title: Protein phosphatase 2C inactivates F-actin binding of human platelet moesin.
abstract: During activation of platelets by thrombin phosphorylation of Thr(558) in the C-terminal domain of the membrane-F-actin linking protein moesin increases transiently, and this correlates with protrusion of filopodial structures. Calyculin A enhances phosphorylation of moesin by inhibition of phosphatases. To measure this moesin-specific activity, a nonradioactive enzyme-linked immunosorbent assay method was developed with the synthetic peptide Cys-Lys(555)-Tyr-Lys-Thr(P)-Leu-Arg(560) coupled to bovine serum albumin as the substrate and moesin phosphorylation state-specific polyclonal antibodies for the detection and quantitation of dephosphorylation. Calyculin A-sensitive and -insensitive protein-threonine phosphatase activities were detected in platelet lysates and separated by DEAE-cellulose chromatography. The calyculin A-sensitive enzyme was identified as a type 1 protein phosphatase. The calyculin A-insensitive enzyme activity was purified to homogeneity by phenyl- Sepharose, protamine-, and phosphonic acid peptide-agarose chromatography and characterized biochemically and immunologically as a 53-kDa protein(s) and a type 2C protein phosphatase (PP2C). Phosphorylation of Thr(558) is necessary for F-actin binding of moesin in vitro. The purified enzyme, as well as bacterially made PP2Calpha and PP2Cbeta, efficiently dephosphorylate(s) highly purified platelet phospho-moesin. This reverses the activating effect of phosphorylation, and moesin no longer co-sediments with actin filaments. In vivo, regulation of these phosphatase activities are likely to influence dynamic interactions between the actin cytoskeleton and membrane constituents linked to moesin.
authors.affiliation: Department of Environmental Biology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Hishiya
authors.forename: M
authors.initials: M
authors.lastname: Ohnishi
authors.forename: S
authors.initials: S
authors.lastname: Tamura
authors.forename: F
authors.initials: F
authors.lastname: Nakamura
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D008387:Marine Toxins; D008840:Microfilament Proteins / Q000378:metabolism*; D010080:Oxazoles / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D008840:Microfilament Proteins; D010080:Oxazoles; D029701:Saccharomyces cerevisiae Proteins; C071058:moesin; C059041:calyculin A; C000607456:PPM1A protein, human; C000607457:PPM1B protein, human; C000607460:PPM1G protein, human; C087358:PTC1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C