The stimulatory and inhibitory effects of several compounds and lignans isolated from the water extract of Taxus yunnanensis on the phosphorylation of three functional brain proteins (bovine myelin basic protein (bMBP), recombinant human tau protein (rhTP) and rat collapsin response mediator protein-2 (rCRMP-2)) by glycogen synthase kinase-3β (GSK-3β) were quantitatively compared in vitro, using ( )-epigallocatechin-3-gallate [( )EGCG] as a positive control. We found that (i) three selected Taxus lignans [(3S,4R)-4′-hydroxy-6,3′-dimethoxyisoflavan-4-ol,(7R)-7-hydroxytaxiresinol and tanegool] highly stimulated the autophosphorylation of GSK-3β and the GSK-3β-mediated phosphorylation of two basic brain proteins [bMBP (pI 11.3) and rhTP (pI 8.2)], but inhibited dose-dependently the phosphorylation of an acidic protein (rCRMP-2, pI 6.0) by the kinase; (ii) these three Taxus lignans showed binding-affinities with bMBP as well as rhTP, but had low affinities with rCRMP-2; (iii) the binding of tanegool and (7R)-7-hydroxytaxiresinol to these two basic proteins induced their novel potent phosphorylation sites for GSK-3β; and (iv) these three Taxus lignans, but not EGCG, induced Tyr-phosphorylation of GSK-3β in vitro. These results provided here suggest that (i) these three Taxus lignans act as novel effective activators for GSK-3β and the GSK-3β-mediated phosphorylation of their binding basic proteins (rhTP and bMBP); and (ii) tanegool (IC 50 1 μM) is an effective inhibitor for the phosphorylation of rCRMP-2 by the kinase in vitro.Key words Taxus lignan; glycogen synthase kinase-3β; stimulatory effect; protein phosphorylation Recently, we reported that (i) plant polyphenol epigallocatechin-3-gallate (EGCG), not quercetin and luteolin, highly stimulates the phosphorylation of human recombinant tau protein (hrTP, pI=8.2) and bovine myelin basic protein (bMBP, pI=11.3) by glycogen synthase kinase-3β (GSK-3β); (ii) EGCG has a binding-affinity with rhTP as well as bMBP; and (iii) the direct binding of EGCG to these two basic brain proteins (bMBP and rhTP) induces their novel potent phosphorylation sites for GSK-3β, and leads to highly stimulate their phosphorylation by the kinase in vitro.1) Also, we previously reported that phosphatidylinositol (PI) and two sulfated lipids [sulfatide and heparin (SH)] function as effective stimulators for the autophosphorylation of GSK-3β and the GSK-3β-mediated phosphorylation of SH-binding proteins 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. 51 kDa) and GSK-3β (approx. 47 kDa, pI=8.98)], which possess similar biochemical characteristics and substrate specificities.3-5) GSK-3 is originally ...