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Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for P53-Mediated Cellular Functions

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Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for P53-Mediated Cellular Functions Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Lee, Jihye, Guillaume Adelmant, Jarrod A. Marto, and Dong-Hyun Lee. 2015. “Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions.” Molecules and Cells 38 (8): 697-704. doi:10.14348/molcells.2015.0066. http:// dx.doi.org/10.14348/molcells.2015.0066. Published Version doi:10.14348/molcells.2015.0066 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:22857022 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Mol. Cells 2015; 38(8): 697-704 http://dx.doi.org/10.14348/molcells.2015.0066 Molecules and Cells http://molcells.org Established in 1990 Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions Jihye Lee, Guillaume Adelmant1, Jarrod A. Marto1, and Dong-Hyun Lee* Deleted in breast cancer-1 (DBC1) contributes to the regu- ATM/ATR kinases (Zannini et al., 2012) and it plays a critical lation of cell survival and apoptosis. Recent studies role in maintaining genomic stability and cellular integrity follow- demonstrated that DBC is phosphorylated at Thr454 by ing genotoxic stress (Kim et al., 2008; 2009a; Magni et al., 2014; ATM/ATR kinases in response to DNA damage, which is a Park et al., 2014; Zannini et al., 2012; Zheng et al., 2013). Upon critical event for p53 activation and apoptosis. However, DNA damage conditions, DBC1 on Thr454 is phosphorylated, how DBC1 phosphorylation is regulated has not been which promotes acetylation-mediated p53 activation through studied. Here we show that protein phosphatase 4 (PP4) inducing the interception of NAD-dependent deacetylase dephosphorylates DBC1, regulating its role in DNA dam- sirtuin-1 (SIRT1) from p53 and triggers apoptosis. When the age response. PP4R2, a regulatory subunit of PP4, medi- phosphonull DBC1T454A mutant (T454A) is present in cells, ates the interaction between DBC1 and PP4C, a catalytic stress-induced apoptosis is significantly reduced, compared to subunit. PP4C efficiently dephosphorylates pThr454 on cells expressing DBC1 WT (Park et al., 2014; Zannini et al., DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells 2012; Zheng et al., 2013). Park et al. (2014) showed that the alters the kinetics of DBC1 phosphorylation and p53 acti- expression of the phosphomimetic DBC1 T454E mutant vation, and increases apoptosis in response to DNA dam- (T454E) increased the ability of the interaction with E3 SUMO- age, which are compatible with the expression of the protein ligase PIAS4, which is an indispensable event for DBC1 phosphomimetic DBC-1 mutant (T454E). These suggest sumoylation and p53-mediated apoptosis. In contrast, the that the PP4-mediated dephosphorylation of DBC1 is nec- T454A mutant significantly decreased its interaction with PIAS4. essary for efficient damage responses in cells. Therefore, the phosphorylation of DBC1 following genotoxic stress in cells is a crucial step and must be tightly regulated to maintain cellular integrity. INTRODUCTION Recently, we and others have identified the role of the protein 1 phosphatase 4 (PP4) in DDR (Chowdhury et al., 2008; Lee and Genotoxic stress inducing DNA breaks and replication stress Lee, 2014; Lee et al., 2010; 2012; 2014; Nakada et al., 2008; stimulates genomic instability and cellular transformation. To Wang et al., 2008). PP4 dephosphorylated the essential proteins, prevent these detrimental consequences, eukaryotic cells have including replication protein A 2 (RPA2), KAP-1, and 53BP1 after evolved an elaborate and complex response system called DNA damage and these dephosphorylation events were critical DNA damage response (DDR), which is mostly initiated by the for the efficient repair of DNA double-strand breaks (DSBs) phosphatidylinositol 3-kinase (PI3)-like family of kinases, includ- (Lee and Lee, 2014; Lee et al., 2010; 2012; 2014). To study the ing DNA-dependent protein kinase (DNA-PK) catalytic subunit, functions of PP4 in depth, we performed tandem affinity purifi- ataxia telangiectasia mutated (ATM), and ataxia telangiectasia- cation followed by mass spectrometry. We identified DBC1, and Rad3-related (ATR) (Ciccia and Elledge, 2010; Lee and which was hyperphosphorylated in the absence of PP4C follow- Chowdhury, 2011; Matsuoka et al., 2007; Mu et al., 2007; ing DNA damage. Here we elucidated the importance of PP4C- Smith et al., 2010). Recently, DBC1 (also named p30 DBC, mediated dephosphorylation of DBC1 and focused on the func- KIAA1967, or CCAR2) was identified as a new target of tional impact of DBC1 dephosphorylation on human cells. MATERIALS AND METHODS Department of Biological Sciences, College of Science, Chonnam Na- 1 tional University, Gwangju 500-757, Korea, Department of Biological Cell culture, antibodies, and reagents Chemistry Molecular Pharmacology, Harvard Medical School, Depart- HeLa S3, U2OS, and RPE1 cells were grown in DMEM sup- ment of Cancer Biology and Blais Proteomics Center, Dana-Farber plemented with 10% (v/v) FBS. In addition to U2OS, RPE1 Cancer Institute, Boston, MA 02115, USA *Correspondence: [email protected] cells contain an intact p53 checkpoint and were therefore used for study on p53. Antibodies used were against PP4R1 (Bethyl), Received 13 March, 2015; revised 30 May, 2015; accepted 8 June, PP4R2 (Bethyl), PP4R3α (Bethyl), PP4R3β (Bethyl), PP4C 2015; published online 21 July, 2015 (Bethyl), DBC1 (Bethyl), pT454-DBC1 (Cell Signaling), p53 (Santa Cruz), p53-Ac-K382 (Cell Signaling), SIRT1 (Sigma), Keywords: deleted in breast cancer-1, depho-sphorylation, DNA dam- Flag-tag (Sigma) and Actin (Millipore). Etoposide was obtained age response, protein phosphatase 4 from Sigma-Aldrich. ATM inhibitor (KU59403) was obtained eISSN: 0219-1032 The Korean Society for Molecular and Cellular Biology. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. PP4-Mediated Dephosphorylation of DBC1 Jihye Lee et al. from KuDOS Pharmaceuticals. (Roche Applied Science, Germany), according to the manufac- turer’s instructions. Briefly, U2OS cells were fixed for 10 min siRNAs and plasmids with fixative (4% formaldehyde) and permeablized for 5 min Cells were transfected with siRNA duplexes (Invitrogen) using with 0.2% Triton X-100. Cells were rinsed with PBS and incu- RNAiMAX (Invitrogen). The PP4C siRNAs were as follows: bated with TUNEL reaction mixture at 37°C for 1 h. Finally, cells siRNA #1, sense: 5′-CGCUAAGGCCAGAGAGA UCUUGGUA- were analyzed under Zeiss Axioplan microscope at 450-500 3′, antisense: 5′-UACCAAGAUCUCUCUG GCCUUAGCG-3; nm. Apoptotic index (AI) was determined as the percentage of siRNA #2, sense: 5′-GGACAAUCGACCGAAAGCAAGAGGU- TUNEL positive cells in 10 randomly selected fields (~200 cell 3′, antisense: 5′-ACCUCUUGC UUUCGGUCGAUUGUCC-3′. counts). The PP4R2 siRNAs were as follows: siRNA #1, sense: 5′- CCAAGCUAUACUGAGAGGUCUAAUA-3′; antisense: 5′-CC- Clonogenic assay AGGCCACUUAAUC GACCAAAGGU-3′. DBC1 phophomu- U2OS (0.3 × 106 cells/well) cells were transfected with siRNAs tants were constructed by QuikChange II XL site-directed mu- against PP4C or PP4R2, or FH-DBC1 WT or phosphomutants. tagenesis kit (Stratagene) according to the manufacturer’s in- After 2 days, 1000 cells were seeded on 6-well plates in quadru- structions. Primers used were the following: T454E-F, 5′-GA- plicate and incubated overnight. Cell were irradiated at indicated GGCAGCTCCCCCA GAGCAGGAGGCACAAGGG-3′; T454E- doses on the following day and allowed to form colonies for 2 R, 5′-CCCTTGTGCCTCCTGCTCTGGGG GAGCTGCCTC-3′; weeks before being stained by 0.1% crystal violet solution for T454A-F: 5′-AGAGGCAGCTCCCCCAGCCCAGGAGG-3′; evaluation. Surviving colonies of > 1 mm diameter were counted. T454A-R: 5′-CCTCCTGGGCTGGGGGAGCTGCCTC T-3′. Mass spectrometry analysis of affinity-purified PP4 Co-immunoprecipitation complexes HeLa S3 or U2OS cells expressing FH-DBC1 WT or pho- Sample preparation, liquid chromatography/tandem mass sphomutants, were lysed in buffer containing 50 mM Tris-HCl spectrometry analysis, database searches and identification of (pH 7.5), 250 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40 and proteins associated with PP4C were performed with slight mod- protease inhibitor cocktail (Roche). Anti-Flag-agarose (Sigma) ifications according to a recently described method (Wang et al., was incubated with lysate at 4°C for 16 h. Immunocomplexes 2013). Briefly, purified protein complexes were denatured and were washed three times with buffer containing 50 mM Tris-HCl reduced by incubation at 56°C for 30 min in 10 mM DTT and (pH 7.5), 250 mM NaCl, 5 mM EDTA and 0.5% (v/v) NP-40. 0.1% RapiGest (Waters). Protein digestion was carried out The immunoprecipitated proteins were resolved by SDS-PAGE overnight at 37°C after adding 500 ng of trypsin and adjusting and analyzed by immunoblot. the pH to 8.0. RapiGest was removed from solution following the manufacturer’s protocol and tryptic peptides were purified Immunofluorescence by batch-mode reverse-phase C18 chromatography (Poros Cells plated on glass slides were fixed for 10 min with fixative 10R2, Applied Biosystems) using 40 μl of a 50% bead slurry in (3%(w/v) PFA, 2%(w/v) sucrose and 1 X PBS) and per- RP buffer A (0.1% trifluoroacetic acid), washed with 100 μl of meabilized for 1 min with 0.2% (v/v) Triton X-100 in PBS. Cells the same buffer and eluted with 50 μl of RP buffer B (40% ace- were rinsed with PBS and incubated with primary antibody tonitrile in 0.1% trifluoroacetic acid). After vacuum concentration, diluted in PBS with 2% (w/v) FBS for 1 h at room temperature peptides were solubilized in 20 μl of 20 mM sodium phosphate (RT).
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