INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 34: 1713-1719, 2014 Oxidized S100A4 inhibits the activation of protein phosphatase 5 through S100A1 in MKN‑45 gastric carcinoma cells MITSUMASA TSUCHIYA1*, FUMINORI YAMAGUCHI2*, SEIKO SHIMAMOTO1, TOMOHITO FUJIMOTO1, HIROSHI TOKUMITSU1, MASAAKI TOKUDA2 and RYOJI KOBAYASHI1 Departments of 1Signal Transduction Sciences and 2Cell Physiology, Kagawa University Faculty of Medicine, Kagawa 761‑0793, Japan Received March 8, 2014; Accepted September 23, 2014 DOI: 10.3892/ijmm.2014.1947 Abstract. S100 proteins bind to numerous target proteins, as Introduction well as other S100 proteins and activate signaling cascades. S100 proteins can be modified by various post‑translational S100A4 protein is a member of the EF-hand calcium modifications, such as phosphorylation, methylation and acety- ion‑binding protein family, of which >25 members have lation. In addition, oxidation is important for modulating their been found in humans (1). They are 25‑65% homologous at activities. Previous studies have shown that S100A1 interacts the amino acid level, while the sequence of the linker (hinge) with S100A4 in vitro and in vivo. Due to this potential cross-talk region and the C-terminal extension are the most variable among the S100 proteins, the aim of the present study was to among the S100 proteins. Each S100 monomer contains two examine whether S100A4 modulates the activity of S100A1. EF-hand Ca2+-binding sites. Ca2+-binding causes a conforma- S100A4 was readily oxidized and formed disulfide‑linked tional change and exposure of a hydrophobic surface allowing dimers and oligomers. Although non‑oxidized S100A4 bound interaction with target proteins (2). They do not possess to protein phosphatase 5 (PP5), the Cu‑oxidized S100A4 failed enzymatic activity, but rather regulate the activity of target to bind PP5. Instead, the Cu‑oxidized S100A4 directly inter- proteins. Several proteins have been identified as S100A4 acted with S100A1 and prevented PP5 activation. Hydrogen targets, including liprin β1 (3), methionine aminopeptidase (4), peroxide induced S100A4 oxidation in MKN-45 gastric the p53 tumor suppressor protein (5) and the heavy chain of adenocarcinoma cells and decreased S100A1-PP5 interaction, non‑muscle myosin II (6). In addition, S100A4 is capable of resulted in the inhibition of PP5 activation by S100A1. These forming heterodimers with S100A1 (7,8), which is likely to data indicate that oxidized S100A4 regulates PP5 activity in a increase its functional potential. Previous studies have shown unique manner under oxidative stress conditions. that S100A4 interacts with S100A1 via heterodimer formation and that S100A1 can antagonize function of S100A4 (9,10). It has been a general observation that two‑hybrid screenings utilizing S100 proteins have primarily detected other S100 family members as targets (11‑13). Correspondence to: Professor Ryoji Kobayashi, Department We have previously demonstrated that S100A1, A2, A6 of Signal Transduction Sciences, Kagawa University Faculty of and S100B interact with the tetratricopeptide repeat (TPR) Medicine, 1750‑1 Ikenobe, Kagawa 761‑0793, Japan 2+ E‑mail: [email protected]‑u.ac.jp domain of PP5 in a Ca ‑dependent manner and significantly activate its phosphatase activity (14). PP5 is a member of the *Contributed equally phosphoprotein phosphatase (PPP) family of a serine/threo- nine phosphatase (15). PP5 contains a C‑terminal catalytic Abbreviations: ASK1, apoptosis stimulating kinase 1; CaM, domain and N-terminal three TPR motifs that are unique in calmodulin; CBB, Coomassie brilliant blue; DTT, dithiothreitol; the PPP family (16,17). The TPR motif consists of 34 amino H2O2, hydrogen peroxide; HMW, higher molecular weight; His, acid sequences, and between 3 and 16 copies of the motif are 6xHistidine; HRP, horseradish peroxidase; NF‑κB, nuclear factor-κB; arranged in the proteins as tandem arrays (18,19). This motif PBS, phosphate-buffered saline; PP5, protein phosphatase 5; PPP, can act as an interaction scaffold for protein complex forma- phosphoprotein phosphatase; ROS, reactive oxygen species; SPR, tion. PP5 is a negative regulator of the apoptosis stimulating surface plasmon resonance; TNF-α, tumor necrosis factor α; TPR, tetratricopeptide repeat; Tricine, N‑[2‑hydroxy‑1,1‑bis (hydroxymethyl) kinase 1 (ASK1) and modulates the apoptosis under oxidative ethyl] glycine; TTBS, Tris‑buffered saline with 0.05% Tween‑20 stress conditions (20). Gastric epithelium is constantly exposed to reactive oxygen species (ROS) and activation of ASK1 by Key words: oxidative stress, S100 protein, protein phosphatase 5, Helicobacter pylori under oxidative stress was reported previ- MKN-45 ously (21). The purpose of the present study was to investigate whether S100A4 affects S100A1 function (i.e., PP5 activation) under oxidative conditions. The oxidized form of S100A4, but not 1714 TSUCHIYA et al: INHIBITION OF S100A1-PP5 INTERACTION AND PP5 ACTIVATION BY OXIDIZED S100A4 the native S100A4 dimer, was found to inhibit the activation of treated with or without 2 mM H2O2 and 0.5 µM ionomycin for PP5 by S100A1 in vitro and in MKN-45 cells. 90 min. Cells were washed once with PBS and lysed in a buffer consisting of 50 mM Tris‑HCl (pH 7.5), 150 mM NaCl, 0.5% Materials and methods Triton X‑100, and 0.5% Nonidet P‑40 with protease inhibitor mixture (Roche). The samples were sonicated and centrifuged Materials. Nickel-nitrilotriacetic acid-agarose was purchased at 16,600 x g for 10 min. Supernatants were incubated with from Qiagen (Hilden, Germany). Rabbit anti-S100A1 anti- 30 µl of anti‑FLAG antibody‑agarose in the presence of body (NB100‑91955) was obtained from Novus Biologicals 1 mM CaCl2 for 2 h at room temperature. Following extensive (Littleton, CO, USA). Sheep anti‑S100A4 antibody (AF4138) washing, the beads were used either for the western blotting or was obtained from R&D Systems (Minneapolis, MN, USA). in vitro phosphatase assay. Mouse anti-FLAG-horseradish peroxidase (HRP) antibody (A8592) and other chemicals were purchased from Sigma Surface plasmon resonance (SPR). Protein binding (St. Louis, MO, USA). Goat-anti-rabbit IgG- HRP-linked anti- analysis was performed using an SPR Biacore 2000 body (7074) was obtained from Cell Signaling (Beverly, MA, system (GE Healthcare Institute, Waukesha, WI, USA). USA) and Donkey‑anti‑sheep IgG‑HRP‑conjugated antibody N‑ethyl‑N'‑(3‑diethylaminopropyl) carbodiimide, (HAF016) was purchased from R&D Systems. N‑hydroxysuccinimide and ethanolamine‑HCl (GE Healthcare) were used for amine coupling of PP5 or S100A1 Plasmids and recombinant proteins. Human PP5 [GenBank: to the dextran surface of the CM5 chip. His-PP5 [2500 RU NM_006247] was cloned to pET16a and pME18S‑FLAG (pH 4.2)] or S100A1 [1400 RU (pH 4.5)] were immobilized vector, and rat S100A1 [Genbank: NM_001007636] and rat in 10-mM ammonium acetate. For all the procedures, HBS-P S100A4 [Genbank: NM_012618] were cloned to pET11a buffer [20 mM HEPES (pH 7.4), 150 mM NaCl and 0.005% plasmids as previously reported (14). Histidine‑tagged PP5 Tween‑20] with 1 mM CaCl2 were used at a flow rate of (His‑PP5) protein was expressed and purified according to the 20 µl/min. Various concentrations of recombinant S100 manufacturer's instructions. S100A1 and S100A4 proteins were proteins were injected. The ligand-coupled sensor chips were expressed and purified as previously described (22,23). Purified regenerated between protein injections with a brief (60 sec) recombinant S100A4 proteins were diluted (0.5 mg/ml) and wash with HBS‑P buffer containing 2.5 mM ethyleneglycol‑ maintained at -30˚C in a freezer for 3 months (air‑oxidized). bis‑(2‑aminoethylether)‑tetra acetic acid (EGTA) and 0.75% Cu‑oxidized S100A4 protein was prepared in accordance with n‑octyl‑β‑D‑glucopyranoside. Response curves were prepared a previous study (24). Briefly, S100A4 (10 µM) was incubated for fitting by subtraction of the signal generated simultane- with 80 µM of CuCl2 in 20 mM Tris‑HCl buffer (pH 7.6) for 2 h ously on the control flow cell. Biacore sensorgrams were at 37˚C. Subsequent to stopping the reaction with the copper analyzed using BIAevaluation 4.1 software (GE Healthcare). chelator diethylenetriamine‑N,N,N',N',N'-pentaacetic acid, the product solution was desalted using centriprep-3 (Amicon, Inc., Native PAGE, Tricine SDS‑PAGE and western blotting. Native Beverly, MA, USA), dialyzed against 20 mM Tris‑HCl buffer PAGE analysis was performed based on a previous study (25). (pH 7.6). For Tricine SDS‑PAGE analysis, samples were treated Tricine SDS-PAGE gel electrophoresis was performed under with (reducing conditions) or without (non-reducing conditions) reducing and non-reducing conditions. The gels were either 10 mM dithiothreitol (DTT) prior to electrophoresis. stained with Coomassie Brilliant Blue (CBB) or used for western blotting. For western blot analysis, proteins were blotted Cell culture and H2O2 treatment. MKN-45 cells were purchased onto nitrocellulose membranes and blocked with 5% skimmed from the Japanese Collection of Research Bioresources (Osaka, milk in Tris‑buffered saline with 0.05% Tween‑20 (TTBS). The Japan) and maintained in RPMI‑1640 (Sigma) supplemented membranes were incubated with an anti‑S100 antibody in the with 10% fetal bovine serum and 1% penicillin and strepto- same buffer overnight. Membranes were washed with TTBS mycin in a humidified 5% CO2 incubator. For the oxidative and incubated with an HRP‑labeled second antibody for 2 h. To stress experiment, 5.5x105 cells were plated on a 10-cm dish detect the FLAG‑tagged PP5, the anti‑FLAG‑HRP antibody and cultured for 3 days in standard medium. Medium was was used. The samples were washed and signals were detected aspirated and washed with phosphate-buffered saline (PBS) using Immobilon Western Chemiluminescent HRP Substrate and cells were exposed to the indicated concentrations of H2O2 (Millipore, Billerica, MA, USA). ranging from 0 to 2 mM in the serum-free medium for 90 min. Following exposure to oxidative stress, these cells were washed In vitro phosphatase assay.