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Regulation of Protein Citrullination Through P53/PADI4 Network in DNA Damage Response

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Regulation of Protein Citrullination Through P53/PADI4 Network in DNA Damage Response Published OnlineFirst October 20, 2009; DOI: 10.1158/0008-5472.CAN-09-2280 Molecular Biology, Pathobiology, and Genetics Regulation of Protein Citrullination through p53/PADI4Network in DNA Damage Response Chizu Tanikawa,1 Koji Ueda,3 Hidewaki Nakagawa,3 Nobuaki Yoshida,2 Yusuke Nakamura,1 and Koichi Matsuda1 1Laboratory of Molecular Medicine, Human Genome Center, 2Division of Gene Expression and Regulation, Institute of Medical Science, the University of Tokyo; and 3Laboratory for Biomarker Development, Center for Genomic Medicine, RIKEN, Tokyo, Japan Abstract modulation of PTMs. However, the role of p53 in the regulation of Upon a wide range of cellular stresses, p53 is activated and PTMs has not yet been elucidated. inhibits malignant transformation through the transcription- Citrullination (also referred to as deimination) is one form of PTMs which converts an arginine residue in protein into a citrulline al regulation of its target genes related to apoptosis, cell 2+ cycle arrest, and DNA repair. However, its involvement in residue (5). This reaction is mediated by a Ca -dependent enzyme posttranslational modifications of proteins has not yet been called peptidylarginine deiminase. Citrullination of proteins results well characterized. Here, we report the novel role of p53 in in a loss of positive charge and causes a significant biochemical the regulation of protein citrullination. p53 transactivated change. For example, PADI4 was shown to convert monomethyl- peptidylarginine deiminase type 4 (PADI4) through an intro- arginine residues of histone H3 and H4 to citrullines and negatively nic p53-binding site. The PADI4 gene encodes an enzyme regulate gene expression (6, 7). catalyzing the citrullination of arginine residues in proteins, On the other hand, citrullinated peptides/proteins are recog- – and ectopic expression of p53 or PADI4 induced protein nized as non self-proteins and subsequently activate immune citrullination. In addition, various proteins were citrullinated systems. Citrullinated myelin basic protein in the brain was consid- in response to DNA damage, but knockdown of PADI4 or p53 ered as a putative autoantigen associated with multiple sclerosis remarkably inhibited their citrullination, indicating the (8). Autoantibodies against citrullinated peptides derived from regulation of protein citrullination in a p53/PADI4-dependent filaggrin or fibrin have been established as specific markers for – manner. We found that PADI4 citrullinated the histone chap- rheumatoid arthritis (9 11). We have also revealed a functional erone protein, nucleophosmin (NPM1), at the arginine 197 haplotype of the PADI4 gene associated with rheumatoid arthritis residue in vivo under physiologic conditions. Citrullination (12). Hence, protein citrullination is now considered to play a of NPM1 by PADI4 resulted in its translocation from the pivotal role in various human diseases (13), but the role(s) of nucleoli to the nucleoplasm, whereas PADI4 did not alter citrullination in carcinogenesis, if any, have not yet been reported. the localization of mutant NPM1 (R197K). Furthermore, Among five members belonging to the peptidylarginine deimi- – ectopic expression of PADI4 inhibited tumor cell growth, nase family (14 16), we found PADI3 and PADI4 to be transacti- and concordantly, the knockdown of PADI4 attenuated vated by ectopic expression of p53 in U373MG cells through our p53-mediated growth-inhibitory activity, demonstrating the cDNA microarray analysis (17, 18). Therefore, we investigated the significance of PADI4-mediated protein citrullination in the possible roles of p53 in the regulation of protein citrullination in p53 signaling pathway. [Cancer Res 2009;69(22):8761–9] this study. Introduction Materials and Methods Mutations in the p53 gene are the most common genetic alter- cDNA microarray. cDNA microarray analysis was performed as de- ation observed in human cancers (1). Because ∼90% of them were scribed previously (19). Briefly, U373MG cells were infected with viral solu- detected within its DNA-binding domain (2, 3), the crucial function tions and incubated at 37°C until the time of harvest. Polyadenylated RNA were isolated from U373MG cells using standard protocols. Each RNA sam- of p53 is considered as a sequence-specific transcription factor. ple was labeled and hybridized to a microarray consisting of 36,864 cDNA Recently, extensive analyses were conducted to clarify the role of fragments. The results of the microarray analysis were deposited at the posttranslational modifications (PTM) in diverse cellular signaling Gene Expression Omnibus database (accession no. GSE14953).4 pathways. Proteome analyses using two-dimensional gel electro- Plasmid construction. Plasmids expressing PADI3 and PADI4 were phoresis showed that activated p53 altered the amounts of various kindly provided by Dr. Akari Suzuki (RIKEN, Yokohama, Japan). Catalytical- protein spots without affecting their mRNA levels (4), suggesting ly inactive mutant PADI4 (D350A and D473A) was generated using the that p53 could change the biological properties of proteins through QuickChange site-directed mutagenesis kit (Stratagene). Primers are shown in Supplementary Table S1. Cell culture and transfections. Each cell line was purchased from American Type Culture Collection, Lonza Biologics, Inc., or Japanese Col- − − Note: Supplementary data for this article are available at Cancer Research Online lection of Research Bioresources. HCT116 (p53+/+ and p53 / ) cell lines (http://cancerres.aacrjournals.org/). were gifts from B. Vogelstein (Johns Hopkins University, Baltimore, MD). Requests for reprints: Koichi Matsuda, Laboratory of Molecular Medicine, Cells were transfected with plasmids using FuGENE6 (Roche). Replica- Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato, tion-deficient recombinant viruses Ad-p53 or Ad-LacZ, expressing p53 or Tokyo 1088639, Japan. Phone: 81-35449-5372; Fax: 81-35449-5433; E-mail: koichima@ ims.u-tokyo.ac.jp. ©2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-09-2280 4 http://www.ncbi.nlm.nih.gov/geo/ www.aacrjournals.org 8761 Cancer Res 2009; 69: (22). November 15, 2009 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst October 20, 2009; DOI: 10.1158/0008-5472.CAN-09-2280 Cancer Research Figure 1. Regulation of protein citrullination by p53. A, quantitative PCR analysis of five PADI isozymes mRNA in U373MG cells infected with Ad-p53 or Ad-LacZ at a multiplicity of infection of 8. β2-Microglobulin was used for the normalization of expression levels. Columns, relative expressions of PADI isozymes; bars, SD (n = 2). B, U373MG (p53 mutant) cells were infected with adenovirus expressing p53 or lacZ at a multiplicity of infection of 20. At 36 h after treatment, whole cell extracts were subjected to immunoblotting with anti–modified citrulline (MC), anti-p53, anti-p21WAF1, or anti–β-actin antibody. *, exogenously expressed p53. Arrowheads, citrullinated proteins. C, U-2 OS cells were treated with ADR at the indicated doses (left). At 6 h after transfection of each siR- NA, U-2 OS cells were treated with 2 μg/mL of ADR for 2 h (right). siEGFP was used as a control. At 36 h after ADR treatment, whole cell extracts were subjected to immunoblotting with anti-MC antibody. LacZ, respectively, were generated and purified, as described previously (20, rabbits. The positive antisera were further purified by immune-affinity pu- 21). U373MG cells were infected with viral solutions at various amounts of rification using a NPM1 Cit197 peptide. Polyclonal anti–modified citrulline multiplicity of infection and incubated at 37°C until the time of harvest. For antibody (17–347) was purchased from Upstate and the modification of treatment with genotoxic stress, cells were continuously incubated with citrulline residues was carried out according to the instructions of the sup- Adriamycin (ADR) for 2 h as indicated in the respective figure legends. plier. Anti–β-actin monoclonal antibody (A5441) and anti-Flag monoclonal Small interfering RNA (siRNA) oligonucleotides, commercially synthesized (F3165) antibody were purchased from Sigma. Anti-p53 monoclonal anti- by Sigma Genosis, were transfected with LipofectAMINE 2000 reagent bodies (OP140 and OP43) and anti-p21WAF1 monoclonal antibody (OP64) (Invitrogen) for 4 h. Sequences of oligonucleotides are shown in Supple- were purchased from Calbiochem. Anti-B23 (NPM1) polyclonal (sc-6013) mentary Table S1. For treatment with calcium ionophore, cells were incu- and monoclonal (sc-32256) antibodies, anti-HA monoclonal (sc-7392) and bated with 5 μmol/L of A23187 (Calbiochem) for 1 h at 37°C. polyclonal (sc-805) antibodies, anti-Myc monoclonal antibody (sc-40), anti- Quantitative real-time PCR. Quantitative real-time PCR was con- HSP90 monoclonal antibody (sc-13119), anti-PARP1 monoclonal antibody ducted with the SYBR Green IMaster on a LightCycler 480 (Roche). Primer (sc-8007), and anti–glutathione S-transferase (GST) monoclonal antibody sequences are indicated in Supplementary Table S1. (sc-138) were purchased from Santa Cruz Biotechnology. Anti-PADI4 poly- Chromatin immunoprecipitation assay. Chromatin immunoprecipita- clonal antibody (ab50332) and anti-nucleolin polyclonal antibody (ab22758) tion (ChIP) assay was performed using ChIP Assay kit (Upstate Biotechnol- were purchased from Abcam. ogy) as described previously (18). PCR amplifications of PADI4 intron 1, In vitro
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