The Intermediate Filament Protein Vimentin Is a New Target for Epigallocatechin Gallate

Svetlana Ermakova, Bu Young Choi, Hong Seok Choi, Bong Seok Kang, Ann M. Bode, Zigang Dong
2005 Journal of Biological Chemistry  
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
more » ... Western blot analysis using an anti-vimentin antibody. Experiments using a pull-down assay with [ 3 H]EGCG demonstrate binding of EGCG to vimentin with a K d 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-tumorpromoting 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 (1, 2). The inhibition of tumorigenesis by green or black tea preparations was demonstrated in animal models at various organ sites (3-5). The structures of the four major catechins, (Ϫ)-epigallocatechin gallate (EGCG), 1 (Ϫ)-epigallocatechin (EGC), (Ϫ)-epicatechin gallate (ECG), and (Ϫ)-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 (4, 6, 7). The inhibitory activity of EGCG against tumorigenesis has been demonstrated. The mechanisms responsible for these cancer-preven-tive 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 (8) ; also fatty acid synthase (Fas) (9), laminin, and the 67-kDa laminin receptor (10, 11). 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 (12), to identify novel proteins that bind with EGCG. The results indicated that EGCG binds with the intermediate filament (IF) protein, vimentin with high affinity (K d ϭ 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 (13, 14) . 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 (15) (16) (17) . The proteolytic derivatives indicate that the aminoterminal domain, but not the carboxyl-terminal domain, has a direct effect on filament stability and polymerization (18). 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 (19). 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 (20 -25).
doi:10.1074/jbc.m414185200 pmid:15713670 fatcat:l67ii36qnfhpdeqg2s2csgj3mi