC1, or IgG. Protein level was assessed by Western blot. (C) Interaction of DBC1 with PP4 complex is PP4R2-mediated. U2OS cells stably expressing empty vector (FH-Empty) or FH-tagged DBC1, or untransfected U2OS cells were subjected to immunoprecipitation using anti-FLAG beads or antibody against DBC1 after IR. PP4R2 or control siRNAs were transfected in cells 72 h prior to immunoprecipitation. The immunoprecipitate was probed with antibodies against PP4C and PP4R2 as indicated. (D) Interaction between DBC1 and PP4 is independent of DNA damage. HeLa cells transiently expressing empty vector (FH-Empty) or FH-tagged DBC1, or untransfected Hela cells were subjected to immunoprecipitation using anti-FLAG beads or antibody against DBC1 at indicated time points before or after IR treatment. Un, Untreated; IR, ionizing radiation.
Fig. 2.
Fig. 2.
DBC1 phosphorylation is regulated by PP4C/PP4R2 complex. (A) Depletion of PP4C or PP4R2 induces hyperphosphorylation of DBC1 on T454. U2OS cells transfected with siRNAs against PP4C or PP4R2 were irradiated and harvested at the indicated times and phospho-DBC1 was assessed by immunoblot using phospho-DBC1 antibody (p-T454). The kinetics of pT454-DBC1 formation was monitored after irradiation. (B) PP4C or PP4R2 depletion attenuates pT454-DBC1 turnover after IR. U2OS cells were transfected with control, PP4R2, or PP4C siRNAs. After 72 h, cells were irradiated, fixed at the indicated times, and immunostained for pT454-DBC1 (green), γH2AX (red) and DAPI (blue). γH2AX served as control showing the DNA damage (DSBs) in cells. Cells expressing PP4C D82A mutant or without transfection were used as control. (C) The average pT454-DBC1 signal intensity per nucleus was quantified using ImageJ software. Data are expressed as mean ± SD; n = 3. *P = 0.0015 (PP4C siRNA), **P = 0.0034 (PP4R2 siRNA), ***P = 0.0067 (PP4C D82A).
Fig. 3.
Fig. 3.
PP4 has an impact on p53 activity through the dephosphorylation of DBC1. (A) Depletion of PP4C or PP4R2 enhances p53 activity, compatible with DBC1 T454E expression. RPE1 cells transfected with siRNAs against PP4C or PP4R2 were irradiated and harvested at the indicated times (Left panel) and U2OS cells expressing Flag-DBC1 WT, T454E, or T454A were irradiated and harvested at the indicated times (Right panel). Ac-p53 was assessed by immunoblot using acetylated p53 antibody (Ac-K382). (B) Depletion of PP4R2 enhances the interaction between DBC1 and SIRT1. RPE1 cells expressing empty vector (FH-Empty), FH-tagged DBC1 were subjected to immunoprecipitation using anti-FLAG beads after IR. PP4R2 siRNAs or control siRNAs were transfected in cells 72 h prior to immunoprecipitation. The immunoprecipitate was probed with antibodies against SIRT1 and PP4R2 as indicated. (C) In vitro dephosphorylation assay. PP4C WT, PP4C D82A, and PP4R2 were purified using the baculoviral system and were serially diluted in the phosphatase reaction. λ phosphatase served as positive control for the reaction. PP4C dephosphorylates phospho-DBC1 on T454 in a dose-dependent manner. Phosphatase reactions were probed with indicated antibodies.
Fig. 4.
Fig. 4.
Functional impact of PP4-mediated dephosphorylation of pT454 DBC1 (A) Hyperphosphorylation of DBC1 induced by PP4C/PP4R2 depletion increases cellular apoptosis. U2OS cells were transfected with siRNAs against PP4C or PP4R2 (Left panel), or expressed with DBC1 WT, T454A, or T454E (Right panel). The 48 h after etoposide treatment, apoptotic cells were analyzed under fluorescence microscope by counting > 200 cells. Data represent average and s. d. of three independent experiments. Immunoblots performed to confirm siRNA efficiency and expression of FH-DBC1 constructs are shown. Data are expressed as mean ± SD; n = 3. *P = 0.0021, **P = 0.0061, #P = 0.0083, ##P = 0.018, §P = 0.028, §§P = 0.017. (B) Radiosensitivity of DBC1 phosphomutants. U2OS cells were transfected with siRNAs against PP4C or PP4R2 (Left panel), or expressed with DBC1 WT, T454A, or T454E (Right panel). At 72 h after transfection, cells were irradiated at the indicated doses, and viability was evaluated by clonogenic survival. Immunoblots performed to confirm siRNA efficiency and expression of FHDBC1constructs are shown. Data are expressed as mean ± SD; n = 3.*P = 0.014 (3 Gy), *P = 0.0027 (6 Gy), **P = 0.0057 (3 Gy), **P = 0.0035 (6 Gy), #P = 0.019 (3 Gy), #P = 0.0082 (6 Gy), ##P = 0.039 (3 Gy), ##P = 0.029 (6 Gy).

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