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• W2097795264 abstract "Epigallocatechin gallate (EGCG) is the major active polyphenol in green tea. Protein interaction with EGCG is a critical step in the effects of EGCG on the regulation of various key proteins involved in signal transduction. We have identified a novel molecular target of EGCG using affinity chromatography, two-dimensional electrophoresis, and mass spectrometry for protein identification. Spots of interest were identified as the intermediate filament, vimentin. The identification was confirmed by Western blot analysis using an anti-vimentin antibody. Experiments using a pull-down assay with [3H]EGCG demonstrate binding of EGCG to vimentin with a Kd of 3.3 nm. EGCG inhibited phosphorylation of vimentin at serines 50 and 55 and phosphorylation of vimentin by cyclin-dependent kinase 2 and cAMP-dependent protein kinase. EGCG specifically inhibits cell proliferation by binding to vimentin. Because vimentin is important for maintaining cellular functions and is essential in maintaining the structure and mechanical integration of the cellular space, the inhibitory effect of EGCG on vimentin may further explain its anti-tumor-promoting effect. Epigallocatechin gallate (EGCG) is the major active polyphenol in green tea. Protein interaction with EGCG is a critical step in the effects of EGCG on the regulation of various key proteins involved in signal transduction. We have identified a novel molecular target of EGCG using affinity chromatography, two-dimensional electrophoresis, and mass spectrometry for protein identification. Spots of interest were identified as the intermediate filament, vimentin. The identification was confirmed by Western blot analysis using an anti-vimentin antibody. Experiments using a pull-down assay with [3H]EGCG demonstrate binding of EGCG to vimentin with a Kd of 3.3 nm. EGCG inhibited phosphorylation of vimentin at serines 50 and 55 and phosphorylation of vimentin by cyclin-dependent kinase 2 and cAMP-dependent protein kinase. EGCG specifically inhibits cell proliferation by binding to vimentin. Because vimentin is important for maintaining cellular functions and is essential in maintaining the structure and mechanical integration of the cellular space, the inhibitory effect of EGCG on vimentin may further explain its anti-tumor-promoting effect. A number of epidemiological studies have shown that the consumption of green tea may protect against many cancer types, including lung, prostate, and breast (1Kelloff G.J. Crowell J.A. Steele V.E. Lubet R.A. Malone W.A. Boone C.W. Kopelovich L. Hawk E.T. Lieberman R. Lawrence J.A. Ali I. Viner J.L. Sigman C.C. J. Nutr. 2000; 130: 467S-471SCrossref PubMed Google Scholar, 2Ahmad N. Mukhtar H. Nutr. Rev. 1999; 57: 78-83Crossref PubMed Scopus (348) Google Scholar). The inhibition of tumorigenesis by green or black tea preparations was demonstrated in animal models at various organ sites (3Liao S. Umekita Y. Guo J. Kokontis J.M. Hiipakka R.A. Cancer Lett. 1995; 96: 239-243Crossref PubMed Scopus (288) Google Scholar, 4Yang C.S. Maliakal P. Meng X. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (829) Google Scholar, 5Lambert J.D. Yang C.S. J. Nutr. 2003; 133: 3262S-3267SCrossref PubMed Google Scholar). The structures of the four major catechins, (–)-epigallocatechin gallate (EGCG), 1The abbreviations used are: EGCG, (–)-epigallocatechin 3-gallate; EC, (–)-epicatechin; ECG, (–)-epicatechin-3-gallate; EGC, (–)-epigallocatechin; PKA, cAMP-dependent protein kinase; Cdc2 kinase, cyclindependent kinase 2; MALDI, matrix-assisted laser desorption/ionization; TOF, time of flight; MS, mass spectrometry; PMSF, phenylmethylsulfonyl fluoride; IF, intermediate filament; FBS, fetal bovine serum; siRNA, small interfering RNA; GST, glutathione S-transferase; GFP, green fluorescent protein; PBS, phosphate-buffered saline. 1The abbreviations used are: EGCG, (–)-epigallocatechin 3-gallate; EC, (–)-epicatechin; ECG, (–)-epicatechin-3-gallate; EGC, (–)-epigallocatechin; PKA, cAMP-dependent protein kinase; Cdc2 kinase, cyclindependent kinase 2; MALDI, matrix-assisted laser desorption/ionization; TOF, time of flight; MS, mass spectrometry; PMSF, phenylmethylsulfonyl fluoride; IF, intermediate filament; FBS, fetal bovine serum; siRNA, small interfering RNA; GST, glutathione S-transferase; GFP, green fluorescent protein; PBS, phosphate-buffered saline. (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG), and (–)-epicatechin (EC), are shown in Fig. 1. EGCG is the major polyphenol in green tea and may account for 50–80% of the total catechins in tea (4Yang C.S. Maliakal P. Meng X. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (829) Google Scholar, 6Yang C.S. Wang Z.Y. J. Natl. Cancer Inst. 1993; 85: 1038-1049Crossref PubMed Scopus (1006) Google Scholar, 7Dong Z. Biofactors. 2000; 12: 17-28Crossref PubMed Scopus (77) Google Scholar). The inhibitory activity of EGCG against tumorigenesis has been demonstrated. The mechanisms responsible for these cancer-preventive effects of tea are not very well understood but are being intensively investigated. Searching for the EGCG “receptor” or high affinity proteins that bind to EGCG is the first step to understanding the molecular and biochemical mechanisms of the anticancer effects of tea polyphenols. A few proteins that can directly bind with EGCG have been identified, including plasma proteins: fibronectin, fibrinogen, and histidine-rich glycoprotein (8Sazuka M. Isemura M. Isemura S. Biosci. Biotechnol. Biochem. 1998; 62: 1031-1032Crossref PubMed Scopus (33) Google Scholar); also fatty acid synthase (Fas) (9Hayakawa S. Saeki K. Sazuka M. Suzuki Y. Shoji Y. Ohta T. Kaji K. Yuo A. Isemura M. Biochem. Biophys. Res. Commun. 2001; 285: 1102-1106Crossref PubMed Scopus (113) Google Scholar), laminin, and the 67-kDa laminin receptor (10Suzuki Y. Isemura M. Cancer Lett. 2001; 173: 15-20Crossref PubMed Scopus (62) Google Scholar, 11Tachibana H. Koga K. Fujimura Y. Yamada K. Nat. Struct. Mol. Biol. 2004; 11: 380-381Crossref PubMed Scopus (578) Google Scholar). Plasma proteins may act as carrier proteins for EGCG. Fas might trigger the cascade of Fas-mediated apoptosis, and the fact that EGCG can bind and regulate biological functions of the 67 laminin receptor has possible implications for prion-related diseases. However, the biologic and physiologic significance for the anticancer effects of tea polyphenols is not clear. Identification of new proteins binding with EGCG should help in the design of new strategies to prevent cancer. Mass spectrometry-based proteomic analysis is a powerful tool to identify proteins binding with EGCG. We used the JB6 mouse epidermal cell line, a system that has been used extensively as an in vitro model for tumor promotion studies (12Cheng T.J. Tseng Y.F. Chang W.M. Chang M.D. Lai Y.K. J. Cell. Biochem. 2003; 89: 589-602Crossref PubMed Scopus (21) Google Scholar), to identify novel proteins that bind with EGCG. The results indicated that EGCG binds with the intermediate filament (IF) protein, vimentin with high affinity (Kd = 3.3 nm). Vimentin, one of the type III IF proteins, is a major component of IFs and is expressed during development in a wide range of cells, including mesenchymal cells and in a variety of cultured cell lines and tumors (13Matsuzawa K. Kosako H. Inagaki N. Shibata H. Mukai H. Ono Y. Amano M. Kaibuchi K. Matsuura Y. Azuma I. Inagaki M. Biochem. Biophys. Res. Commun. 1997; 234: 621-625Crossref PubMed Scopus (64) Google Scholar, 14Okayama N. Fowler M.R. Jennings S.R. Specian R. Alexander B. Jackson T.H. Oshima T. Shannon T. Alexander J.S. In Vitro Cell Dev. Biol. Anim. 2000; 36: 228-234Crossref PubMed Google Scholar). IFs are essential for structure and mechanical integration of the cellular space and a variety of cellular functions such as mitosis, locomotion, and organizational cell architecture, and vimentin is readily phosphorylated by numerous protein kinases, thereby regulating their function (15Chou Y.H. Goldman R.D. J. Cell Biol. 2000; 150: F101-F106Crossref PubMed Google Scholar, 16Helfand B.T. Chang L. Goldman R.D. Annu. Rev. Cell Dev. Biol. 2003; 19: 445-467Crossref PubMed Scopus (84) Google Scholar, 17Yoon M. Moir R.D. Prahlad V. Goldman R.D. J. Cell Biol. 1998; 143: 147-157Crossref PubMed Scopus (210) Google Scholar). The proteolytic derivatives indicate that the amino-terminal domain, but not the carboxyl-terminal domain, has a direct effect on filament stability and polymerization (18Ando S. Tanabe K. Gonda Y. Sato C. Inagaki M. Biochemistry. 1989; 28: 2974-2979Crossref PubMed Scopus (145) Google Scholar). We used the Swiss-Prot program and a program from the National Genomic Information center to search the MALDI-TOF data base for peptides important in binding with EGCG. The peptide (SLYSSSPGGAYVTR) contains two phosphorylation sites of vimentin, serine 50 and serine 55. Our results in vivo demonstrated that EGCG inhibited the phosphorylation of vimentin at serines 50 and 55 in a dose-dependent manner. In vitro studies revealed that the phosphorylation of IF proteins by various types of serine/threonine protein kinases induces disassembly of the filament structure (19Yasui Y. Goto H. Matsui S. Manser E. Lim L. Nagata K. Inagaki M. Oncogene. 2001; 20: 2868-2876Crossref PubMed Scopus (78) Google Scholar). Vimentin is an excellent substrate for Cdc2 and PKA. Cdc2 phosphorylates vimentin at serine 55, and serine 50 is a phosphorylation site of PKA. In this study, we provide evidence that EGCG inhibited phosphorylation of vimentin by Cdc2 and PKA. EGCG is known to inhibit cell proliferation of various cancer cell lines (20Chen A. Zhang L. J. Biol. Chem. 2003; 278: 23381-23389Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 21Chung L.Y. Cheung T.C. Kong S.K. Fung K.P. Choy Y.M. Chan Z.Y. Kwok T.T. Life Sci. 2001; 68: 1207-1214Crossref PubMed Scopus (165) Google Scholar, 22Gupta S. Ahmad N. Nieminen A.L. Mukhtar H. Toxicol. Appl. Pharmacol. 2000; 164: 82-90Crossref PubMed Scopus (255) Google Scholar, 23Yang G.Y. Liao J. Kim K. Yurkow E.J. Yang C.S. Carcinogenesis. 1998; 19: 611-616Crossref PubMed Scopus (648) Google Scholar, 24Lea M.A. Xiao Q. Sadhukhan A.K. Cottle S. Wang Z.Y. Yang C.S. Cancer Lett. 1993; 68: 231-236Crossref PubMed Scopus (95) Google Scholar, 25Uesato S. Kitagawa Y. Kamishimoto M. Kumagai A. Hori H. Nagasawa H. Cancer Lett. 2001; 170: 41-44Crossref PubMed Scopus (74) Google Scholar). Our data show that inhibition of vimentin phosphorylation by EGCG was directly associated with a decrease in cell proliferation. We hypothesize that the binding of EGCG to vimentin could be very important in a cascade of vimentin-mediated cell proliferation. Materials—Eagle's minimal essential medium, fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); RPMI 1640 modified medium (2 mml-glutamine, 10 mm HEPES, 1mm sodium pyruvate, 4500 mg/liter glucose, and 1500 mg/liter sodium bicarbonate) and human insulin were from American Type Culture Collection (Manassas, VA); l-glutamine was from Invitrogen; Kodak MS film was obtained from Sigma; CNBr-Sepharose 4B, ECL plus kit, glutathione-Sepharose 4B, and [γ-32P]ATP were purchased from Amersham Biosciences; and rec-Protein G-Sepharose 4B was obtained from Zymed Laboratories Inc. (South San Francisco, CA). [3H]EGCG (13 Ci/mmol in ethanol containing 8 mg/ml ascorbic acid) was a gift from Dr. Yukihiko Hara (Food Research Laboratory, Mitsui Norin Co. Ltd., Fujieda, Shizuoka, Japan). The mouse monoclonal (VI-01) vimentin antibody was purchased from Abcam (Cambridge, MA); the anti-phosphorylated vimentin mouse monoclonal (Ser50) and (Ser55) antibodies were from MBL (Woburn, MA); the secondary goat anti-mouse horseradish peroxidase-conjugated antibody and fluorescein isothiocyanate-conjugated antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The two-well culture slide was purchased from BD Biosciences (Palo Alto, CA). Recombinant human vimentin was obtained from Research Diagnostics, Inc. (Flanders, NJ). Cdc2 protein kinase and the CellTiter 96® AQueous One Solution cell proliferation assay kit were purchased from Promega (Madison, WI). ReadyPrep two-dimensional starter kit, IPG non-linear strips, pH 5–8 and 4–7. 10–20% SDS polyacrylamide gels, the ReadyPrep two-dimensional starter kit, and SYPRO Ruby protein gel stain were purchased from Bio-Rad. PKA protein kinase and kemptide were from Upstate (Waltham, MA). EGCG, EC, ECG, and EGC were generous gifts from Dr. Chi-Tang Ho (Rutgers University, Piscataway, NJ). Cell Culture—The JB6 Cl41 mouse epidermal cell line was grown in monolayers with minimal essential medium supplemented with 5% (v/v) heat-inactivated FBS, 2 mml-glutamine, and 25 μg/ml gentamicin. The cell lines were cultured at 37 °C in a humidified atmosphere of 5% CO2. Affinity Chromatography—EGCG was first coupled to the CNBr-activated Sepharose 4B matrix, and the binding between vimentin and EGCG was examined by affinity chromatography according to the manufacturer's instructions. The cell lysates from JB6 Cl41 cells cultured in 10-cm dishes were disrupted in lysis buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1 mm dithiothreitol, 0.01% Nonidet P-40, 0.02 mm PMSF, 1× protease inhibitor mixture). The lysate was sonicated and centrifuged at 15000 × g for 30 min, and the supernatant fraction (10 mg/8 ml) was applied to the EGCG-Sepharose 4B column (12 × 8 mm) at 4 °C. The mobile phase was T buffer (50 mm Tris-HCl pH 7.5, 100 mm NaCl, 10 mm 6-aminohexanoic acid, 1 mm PMSF, 1 mm benzamidine hydrochloride, and 1 mm EDTA) running at a flow rate of 0.5 ml/min. The bound proteins were then eluted with T1 buffer (4 m urea, 1 m NaCl in T buffer). Protein elution was monitored in fractions using the Bio-Rad protein assay. Two-dimensional Gel Electrophoresis—The samples were precipitated with trichloroacetic acid, and the ReadyPrep two-dimensional Starter kit was used as outlined in the instruction manual. The samples were then applied onto the IPG nonlinear strips, pH 5–8, for the first dimension separation. The second-dimensional separation of eluted proteins was performed by 10–20% SDS-PAGE. The gels and proteins were stained with SYPRO Ruby protein gel stain or transferred onto nitrocellulose membranes. MALDI-TOF Analysis—The protein spot was excised from a polyacrylamide gel and digested overnight with trypsin. The extracted peptides were mixed with the matrix α-cyano-4-hydroxycinammic acid (Aldrich), spotted onto a MALDI plate, and analyzed with a Voyager DePro mass spectrometer (Applied Biosystems, Birmingham, AL). The peptide masses were entered into Mascot (www.matrixscience.com/), and the NCBI data base was searched to identify the protein. In Vitro Pull-down Assay—Recombinant vimentin (2 μg) or a JB6 Cl41 cellular supernatant fraction (600 μg) was incubated with the EGCG-Sepharose 4B (or Sepharose 4B as control) beads (100 μl, 50% slurry) in reaction buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1 mm dithiothreitol, 0.01% Nonidet P-40, 2 μg/ml bovine serum albumin, 0.02 mm PMSF, 1× protease inhibitor mixture). After incubation with gentle rocking overnight at 4 °C, the beads were washed five times with buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1 mm dithiothreitol, 0.01% Nonidet P-40, 0.02 mm PMSF), and proteins bound to the beads were analyzed by immunoblotting. Immunoprecipitation—JB6 Cl41 cells were disrupted in buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1 mm dithiothreitol, 0.01% NP-40, 0.02 mm PMSF, 1× protease inhibitor), and cell lysate (600 μg) was used for immunoprecipitation as described (26He Z. Ma W.Y. Hashimoto T. Bode A.M. Yang C.S. Dong Z. Cancer Res. 2003; 63: 4396-4401PubMed Google Scholar). SDS-PAGE and Western Blotting—The samples were analyzed by SDS-PAGE (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) Google Scholar) and Western blotting as described (28Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44843) Google Scholar) with some modification as described previously (26He Z. Ma W.Y. Hashimoto T. Bode A.M. Yang C.S. Dong Z. Cancer Res. 2003; 63: 4396-4401PubMed Google Scholar). Construction of Vimentin Plasmids—A cDNA fragment encoding vimentin was inserted in-frame into the BamHI/EcoRI sites of the pcDNA3.1/V5-His vector (Invitrogen) or pGEX5X1 (Amersham Biosciences) to produce the V5 epitope-tagged construct, pcDNA3.1/V5-vimentin, or pGEX5X1-vimentin. All of the positive clones containing cDNA inserts were identified by restriction enzyme mapping and sequenced at Genewiz, Inc. (North Brunswick, NJ). Bacterial Expression and Purification of the GST-Vimentin Fusion Protein—The GST-vimentin fusion protein was expressed in Escherichia coli BL21 and purified as described (12Cheng T.J. Tseng Y.F. Chang W.M. Chang M.D. Lai Y.K. J. Cell. Biochem. 2003; 89: 589-602Crossref PubMed Scopus (21) Google Scholar). The GST-vimentin was used for affinity binding and in vitro kinase assays. GST-Vimentin Affinity Binding Assay Estimation for Kd—For the GST-vimentin affinity binding assay, expressed GST fusion proteins were incubated for immobilization with glutathione-Sepharose 4B beads for 1 h at room temperature. The affinity binding assay was carried out overnight at 4 °C in a 500-μl reaction mixture containing reaction buffer (see “In Vitro Pull-down Assay”) with 1 μg of GST-vimentin-Sepharose 4B or GST-Sepharose 4B and 0.5 μCi of [3H]EGCG. For analyzing concentration-dependent uptake, 39 pm to 50 μm concentrations of EGCG were applied. The Kd value was determined through nonlinear regression analysis using the Prizm 4.0 software program (Graphpad Inc., San Diego, CA). Assay of Phosphorylation of Vimentin at Serine 50 and Serine 55— JB6 Cl41 cells were seeded in 10-cm dishes (4 × 105). After culturing at 37 °C (5% FBS) for 12 h, the cells were treated with different concentrations of EGCG (1, 5, 10, or 20 μm). 72 h following treatment, the cells were lysed, and equal amounts of protein were separated by SDS-PAGE. The blots were probed with phospho-specific antibodies to vimentin. Kinase Assay—The Cdc2 kinase assay was performed at 30 °C for 1 h in a 50-μl reaction mixture containing kinase buffer (50 mm Tris, pH 7.5, 10 mm MnCl2, 1 mm EGTA, 2 mm dithiothreitol and 0.01% Brij), 1 μl (20 units) of enzyme, 10 μm of the kinase substrates (histone H1) or 4 μg of GST-vimentin, EGCG (1–20 μm) or EC/ECG/EGC (20 μm), 10 μm ATP, and 1 μCi of [γ-32P]ATP. The PKA kinase assay was performed at 30 °C for 1 h in a 50-μl reaction mixture containing kinase buffer, 0.5 μl (1 ng) of enzyme, 250 μm of the kinase substrates (kemptide), or 4 μg of GST-vimentin and EGCG (1–20 μm). The reactions were stopped by adding an equal volume of 5× SDS sample buffer. After boiling for 5 min, one-half of the sample was separated by 12% SDS-PAGE, and the phosphorylated proteins were visualized by x-ray film. siRNA Preparation and Vector Construction—Two pairs of hairpin siRNA oligonucleotides containing BamHI and HindIII sites were designed as described before (29Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8107) Google Scholar). To design target-specific siRNA templates, we selected sequences of the type AA(N19)NN or AA(N19)UU from the open reading frame of the targeted mRNA to obtain 19-nucleotide sense and antisense strands. This strategy for choosing siRNA target sites is based on the previous observation by Elbashir et al. (30Elbashir S.M. Martinez J. Patkaniowska A. Lendeckel W. Tuschl T. EMBO J. 2001; 20: 6877-6888Crossref PubMed Scopus (1199) Google Scholar) that siRNA with 3′ overhanging UU dinucleotides are the most effective. Hairpin siRNA template oligonucleotides were chemically synthesized using a DNA synthesizer and deprotected and gel-purified as purchased from Sigma Genosys. Selected siRNA target sequences were aligned to the genome data base in a BLAST search to ensure sequences without significant homology to other genes. The sense siRNA template sequence for vimentin was 5′-GATCCGTACCAAGATCTGCTCAATGTTCAAGAGACATTGAGCAGATCTTGGTATTTTTTGGAAA-3′, and the antisense siRNA template sequence was 5′-AGCTTTTCCAAAAAATACCAAGATCTGCTCAATGTCTCTTGAACATTGAGCAGATCTTGGTACG-3′. Sense and antisense oligonucleotides were annealed and cloned into the pSilencer 3.1-H1 neo vector (Ambion) at the BamHI and HindIII sites as described in the manufacturer's instructions. The siRNA sequence targeting vimentin (NM_011701) was from positions 1147–1165 relative to the start codon. As a negative siRNA control, we used two kinds of circular plasmids encoding a hairpin siRNA sequence targeting the green fluorescent protein (siRNA GFP) and a scrambled siRNA, the sequence of which lacks significant sequence homology to the mouse, human, or rat genome data base. The resulting siRNA-expressing plasmids were used for both transient and stable transfections. Transfection and Stable Cell Lines—JB6 Cl41 cells were maintained in minimal essential medium supplemented with 5% (v/v) heat-inactivated FBS, 2 mml-glutamine, and 25 μg/ml gentamicin at 37 °C in a humidified atmosphere of 5% CO2. Stable cell lines were obtained by transfecting 2 × 105 cells in 6-cm dishes with 4 μg of the expression plasmids. The cell lines were selected and subsequently maintained in medium containing 200 μg/ml zeocin (Invitrogen). Immunofluorescence—Transfected cells grown on glass chambers were fixed in 4% formaldehyde in PBS for 15 min and extracted in 0.5% Triton X-100 in PBS for 10 min at room temperature to allow subsequent antibody penetration. After extensive washing in PBS, the fixed and extracted cells were incubated with primary antibodies at 37 °C in a humidified chamber for 1 h, washed three times in PBS, and then incubated with the fluorescein isothiocyanate-conjugated secondary antibody for an additional hour at 37 °C. The cells on coverslips were viewed using an LEI-750D CE microscope (Leica, Bannockburn, IL) equipped with epifluorescence optics. Cell Proliferation Assay—To assess proliferation, control (GFP siRNA) or vimentin siRNA cells (3 × 103) were seeded in 96-well tissue culture plates and cultured at 37 °C in a 5% CO2 incubator. Cellular proliferation was then estimated at 24, 48, and 72 h using the CellTiter 96 AQueous One Solution cell proliferation assay kit (Promega) according to the manufacturer's instructions. The assay solution is added to each well, and absorbance (492 nm and 690 nm background) is read with a 96-well plate reader. Absorbance at 492 nm is directly proportional to the number of living cells. Then to test whether EGCG had an effect on proliferation of control (GFP siRNA) or vimentin siRNA cells (3 × 103), the cells were seeded in 96-well tissue culture plates for 12 h and then treated with different concentrations of EGCG (1, 5, 10, 15, or 20 μm). The cells were cultured for an additional 72 h, and then 20 μlof the CellTiter 96 AQueous One Solution were added to each well. The cells were incubated for an additional hour, and then absorbance (492 nm and 690 nm background) was measured using a 96-well plate reader as above. Data Analysis—The figures shown in this paper are representative of at least three independent experiments with similar results. The statistical differences were evaluated using Student's t test and considered significant at p ≤ 0.05. Identification of Vimentin as an EGCG-binding Protein— Proteins from JB6 Cl41 cell lysates were subjected to affinity chromatography using EGCG-Sepharose 4B (Fig. 2). Fractions containing proteins binding with EGCG were analyzed by two-dimensional electrophoresis and MALDI-TOF-MS to identify proteins that directly bind with EGCG. Experimental conditions were first established to obtain a reproducible pattern of spots by two-dimensional electrophoresis (Fig. 3).Fig. 3Two-dimensional electrophoresis of proteins captured from EGCG-Sepharose 4B beads. Cell lysates were applied to an EGCG-Sepharose 4B column, and ECGG-binding proteins were used for analysis by two-dimensional electrophoresis. Spots pertaining to vimentin are indicated in the dotted ring and enlarged in the inset. The numbered protein spots were digested with trypsin, and the resulting peptides were analyzed by mass spectrometry (Table I).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The two-dimensional electrophoresis was performed with different quantities of protein from the affinity column (data not shown) to determine optimal conditions for two-dimensional electrophoresis, and each protein fraction was run in triplicate. The results indicate that three spots were consistently observed (Fig. 3) by two-dimensional electrophoresis stained with SYPRO Ruby protein gel stain. The respective spots were removed and digested with trypsin, and the resulting peptides were subjected to analysis by MALDI-TOF-MS (Table I). A search of the NCBI data base revealed that all three spots corresponded to vimentin, an intermediate filament protein.Table IThe resulting peptide fragments were analyzed by MALDI-TOF-MS for identification of EGCG-binding proteinsPeptide fragmentStart-EndCalculated m/zObserved m/zSpot 1Spot 2Spot 3LQEEMLQR189-1961061.581062.59QESNEYRR294-3011080.491081.581081.54FALDSEAANR295-3041092.521093.58VELQELNDR105-1131114.561115.63FANYIDKVR114-1221124.601125.661124.65LGDLYEEEMR146-1551269.551270.641270.60EEAESTLQSFR197-2071295.601296.67MALDIEIATYR391-4011310.61311.71EKLQEEMLQR187-1961318.661319.67SLYSSSPGGAYVTR51-641443.701444.771444.771444.73TYSLGSALRPSTSR37-501494.821495.831495.82HLREYQDLLNVK379-3901526.821527.88VESLQEEIAFLKK224-2361532.841533.91ILLAELEQLKGQGK130-1431538.901539.911539.85ISLPLPTFSSLNLR411-4241556.891557.951557.941557.86TNEKVELQELNDR101-1131586.791587.85LGDLYEEEMRELR146-1581667.781668.811668.76VEVERDNLAEDIMR171-1841703.811704.79LQDEIQNMKEEMAR365-3781756.801766.84FADLSEAANRNNDALR295-3101775.851776.891776.87DGQVINETSQHHDDLE451-4661835.791836.77ETNLESLPLVDTHSKR425-4401837.951838.981838.88EMEENFALEAANYQDTIGR346-3642215.972217.05NCBI databasegi:55408gi:55408gi:55408Mass55,68955,68955,689Scope189138107 Open table in a new tab Western Blot Confirmation of Vimentin as an EGCG-binding Protein—Further evidence for the binding of vimentin with EGCG was demonstrated with immunoblot analysis. Analysis was done using JB6 cell lysates and EGCG-Sepharose 4B. To create a two-dimensional image of vimentin binding with EGCG, samples subjected to the affinity column were analyzed by two-dimensional electrophoresis under the same conditions as described above, and the proteins were transferred onto nitrocellulose membranes. The spots were confirmed to be vimentin by Western analysis using a mouse monoclonal (VI-01) vimentin antibody (Fig. 4A). These results strongly indicate that vimentin binds with EGCG. To directly test this possibility, we used a combination of a pull-down assay with EGCG-Sepharose 4B and an immunoprecipitation assay with protein G-Sepharose 4B and the vimentin antibody. The lysate from JB6 Cl41 cells was directly applied to the EGCG-Sepharose 4B column, and following elution, Western blot analysis revealed the presence of a 54-kDa protein that was reactive with the vimentin antibody (VI-01) (Fig. 4B, lane 2), confirming the presence of vimentin in the EGCG-bound fraction. Lane 1 shows vimentin from lysate as the control. The lysates were applied to protein G-Sepharose 4B beads and immunoprecipitated with the vimentin antibody (Fig. 4C, lane 2) or were applied to the EGCG-Sepharose 4B column and Sepharose 4B column and eluted (Fig. 4C, lanes 3 and 4, respectively). The results clearly indicate that vimentin is present in mouse cells and that EGCG binds with vimentin in vivo. To characterize the binding interaction, we measured the binding affinity of GST-vimentin and [3H]EGCG using a pull-down assay. The results indicate that vimentin displayed a high affinity for binding with [3H]EGCG (Fig. 4D). GST-Sepharose 4B alone was used only as a negative control, and no binding occurred (data not shown). The Kd value for the binding of EGCG to vimentin was 3.3 nm. EGCG Inhibits Phosphorylation of Vimentin at Ser50 and Ser55 in a Dose-dependent Manner—JB6 Cl41 cells were employed to analyze the EGCG inhibition of phosphorylation of vimentin at serine 50 and serine 55 (Fig. 5A). The dose course study shows that phosphorylation of vimentin gradually decreases following 72 h of treatment with increasing amounts of EGCG (1, 5, 10, or 20 μm) (Fig. 5B), with no effect on total vimentin protein levels (Fig. 5B). These results indicate that phosphorylation of vimentin at serine 50 and serine 55 is inhibited by EGCG in a dose-dependent manner. EGCG Specifically Inhibits the Phosphorylation of Vimentin by Cdc2—Cdc2 kinase activity was analyzed by in vitro kinase assays using GST-vimentin and histone H1 as substrates. First we determined the effect of EGCG on the total phosphorylation of vimentin by Cdc2. As shown in Fig. 5C (top panel), up to 5 μm EGCG did not affect Cdc2 kinase activity, whereas 10 or 20 μm induced a gradual decrease in kinase activity. The results indicate that increasing concentrations of EGCG inhibit Cdc2 kinase-mediated phosphorylation of vimentin. The next question to be answered was whether the inhibition was due to an interference of EGCG with the phosphorylation of vimentin directly or indirectly via Cdc2 kinase. In a second series of experiments, the kinase assay was carried out using GST-vimentin or histone H1 as a substrate for Cdc2 kinase. The results confirm that the addition of EGCG to the k" @default.
• W2097795264 created "2016-06-24" @default.
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• W2097795264 date "2005-04-01" @default.
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• W2097795264 title "The Intermediate Filament Protein Vimentin Is a New Target for Epigallocatechin Gallate" @default.
• W2097795264 cites W1559380160 @default.
• W2097795264 cites W1584847179 @default.
• W2097795264 cites W1611419331 @default.
• W2097795264 cites W1951658941 @default.
• W2097795264 cites W1970266087 @default.
• W2097795264 cites W1970530418 @default.
• W2097795264 cites W1972676329 @default.
• W2097795264 cites W1977499446 @default.
• W2097795264 cites W1978011847 @default.
• W2097795264 cites W1982944995 @default.
• W2097795264 cites W1993956443 @default.
• W2097795264 cites W1994419707 @default.
• W2097795264 cites W1999375857 @default.
• W2097795264 cites W2003895116 @default.
• W2097795264 cites W2014366379 @default.
• W2097795264 cites W2015554867 @default.
• W2097795264 cites W2019913413 @default.
• W2097795264 cites W2026777690 @default.
• W2097795264 cites W2027156218 @default.
• W2097795264 cites W2032729098 @default.
• W2097795264 cites W2034114619 @default.
• W2097795264 cites W2035313943 @default.
• W2097795264 cites W2048848497 @default.
• W2097795264 cites W2050451392 @default.
• W2097795264 cites W2052090797 @default.
• W2097795264 cites W2057232203 @default.
• W2097795264 cites W2058676147 @default.
• W2097795264 cites W2059322936 @default.
• W2097795264 cites W2063611358 @default.
• W2097795264 cites W2074317678 @default.
• W2097795264 cites W2080234559 @default.
• W2097795264 cites W2087182820 @default.
• W2097795264 cites W2087334753 @default.
• W2097795264 cites W2100837269 @default.
• W2097795264 cites W2101108802 @default.
• W2097795264 cites W2103908920 @default.
• W2097795264 cites W2122412289 @default.
• W2097795264 cites W2124290816 @default.
• W2097795264 cites W2124690619 @default.
• W2097795264 cites W2126048177 @default.
• W2097795264 cites W2127718764 @default.
• W2097795264 cites W2130150098 @default.
• W2097795264 cites W2134841682 @default.
• W2097795264 cites W2154846674 @default.
• W2097795264 cites W2169058970 @default.
• W2097795264 cites W2614443566 @default.
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• W2097795264 doi "https://doi.org/10.1074/jbc.m414185200" @default.
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