Epigallocatechin‐3‐gallate and penta‐O‐galloyl‐β‐d‐glucose inhibit protein phosphatase‐1

Abstract

Protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) are responsible for the dephosphorylation of the majority of phosphoserine ⁄ threonine residues in cells. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG) and 1,2,3,4,6-penta-O-galloyl-b-D-glucose (PGG), polyphenolic constituents of green tea and tannins, inhibit the activity of the PP1 recombinant d-isoform of the PP1 catalytic subunit and the native PP1 catalytic subunit (PP1c) with IC 50 values of 0.47-1.35 lM and 0.26-0.4 lM, respectively. EGCG and PGG inhibit PP2Ac less potently, with IC 50 values of 15 and 6.6 lM, respectively. The structure-inhibitory potency relationships of catechin derivatives suggests that the galloyl group may play a major role in phosphatase inhibition. The interaction of EGCG and PGG with PP1c was characterized by NMR and surface plasmon resonance-based binding techniques. Competitive binding assays and molecular modeling suggest that EGCG docks at the hydrophobic groove close to the catalytic center of PP1c, partially overlapping with the binding surface of microcystin-LR or okadaic acid. This hydrophobic interaction is further stabilized by hydrogen bonding via hydroxyl ⁄ oxo groups of EGCG to PP1c residues. Comparative docking shows that EGCG binds to PP2Ac in a similar manner, but in a distinct pose. Long-term treatment (24 h) with these compounds and other catechins suppresses the viability of HeLa cells with a relative effectiveness reminiscent of their in vitro PP1c-inhibitory potencies. The above data imply that the phosphatase-inhibitory features of these polyphenols may be implicated in the wide spectrum of their physiological influence.Abbreviations CLA, calyculin-A; EC, (-)-epicatechin; ECG, (-)-epicatechin-3-gallate; EGC, (-)-epigallocatechin; EGCG, (-)-epigallocatechin-3-gallate; ERK, extracellular signal-related kinase; GCG, (-)-gallocatechin-3-gallate; JNK, c-Jun NH 2 -terminal kinase; MC-LR, microcystin-LR; MLC20, 20-kDa myosin light chain; MP, myosin phosphatase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; MYPT1, myosin phosphatase target subunit-1; OA, okadaic acid; PGG, 1,2,3,4,6-penta-O-galloyl-b-D-glucose; PP1, protein phosphatase-1; PP1c, protein phosphatase-1 catalytic subunit; PP1cd, hexahistidine-tagged recombinant protein phosphatase-1 catalytic subunit d-isoform; PP2A, protein phosphatase-2A; PP2Ac, protein phosphatase-2A catalytic subunit; RU, response unit; SPR, surface plasmon resonance; STD, saturation transfer difference; TM, tautomycin.

Long-Term Oral Administration of Theaphenon-E Improves Cardiomyocyte Mechanics and Calcium Dynamics by Affecting Phospholamban Phosphorylation and ATP Production

“…Of note EGCG, possibly through its galloyl group, inhibits the activity of PP1 at physiological achievable concentrations. Comparative docking assay shows that EGCG binds to PP2A in a similar manner, but in a distinct domain [53,54]. This inhibitory effect of the EGCG on phosphatases might be invoked to explain the higher level of PLB phosphorylation in GTE cardiomyocytes.…”

Pentagalloyl Glucose and Its Functional Role in Vascular Health: Biomechanics and Drug-Delivery Characteristics

“…This liposome encapsulation method has been utilized for epigallocatechin-3-gallate (EGCG) 201 (a polyphenolic compound similar to PGG 85 ) and IC 50 of PGG, which is almost half that of EGCG. 16 This technique can be extended for improvement of PGG bioavailability.…”

Interplay of myosin phosphatase and protein phosphatase-2A in the regulation of endothelial nitric-oxide synthase phosphorylation and nitric oxide production

“…In our previous work we showed that EGCG interacted with and inhibited PP1c activity at micromolar concentrations in in vitro phosphatase assays and binding of EGCG to the hydrophobic groove of the PP1c substrate binding region was also established42. However, only moderate extent of phosphatase inhibition occurred when intact cells were incubated with relatively high EGCG concentration (100–500 μM) and it was presumably due to low extent of EGCG permeation through the cell membranes43.…”