Biochemical Characterization of Epigallocatechin-3-gallate as an Effective Stimulator for the Phosphorylation of Its Binding Proteins by Glycogen Synthase Kinase-3&beta; <i>in Vitro</i>

Abstract

Glycogen synthase kinase 3 (GSK-3) is a constitutively active, proline-directed serine (Ser)/threonine (Thr) protein kinase encoded by two isoforms and GSK-3b (approx. 47 kDa)], which possess similar biochemical characteristics and substrate specificities.1-3) GSK-3 has been originally identified as a protein kinase that phosphorylates the rate-limiting enzyme, glycogen synthase (GS), in glycogen synthesis. 4,5) Further detail characterization revealed that (i) GSK-3b has a wide array of substrates, including cytoplasmic enzymes, nuclear transcriptional factors and signal-mediating molecules; and (ii) this kinase plays an important physiological role in the regulation of numerous signalling pathways involved in cell differentiation and morphological development of neurons. 5,6) In the present study, we used two basic brain proteins [bovine myelin basic protein (bMBP, pIϭ11.3) and human tau protein (hTP, pIϭ8.2)] and two acidic proteins [human vimentin (hVM, pIϭ5.1) and rat collapsing response mediator protein-2 (rCRMP-2, pIϭ6.0)] as effective substrates for GSK-3b in vitro, because (i) these proteins, containing multiple potent phosphorylation sites for GSK-3b, function as effective phosphate acceptors for the kinase in vitro; (ii) GSK3b is identified as a key protein kinase responsible for their functional regulation in brain [7][8][9][10] ; and (iii) phosphatidylinositol (PI) and two sulfated lipids [sulfatide and heparin (SH)] function as effective stimulators for autophosphorylation of GSK-3b and the GSK-3b-mediated high phosphorylation of SH-binding proteins, such as MBP and TP, in the highly accumulated levels of these acidic and sulfated modulators in the brain. 11)A number of epidemiological studies have shown that the consumption of green tea may account for the anti-oxdant, anti-inflammatory, anti-carcinogenic, and neuropotective effects.12) The chemical structures of four major catechins [(Ϫ)-epigallocatechin gallate (EGCG), (Ϫ)-epigallocatechin, (Ϫ)-epicatechin gallate and (Ϫ)-epicatechin] in green tea have been determined and EGCG is a major polyphenol in green tea and accounts for 50-80% of the total catechins in the tea.12,13) Several EGCG-binding proteins, such as plasma proteins (fibronectin, fibrinogen and histidine-rich glycoprotein), 14) fatty acid synthase (Fas), 15) 67 kDa laminin receptor (67-LR), 16) platelet derived growth factor (PDGF), 17) and vimentin (type III intermediate filament protein) 18) have been identified. The N-terminal head (50-63: SLYSSSPG-GAYVTR) of VM is identified as a target for EGCG, which inhibits dose-dependently the A-kinase-mediated phosphorylation of the head fragment, containing two phosphorylation sites at Ser-50 for A-kinase and Ser-55 for Cdc2-kinase, in vitro.18) Furthermore, early studies concerning the biological effects of EGCG in vivo revealed that (i) green tea polyphenols exhibit potentially anti-amyloidgenic effect and induce to protect neuron-like cells against amyloid b-mediated toxicity 19) ; (ii) EGCG can induce apoptosis preferentially in can...

The flavonoid quercetin ameliorates Alzheimer's disease pathology and protects cognitive and emotional function in aged triple transgenic Alzheimer's disease model mice

“…Although there are no reports of the mechanism of action of quercetin regarding tauopathy, it has been found that epigallocatechin-3-gallate (EGCG), a potent antioxidant structurally related to quercetin, induced a reduction in potentially toxic sarkosyl-soluble phospho-tau isoforms (Rezai-Zadeh et al, 2008). Additionally, EGCG-inducible phosphorylation sites on GSK-3β have been demonstrated in vitro (Miyai et al, 2010). This mechanism has been recognized to underlie the Morin-flavonoid-induced reduction of tau hyperphosphorylation (Gong et al, 2011).…”

Biology-oriented development of novel lipophilic antioxidants with neuroprotective activity

“…In fact, GSK-3b is signicantly involved in key pathological events, namely Tau hyperphosphorylation, amyloidogenesis, inammatory response and ACh decit. 55 As the modulation of GSK-3b activity was also mediated by a diversity of polyphenolic systems [56][57][58] it was decided to screen HCAs and derivatives (compounds 1, 2 and 7-14) towards GSK-3b, at a xed concentration of 10 mM, using a previously described luminescent technique. Recent studies report the ability of compound 1 and its natural phenethyl ester derivative (CAPE) to modulate the Akt/GSK-3b signalling pathway and GSK-3b activity.…”

Biochemical Characterization of Novel Lignans Isolated from the Wood of Taxus yunnanensis as Effective Stimulators for Glycogen Synthase Kinase-3β and the Phosphorylation of Basic Brain Proteins by the Kinase in Vitro

“…2) From these previous reports, 1,2) we conclude that both EGCG and SH may function as effective stimulators for the GSK-3β-mediated phosphorylation of these two basic brain proteins (bMBP and rhTP), containing multiple potent phosphorylation sites for the kinase in vitro. 1,2) Glycogen synthase kinase 3 (GSK-3) is a constitutively active, proline-directed serine (Ser)/threonine (Thr) protein kinase encoded by two isoforms [GSK-3α (approx.…”

“…EGCG had binding affinities with these two basic proteins, as previously reported. 1) However, the binding abilities of these three Taxus compounds with three protein substrates (bMBP, rhTP and rCRMP-2) did …”

“…1,2) Glycogen synthase kinase 3 (GSK-3) is a constitutively active, proline-directed serine (Ser)/threonine (Thr) protein kinase encoded by two isoforms [GSK-3α (approx. 51 kDa) and GSK-3β (approx.…”