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Protein phosphatase 2C inactivates F-actin binding of human platelet moesin
A Hishiya1, M Ohnishi, S Tamura
1Department of Environmental Biology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
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.
Insights
Platelet activation involves moesin phosphorylation, crucial for cell structure. This study identifies protein phosphatases, including PP2C, that reverse this phosphorylation, regulating actin cytoskeleton dynamics.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Platelet activation by thrombin leads to transient phosphorylation of moesin at Thr(558).
- Moesin phosphorylation correlates with filopodial protrusion, indicating a role in cytoskeletal dynamics.
- Phosphatase activity regulates moesin phosphorylation state, influencing its interaction with the actin cytoskeleton.
Purpose of the Study:
- To develop a method for measuring moesin-specific phosphatase activity.
- To identify and characterize phosphatases involved in moesin dephosphorylation in platelets.
- To investigate the role of moesin dephosphorylation in regulating F-actin binding and cytoskeletal organization.
Main Methods:
- Development of a nonradioactive enzyme-linked immunosorbent assay (ELISA) using a synthetic phosphopeptide substrate.
- Separation and identification of protein-threonine phosphatase activities in platelet lysates using DEAE-cellulose chromatography.
- Purification and biochemical/immunological characterization of calyculin A-insensitive phosphatase activity as PP2C.
Main Results:
- A novel ELISA method was established for quantifying moesin dephosphorylation.
- Both calyculin A-sensitive (Type 1) and -insensitive (Type 2C, PP2C) phosphatase activities were detected.
- Purified PP2C efficiently dephosphorylated platelet phospho-moesin, reversing its F-actin binding capability in vitro.
Conclusions:
- Protein phosphatase 2C (PP2C) plays a significant role in dephosphorylating moesin in platelets.
- Moesin dephosphorylation by PP2C is critical for regulating the interaction between the actin cytoskeleton and membrane components.
- The dynamic regulation of moesin phosphorylation/dephosphorylation by phosphatases influences platelet function and cytoskeletal organization.