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.
Highlights
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]
Materials—Eagle’s minimal essential medium, fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); RPMI 1640 modified medium (2 mM L-glutamine, 10 mM HEPES, 1 mM 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 [␥-32P]ATP were purchased from Amersham Biosciences; and rec-Protein G-Sepharose 4B was obtained from Zymed Laboratories Inc. (South San Francisco, CA). [3H]Epigallocatechin gallate (EGCG) (13 Ci/mmol in ethanol containing 8 mg/ml ascorbic acid) was a gift from Dr Yukihiko Hara
Fractions containing proteins binding with EGCG were analyzed by twodimensional electrophoresis and MALDI-TOF-MS to identify proteins that directly bind with EGCG
Summary
Materials—Eagle’s minimal essential medium, fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); RPMI 1640 modified medium (2 mM L-glutamine, 10 mM HEPES, 1 mM 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 [␥-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 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). 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, 1ϫ protease inhibitor mixture). In Vitro Pull-down Assay—Recombinant vimentin (2 g) or a JB6 Cl41 cellular supernatant fraction (600 g) was incubated with the EGCG-Sepharose 4B (or Sepharose 4B as control) beads (100 l, 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 g/ml bovine serum albumin, 0.02 mM PMSF, 1ϫ 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
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