Protein phosphatase 2C inactivates F-actin binding of human platelet moesin

Abstract

During activation of platelets by thrombin phosphorylation of Thr558 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-Lys555-Tyr-Lys-Thr(P)Leu-Arg560 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 Thr558 is necessary for F-actin binding of moesin in vitro. The purified enzyme, as well as bacterially made PP2Cα and PP2Cβ, efficiently dephosphorylate(s) highly purified platelet phosphomoesin. 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.