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-3β (GSK-3β) were comparatively determined in vitro. We found that (i) EGCG, not quercetin and luteolin, highly stimulated the GSK-3β-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-3β; (iii) only EGCG significantly enhanced autophosphorylation of GSK-3β; 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-3β, and to significantly reduce the K(m) value and increase the V(max) value of these two substrate proteins for the kinase in vitro.