(-)-Epigallocatechin Gallate Regulates CD3-mediated T Cell Receptor Signaling in Leukemia through the Inhibition of ZAP-70 Kinase
2008; Elsevier BV; Volume: 283; Issue: 42 Linguagem: Inglês
10.1074/jbc.m802200200
ISSN1083-351X
AutoresJung‐Hyun Shim, Hong Seok Choi, Angelo Pugliese, Sung‐Young Lee, Jung‐Il Chae, Bu Young Choi, Ann M. Bode, Zigang Dong,
Tópico(s)Atherosclerosis and Cardiovascular Diseases
ResumoThe ζ chain-associated 70-kDa protein (ZAP-70) of tyrosine kinase plays a critical role in T cell receptor-mediated signal transduction and the immune response. A high level of ZAP-70 expression is observed in leukemia, which suggests ZAP-70 as a logical target for immunomodulatory therapies. (-)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified ZAP-70 as an important and novel molecular target of EGCG in leukemia cells. ZAP-70 and EGCG displayed high binding affinity (Kd = 0.6207 μmol/liter), and additional results revealed that EGCG effectively suppressed ZAP-70, linker for the activation of T cells, phospholipase Cγ1, extracellular signaling-regulated kinase, and MAPK kinase activities in CD3-activated T cell leukemia. Furthermore, the activation of activator protein-1 and interleukin-2 induced by CD3 was dose-dependently inhibited by EGCG treatment. Notably, EGCG dose-dependently induced caspase-mediated apoptosis in P116.cl39 ZAP-70-expressing leukemia cells, whereas P116 ZAP-70-deficient cells were resistant to EGCG treatment. Molecular docking studies, supported by site-directed mutagenesis experiments, showed that EGCG could form a series of intermolecular hydrogen bonds and hydrophobic interactions within the ATP binding domain, which may contribute to the stability of the ZAP-70-EGCG complex. Overall, these results strongly indicated that ZAP-70 activity was inhibited specifically by EGCG, which contributed to suppressing the CD3-mediated T cell-induced pathways in leukemia cells. The ζ chain-associated 70-kDa protein (ZAP-70) of tyrosine kinase plays a critical role in T cell receptor-mediated signal transduction and the immune response. A high level of ZAP-70 expression is observed in leukemia, which suggests ZAP-70 as a logical target for immunomodulatory therapies. (-)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified ZAP-70 as an important and novel molecular target of EGCG in leukemia cells. ZAP-70 and EGCG displayed high binding affinity (Kd = 0.6207 μmol/liter), and additional results revealed that EGCG effectively suppressed ZAP-70, linker for the activation of T cells, phospholipase Cγ1, extracellular signaling-regulated kinase, and MAPK kinase activities in CD3-activated T cell leukemia. Furthermore, the activation of activator protein-1 and interleukin-2 induced by CD3 was dose-dependently inhibited by EGCG treatment. Notably, EGCG dose-dependently induced caspase-mediated apoptosis in P116.cl39 ZAP-70-expressing leukemia cells, whereas P116 ZAP-70-deficient cells were resistant to EGCG treatment. Molecular docking studies, supported by site-directed mutagenesis experiments, showed that EGCG could form a series of intermolecular hydrogen bonds and hydrophobic interactions within the ATP binding domain, which may contribute to the stability of the ZAP-70-EGCG complex. Overall, these results strongly indicated that ZAP-70 activity was inhibited specifically by EGCG, which contributed to suppressing the CD3-mediated T cell-induced pathways in leukemia cells. For thousands of years, tea has been the most widely consumed beverage in the world after water. Historically, tea has been credited with various beneficial health effects, including medicinal efficacy in the prevention and treatment of numerous diseases. Thus, longevity and good health have often been associated with the habit of drinking tea (1Yang C.S. Maliakal P. Meng X. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (846) Google Scholar). Four major polyphenolic catechins are found in green tea and include (-)-epicatechin (EC), 3The abbreviations used are: EC, (-)-epicatechin; ECG, (-)-epicatechin 3-gallate; EGC, (-)-epigallocatechin; EGCG, (-)-epigallocatechin 3-gallate; TCR, T cell receptor; IL, interleukin; PTK, protein-tyrosine kinase; CLL, chronic lymphocytic leukemia; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; ERK, extracellular signaling-regulated kinase; PLC, phospholipase C; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); ITAM, immunoreceptor tyrosine-based activation motif; FITC, fluorescein isothiocyanate; SH2, Src homology domain 2; LAT, anti-linker for the activation of T cells. (-)-epicatechin 3-gallate (ECG), (-)-epigallocatechin (EGC), and (-)-epigallocatechin 3-gallate (EGCG). A cup of green tea may contain 100–200 mg of EGCG (2Zaveri N.T. Life Sci. 2006; 78: 2073-2080Crossref PubMed Scopus (708) Google Scholar). Several investigators have reported that green tea exerts cancer preventive activity at a variety of organ sites, including skin, lung, oral cavity, esophagus, stomach, small intestine, colon, pancreas, and mammary gland (1Yang C.S. Maliakal P. Meng X. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 25-54Crossref PubMed Scopus (846) Google Scholar, 3Yang C.S. Wang Z.Y. J. Natl. Cancer Inst. 1993; 85: 1038-1049Crossref PubMed Scopus (1016) Google Scholar, 4Yang D.J. Hwang L.S. J. Chromatogr. A. 2006; 1119: 277-284Crossref PubMed Scopus (93) Google Scholar). However, the mechanisms explaining the cancer preventive activity of tea and tea polyphenols are still not clearly understood. The ζ-associated 70-kDa protein (ZAP-70) is a Syk (spleen tyrosine kinase) family tyrosine kinase, which is associated with the ζ subunit of the T cell receptor (TCR). The ZAP-70 protein is primarily expressed in T cells and natural killer cells and plays an essential role in signaling through the T cell antigen receptor (5Jin L. Pluskey S. Petrella E.C. Cantin S.M. Gorga J.C. Rynkiewicz M.J. Pandey P. Strickler J.E. Babine R.E. Weaver D.T. Seidl K.J. J. Biol. Chem. 2004; 279: 42818-42825Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The TCRs are associated with tyrosine phosphorylation of multiple proteins resulting in activation of various signaling pathways causing alterations in gene expression, increased T cell proliferation, and secretion of cytokines (6Deindl S. Kadlecek T.A. Brdicka T. Cao X. Weiss A. Kuriyan J. Cell. 2007; 129: 735-746Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). CD3 (cluster of differentiation 3) stimulation of the T cell antigen receptor plays a role in tyrosine phosphorylation of a number of cellular substrates. An important substrate of ZAP-70 is the TCR ζ chain, which can mediate the transduction of extracellular stimuli into cellular effector functions (7Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Crossref PubMed Scopus (596) Google Scholar, 8Cantrell D.A. Immunology. 2002; 105: 369-374Crossref PubMed Scopus (65) Google Scholar). ZAP-70 plays a critical role in cell surface expression of T cell antigen receptor-CD3 complex signaling during the early stages of T cell development and differentiation (9Hamblin T.J. Best Pract. Res. Clin. Haematol. 2007; 20: 455-468Crossref PubMed Scopus (58) Google Scholar, 10Gibbs G. Bromidge T. Howe D. Johnson S. Int. J. Lab. Hematol. 2007; 29: 225-227Crossref PubMed Scopus (4) Google Scholar, 11Zanotti R. Ambrosetti A. Lestani M. Ghia P. Pattaro C. Remo A. Zanetti F. Stella S. Perbellini O. Prato G. Guida G. Caligaris-Cappio F. Menestrina F. Pizzolo G. Chilosi M. Leukemia (Baltimore). 2007; 21: 102-109Crossref PubMed Scopus (32) Google Scholar, 12Chan A.C. Irving B.A. Fraser J.D. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9166-9170Crossref PubMed Scopus (338) Google Scholar, 13Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (889) Google Scholar). The ZAP-70 tyrosine kinase is reported to play a critical role in T cell activation and the immune response, and therefore might be a logical target for immunomodulatory therapies (5Jin L. Pluskey S. Petrella E.C. Cantin S.M. Gorga J.C. Rynkiewicz M.J. Pandey P. Strickler J.E. Babine R.E. Weaver D.T. Seidl K.J. J. Biol. Chem. 2004; 279: 42818-42825Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Crespo et al. (14Crespo M. Bosch F. Villamor N. Bellosillo B. Colomer D. Rozman M. Marce S. Lopez-Guillermo A. Campo E. Montserrat E. N. Engl. J. Med. 2003; 348: 1764-1775Crossref PubMed Scopus (1178) Google Scholar) observed that among B cell and T cell lymphoproliferative disorders, a high level of ZAP-70 expression is found in T cell proliferative diseases, acute lymphoblastic leukemia, and a subgroup of chronic lymphocytic leukemia (CLL) (15Buggins A.G. Hirst W.J. Pagliuca A. Mufti G.J. Br. J. Haematol. 1998; 100: 784-792Crossref PubMed Scopus (55) Google Scholar, 16Khan I.H. Mendoza S. Rhyne P. Ziman M. Tuscano J. Eisinger D. Kung H.J. Luciw P.A. Mol. Cell. Proteomics. 2006; 5: 758-768Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These studies suggested that ZAP-70 could be an excellent prognostic biomarker in CLL. Despite advances in T cell leukemia therapy, only a minority of patients achieve long term tumor-free survival with conventional chemotherapy but at the cost of significant, irreversible toxic side effects, which often limit effective treatment (17Bremer E. ten Cate B. Samplonius D.F. de Leij L.F. Helfrich W. Blood. 2006; 107: 2863-2870Crossref PubMed Scopus (47) Google Scholar). Therefore, new therapeutic approaches with enhanced tumor selectivity and more favorable toxicity profiles are urgently needed. EGCG, a major active constituent of green tea, has been shown to stimulate apoptosis and cell cycle arrest in various cancer cell lines, including prostate, colon, lung, leukemias, and lymphomas (3Yang C.S. Wang Z.Y. J. Natl. Cancer Inst. 1993; 85: 1038-1049Crossref PubMed Scopus (1016) Google Scholar, 18Nakazato T. Ito K. Miyakawa Y. Kinjo K. Yamada T. Hozumi N. Ikeda Y. Kizaki M. Haematologica. 2005; 90: 317-325PubMed Google Scholar). However, the anticancer mechanisms and molecular targets of EGCG are poorly understood, especially in T cell-mediated leukemias and lymphomas. Here we demonstrate that EGCG might be a potential immunomodulator for the management of ZAP-70-dependent T cell activation in human leukemias and lymphomas. The effects of EGCG were extensively investigated in a ZAP-70-deficient Jurkat T cell line (P116 cells) and a cell line in which ZAP-70 activity was recovered (P116.cl39 cells). Results suggested that ZAP-70 antagonists could be useful immunomodulatory therapeutic agents. Materials—All media were obtained from American Type Culture Collection (Manassas, VA), and fetal bovine serum was from Gemini Bio-Products (Calabasas, CA). CNBr-Sepharose 4B, glutathione-Sepharose 4B, and [γ-32P]ATP were purchased from Amersham Biosciences. [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). Recombinant ZAP-70 and the poly-(Glu4-Tyr) peptide were from Upstate Biotechnology, Inc. (Charlottesville, VA). The following antibodies were used: anti-ZAP-70 (1E7.2), anti-linker for the activation of T cells (LAT) (FL-233), anti-phospholipase Cγ1 (PCLγ1) (E-12), anti-α-tubulin (TU-02) (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (MOC, Sigma), anti-extracellular signaling-regulated kinase (ERK), anti-phospho-ERK (Thr202/Tyr204), anti-MAPK kinase (MEK), anti-phospho-MEK (Ser217/221), anti-phospho-PLCγ1 (Tyr783), and anti-phosphotyrosine 100 (Cell Signaling Technology, Inc. Charlottesville, VA). The ATP immobilized on agarose 4B was purchased from Fluka (St. Louis). The mouse anti-human CD3 monoclonal antibody, mouse IgG1κ, monoclonal immunoglobulin isotype control, anti-phospho-ZAP-70 (Tyr319), anti-phospho-ZAP-70 (Tyr493), anti-CD3ζ, and anti-phospho-CD3ζ (Tyr142) were purchased from Pharmingen™. The TnT® Quick Coupled transcription/translation system was from Promega (Madison, WI). EGCG, EC, ECG, and EGC were generous gifts from Dr. Chi-Tang Ho (Rutgers University, Piscataway, NJ). Cell Culture—The Jurkat (clone E6–1) human T cell leukemia, ZAP-70-deficient Jurkat mutant P116, and ZAP-70 recovered P116 mutant P116.cl39 cells were kindly provided by Dr. Robert T. Abraham (Department of Immunology, Mayo Clinic, Rochester, MN) (19Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (224) Google Scholar). Jurkat and P116 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, and the P116.cl39 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 0.4 mg/ml G418. Construction of Deletion Mutants and Site-directed Mutagenesis—A cDNA encoding ZAP-70 was generated by PCR and subcloned into the BamHI/EcoRI sites of the pcDNA4/HisMax vector (Amersham Biosciences) to produce the ZAP-70 protein. The deletion mutants of ZAP-70 were generated from ZAP-70 wild type and inserted in-frame into the BamHI/EcoRI sites of the pcDNA4/HisMax vector. The products were cut with BamHI and EcoRI and then subcloned into pcDNA4/HisMax, generating the constructs ZAP-70(1–256), ZAP-70(1–415), and ZAP-70-(1–466) according to the insert sizes. cDNAs encoding the Glu415, Lys369, Asp479, Glu386, and Arg465 mutants of ZAP-70 were generated using the QuikChange® multisite-directed mutagenesis kit (Stratagene, La Jolla, CA) and subcloned into the pcDNA4/HisMax vector as follows: ZAP-70 E415Q, ZAP-70 E415Q/K369R, ZAP-70 E415Q/D479N, ZAP-70 E415Q/K369R/D479N, and ZAP-70 E415Q/K369R/D479N/E386Q/R465K. The constructs were confirmed by DNA sequence analysis (GENEWIZ, South Plainfield, NJ). In Vitro EGCG-Sepharose 4B and ATP-Agarose 4B Pulldown Assays—This method has been described previously (20Ermakova S. Choi B.Y. Choi H.S. Kang B.S. Bode A.M. Dong Z. J. Biol. Chem. 2005; 280: 16882-16890Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Briefly, the cells were transfected with 2 μg of each ZAP-70 point mutant using the jetPEI (Polyplus-Transfection SA, San Marcos, CA) reagent following the manufacturer's suggested protocol. The cellular supernatant fraction (Jurkat, P116, P116.cl39, or transfected cells), recombinant ZAP-70, or plasmids (pcDNA4/HIS/Max-ZAP-70 and deletion mutants of ZAP-70) were translated in vivo with l-[35S]methionine. Respective proteins were incubated with EGCG-Sepharose 4B beads or ATP-agarose 4B beads 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 phenylmethylsulfonyl fluoride, 1× proteinase inhibitor). 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 phenylmethylsulfonyl fluoride), and proteins bound to the beads were analyzed by autoradiography or immunoblotting with the appropriate antibodies. Physical Binding and Kd Measurement—ZAP-70 binding assays were carried out as described (20Ermakova S. Choi B.Y. Choi H.S. Kang B.S. Bode A.M. Dong Z. J. Biol. Chem. 2005; 280: 16882-16890Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) with some modifications. For analyzing concentration-dependent uptake, 1 nm to 10 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). Molecular Modeling of the Interactions of EGCG with ZAP-70—Molecular docking studies were carried out using the Maestro software suite (Maestro, version 7.5, Schrödinger, New York). The EGCG molecule was drawn using the builder tool in Maestro and then optimized for docking in Ligprep. The ZAP-70 crystal structure complexed with staurosporine (Protein Data Bank code 1u59) was prepared for docking following the Glide standard procedure (21Friesner R.A. Banks J.L. Murphy R.B. Halgren T.A. Klicic J.J. Mainz D.T. Repasky M.P. Knoll E.H. Shelley M. Perry J.K. Shaw D.E. Francis P. Shenkin P.S. J. Med. Chem. 2004; 47: 1739-1749Crossref PubMed Scopus (6462) Google Scholar). Grids defining the protein receptor were generated considering the binding mode of staurosporine. Many protein-binding sites undergo structural rearrangements upon ligand binding, the so-called "induced fit" allows the binding site to follow the shape of the ligand with resulting better interactions. Consequently, molecular docking was performed with the induced fit docking protocol (Schrödinger Suite 2006). The induced fit docking is a part of the Maestro software suite that attempts to reproduce the protein conformational rearrangement upon binding. Initially, EGCG was docked with Glide in extra-precision mode using a softened potential: scaling the receptor (0.70) and the ligand (0.50) van der Waals radius. The best 20 EGCG poses were retained. Then the receptor side chains within 5 Å distance from the ligand were predicted and minimized for each protein-ligand complex, and then another round of minimizations was performed on each protein-ligand complex pose. Finally, Glide redocking in extra-precision mode of each protein-ligand complex within 30.0 kcal/mol of the lowest energy structure was performed. Herein the best-docked representative structure is presented. Kinase Assay—The ZAP-70 kinase assay was performed at 30°C for 2 h in a 25-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 μg of recombinant ZAP-70, 5 μg of the kinase substrate (poly(Glu4-Tyr) peptide, biotin conjugate), EGCG (0.5–20 μm), 100 μm ATP, and 1 μCi of [γ-32P]ATP. The reactions were stopped by adding 12.5 μl of termination buffer, and samples were quantified (counts/min) by scintillation counting. Immunoprecipitation and Western Blotting—The P116 and P116.cl39 cells (5 × 105/ml) were grown in 75-cm3 flasks for 24 h and then serum-starved for 14 h at 37 °C. Where indicated, the cells were pretreated with EGCG (4 or 8 μm) for 2 h and then washed three times with phosphate-buffered saline. The cells were stimulated for 30 min at 37 °C with 2 μg/ml mouse IgG1κ monoclonal immunoglobulin isotype control and 2 μg/ml mouse anti-human CD3. The reactions were stopped by adding cold phosphate-buffered saline followed by centrifugation for subsequent lysis. Lysates were incubated overnight at 4 °C with the indicated antibodies and protein A/G Plus-agarose beads. After centrifugation, the beads were washed three times with washing buffer and were resuspended in SDS sample buffer. Eluted immunoprecipitates or whole-cell lysates were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then incubated with appropriate antibodies. Immunocomplexes were detected by subsequent incubation with appropriate horseradish peroxidase-conjugated secondary IgG antibodies and visualized by ECL according to the manufacturer's instructions (Amersham Biosciences). Electrophoretic Mobility Shift Assay—Nuclear extracts were prepared as described previously (22Shim J.H. Cho K.J. Lee K.A. Kim S.H. Myung P.K. Choe Y.K. Yoon D.Y. Proteomics. 2005; 5: 2112-2122Crossref PubMed Scopus (24) Google Scholar). The cells were treated and prepared as described above for Western blotting and then disrupted with a hypotonic buffer. The nuclei pellet was disrupted in a hypertonic buffer, and the nuclear extracts were retained for use in the DNA binding assay. A double-stranded deoxyoligonucleotide corresponding to AP (activator protein)-1 responsive elements (Santa Cruz Biotechnology) was end-labeled with [γ-32P]ATP using T4 kinase. Nuclear extracts (5 μg) were incubated in binding buffer for 15 min with poly(dI·dC) and the 32P-labeled DNA probe. The DNA binding activity was separated from free probe using a TBE Ready Gel Precast Gel (Bio-Rad). Following electrophoresis, the gel was dried and visualized by autoradiography. Measurement of IL-2 Production—Quantification of interleukin (IL)-2 production from P116 and P116.cI39 cells was performed using a commercial enzyme-linked immunosorbent assay system (Pharmingen). The cells (5 × 105 cells/ml) were resuspended in fresh medium in a 48-well plate and incubated for 24 h. The cells were treated with various concentrations of EGCG and 2 μg/ml mouse IgG1κ monoclonal immunoglobulin isotype control or 2 μg/ml mouse anti-human CD3 for 24 or 48 h at 37 °C. The levels of IL-2 in the supernatant fractions were subsequently measured using an enzyme-linked immunosorbent assay kit (Pharmingen) according to the manufacturer's instructions. MTS Assay—Cells (1 × 104) were seeded in a 96-well plate and then incubated for 24 h with different concentrations of EGCG (0, 0.5, 1, 2, or 4 μm) or EC, ECG, or EGC (4 μm) for 72 h. The effect of EGCG on viability was estimated using the Cell-Titer 96 AQueous One Solution cell proliferation assay kit (Promega, Madison, WI) according to the manufacturer's instructions. The assay solution was added to each well, and absorbance (492 nm and 690 background) was read with a 96-well plate reader (Labsystem Multiskan MS, Labsystem, Finland). Annexin V Staining—Apoptosis was performed using the annexin V-FITC apoptosis detection kit as recommended by the manufacturer (MBL International Corp., Watertown, MA). Apoptosis was compared in P116 and P116.cl39 cells that were treated or not treated with 4 or 8 μm EGCG for 24, 48, 72, or 96 h. The cells were harvested and washed with phosphate-buffered saline and incubated for 5 min at room temperature with annexin V-FITC plus propidium iodide following the protocol included in the kit. Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Statistical Analysis—Data are presented as means ± S.D. of triplicate samples from at least three independent experiments. Differences between means were assessed by one-way analysis of variance, and the minimum level of significance was set at p < 0.05. EGCG Binds with ZAP-70 ex Vivo and in Vitro—Expression of ZAP-70 was recently reported to be a reliable prognostic marker among patients with CLL (14Crespo M. Bosch F. Villamor N. Bellosillo B. Colomer D. Rozman M. Marce S. Lopez-Guillermo A. Campo E. Montserrat E. N. Engl. J. Med. 2003; 348: 1764-1775Crossref PubMed Scopus (1178) Google Scholar). In a kinase screening experiment (kinase profiler specificity testing service, Upstate), EGCG (5 μm) was found to inhibit ZAP-70 kinase activity. We therefore studied the potential interaction of EGCG and ZAP-70 in wild type Jurkat cells, Jurkat cells (P116) that do not express ZAP-70, and in P116 cells (P116.cl39) in which ZAP-70 protein expression has been restored (Fig. 1A). The interaction of ZAP-70 and EGCG was determined in an EGCG-Sepharose 4B affinity chromatography experiment combined with immunoblotting with anti-ZAP-70. Results indicated that EGCG formed a complex with ZAP-70 in P116.cl39 cells but not in P116 cells (Fig. 1B). To characterize the physical binding between EGCG and ZAP-70, we measured the binding affinity (Kd) of the complex using a GST pulldown assay and 3H-labeled EGCG. The Kd value of ZAP-70 and EGCG binding was determined to be 0.6207 μmol/liter (Fig. 1C). Identification of the EGCG-binding Site of ZAP-70—The ZAP-70 protein has a kinase domain, an SH2-kinase linker region, an inter-SH2 linker region, a C-terminal SH2 domain, and an N-terminal SH2 domain (6Deindl S. Kadlecek T.A. Brdicka T. Cao X. Weiss A. Kuriyan J. Cell. 2007; 129: 735-746Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). To determine the binding region of ZAP-70 specific for EGCG, we created three serially deleted ZAP-70 mutants s follows: ZAP-70 D1 (amino acids 1–466), ZAP-70 D2 (amino acids 1–415), and ZAP-70 D3 (amino acids 1–256) (Fig. 1D). These mutants were used in combination with the EGCG-Sepharose 4B pulldown assay to determine the area of ZAP-70 to which EGCG binds. Results indicated that EGCG binds strongly with full-length ZAP-70 (ZAP-70FL) and the ZAP-70 D1 (amino acids 1–466) domain. On the other hand, ZAP-70 D2 (amino acids 1–415) showed weak binding with EGCG, whereas ZAP-70 D3 (amino acids 1–256) did not bind with EGCG, which suggested that EGCG binds primarily with the kinase domain (Fig. 1E). Structural Analysis of the Interaction between EGCG and ZAP-70—To better characterize the possible interaction between EGCG and the kinase/ATP binding domain of ZAP-70, we used the x-ray co-crystal structure of ZAP-70 complexed with staurosporine (Protein Data Bank code 1u59) as a starting point for a docking experiment. The accuracy of small molecule/protein docking increases when considering holo-proteins (23McGovern S.L. Shoichet B.K. J. Med. Chem. 2003; 46: 2895-2907Crossref PubMed Scopus (247) Google Scholar), and staurosporine is a rigid and flat molecule that creates an open conformation to the ZAP-70 ATP binding pocket. An open conformation is usually adopted by kinases when bound to small ligands, such as EGCG. In our docking model, EGCG presented a calculated binding affinity of -13.07 kcal/mol. One of the hydroxyl groups of the EGCG A ring, similar to other known kinase inhibitors, appears to form two hydrogen bonds with the kinase hinge region and, in particular, with the backbone carbonyl group of Glu415 and the amide group of Ala417 (Fig. 2A). The A–C ring system acts as an adenine mimic and likewise interacts with the front cleft hydrophobic pocket (Fig. 2B). The gallate moiety (D ring) occupies mainly the hydrophilic and solvent-exposed pocket covered by the G-loop, which is believed to be less important for ligand affinity (24Fabbro D. Ruetz S. Buchdunger E. Cowan-Jacob S.W. Fendrich G. Liebetanz J. Mestan J. O'Reilly T. Traxler P. Chaudhuri B. Fretz H. Zimmermann J. Meyer T. Caravatti G. Furet P. Manley P.W. Pharmacol. Ther. 2002; 93: 79-98Crossref PubMed Scopus (299) Google Scholar). The side chains of lysine (Lys369) and aspartate (Asp479) may form a network of hydrogen bonds with the hydroxyl groups of the B ring. In addition, the side chain of Glu386 is expected to form a hydrogen bond with one of the hydroxyl groups of this ring (Fig. 2, A and B). This docking experiment suggested that disrupting specific hydrogen bonding networks by point mutation should affect the ability of EGCG to bind with ZAP-70 as modeled. Therefore, to continue to assess the importance of the interaction between EGCG and the ZAP-70 kinase/ATP-binding site, we generated a variety of ZAP-70 point mutants, including ZAP-70 E415Q, ZAP-70 E415Q/K369R, ZAP-70 E415Q/D479N, ZAP-70 E415Q/K369R/D479N, and ZAP-70 E415Q/K369R/D479N/E386Q/R465K. The various ZAP-70 mutants were each transfected into HEK 293 cells to determine the effect on ZAP-70 binding with EGCG using EGCG-Sepharose 4B or Sepharose 4B combined with Western blot analysis (Fig. 2C). As predicted, the E415Q substitution did not affect the binding because EGCG forms hydrogen bonds with the main chain atoms in the hinge region. The binding seems to be dependent on the interactions of EGCG with Lys369 and Asp479, which we predicted would form a network of hydrogen bonds with the ligand B ring. The bulkier arginine in the K369R mutant very likely affects potential hydrogen bonding with EGCG but also may alter the shape of the cavity, preventing the ligand from assuming the correct orientation for satisfying all the necessary connections with the binding site. Replacing aspartate 479 with asparagine (D479N) also affects the ability of EGCG to bind. We predicted a hydrogen bond between the B ring and the aspartate side chain. The mutant, in which both Lys369 and Asp479 were substituted, consistently showed inhibition of EGCG binding. Thus, these data suggested that ZAP-70 Asp479 and Lys369 are required for the essential interactions of ZAP-70 with EGCG within the catalytic site. The information from the docking experiment suggested that EGCG might effectively inhibit ATP binding to active ZAP-70. Thus, we examined the effect of EGCG on ATP binding by pulldown assay using ATP-agarose 4B and the active ZAP-70 protein. Results confirmed that the binding of ATP with ZAP-70 decreased with increasing amounts of EGCG (Fig. 3A). This suggested that EGCG might inhibit ZAP-70 kinase activity by competing with ATP binding. We next determined whether EGCG could prevent ZAP-70 phosphorylation and/or suppress kinase activity. Kinase activity was analyzed using an in vitro kinase assay with active ZAP-70 and the preferred kinase substrate, poly(Glu4-Tyr) peptide. Results indicated that increasing concentrations of EGCG markedly suppressed ZAP-70 kinase activity, and 3 μm EGCG significantly inhibited ZAP-70 kinase activity by 50% (Fig. 3B). ZAP-70 is recruited to the phosphorylated CD3 and ζ subunits after TCR stimulation (25Kersh E.N. Shaw A.S. Allen P.M. Science. 1998; 281: 572-575Crossref PubMed Scopus (305) Google Scholar). The immunoreceptor tyrosine-based activation motifs (ITAM) of the signal-transducing antigen receptor subunit (CD3 and ζ) are phosphorylated by Src PTK thus allowing the Syk family PTK ZAP-70 to bind to the ITAM (26Neumeister E.N. Zhu Y. Richard S. Terhorst C. Chan A.C. Shaw A.S. Mol. Cell. Biol. 1995; 15: 3171-3178Crossref PubMed Google Scholar). EGCG had no effect on CD3-induced phosphorylation of CD3ζ (Fig. 3C). TCR-mediated Lck activity leads to phosphorylation of ZAP-70 on Tyr493 in the regulatory loop of the PTK domain resulting in the up-regulation of ZAP-70 kinase activity (27Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (325) Google Scholar). Our data showed that EGCG had no effect on phosphorylation of ZAP-70 (Tyr493) (Fig. 3C). Tyrosine 319 is a key phosphorylation
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