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 ...
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...
Fucoidan is a sulfated polysaccharide extracted from brown seaweed. It has been demonstrated that fucoidan exhibits anti-tumor, anti-allergic, anti-coagulate, anti-microbial, anti-viral, and anti-parasitological effects of fucoidan.
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