Biochemical Characterization of Epigallocatechin-3-gallate as an Effective Stimulator for the Phosphorylation of Its Binding Proteins by Glycogen Synthase Kinase-3β in Vitro

The stimulatory and inhibitory effects of epigallocatechin-3-gallate (EGCG) and its related two compounds (luteolin and quercetin) on the phosphorylation of four proteins [bovine myelin basic protein (bMBP), human recombinant tau protein (hrTP), human recombinant vimentin (hrVM) and rat collapsin response mediator protein-2 (rCRMP-2)] by glycogen synthase kinase-3 beta (GSK-3 beta) were comparatively determined in vitro We found that (1) EGCG, not quercetin and luteolin, highly stimulated the GSK-3 beta-mediated phosphorylation of hrTP and significantly stimulated the phosphorylation of bMBP and hrVM by the kinase, (ii) these three polyphenols inhibited dose-dependently the phosphorylation of rCRMP-2 by GSK-3 beta, (m) only EGCG significantly enhanced autophosphorylation of GSK-3 beta and (iv) EGCG had a binding-affinity with two basic proteins (bMBP and hrTP) and a low affinity with rCRMP-2 rather than hrVM in vitro In addition, the binding of EGCG to these two basic proteins induced to highly stimulate their phosphorylation, including novel potent sites for GSK-3 beta, and to significantly reduce the K-m value and increase the V-max value of these two substrate proteins for the kinase in vitro These results provided here suggest that EGCG acts as an effective stimulator for the GSK-3 beta-mediated phosphorylation of its binding proteins containing EGCG-inducible phosphorylation sites for the kinase in vitro