Phosphorylation of Threonine 558 in the Carboxyl-terminal Actin-binding Domain of Moesin by Thrombin Activation of Human Platelets

doi:10.1074/jbc.270.52.31377

Phosphorylation of Threonine 558 in the Carboxyl-terminal Actin-binding Domain of Moesin by Thrombin Activation of Human Platelets (*)

From the Department of Pathology, Laboratory of Experimental Oncology, Stanford University School of Medicine, Stanford, California 94305-5324

** To whom correspondence and reprint requests should be addressed. Tel.: 415-725-8223; Fax: 415-725-4905.

Abstract

The phosphorylation and localization of the membrane-linking protein moesin was analyzed during early activation of platelets with thrombin. Activated platelets elaborate filopodia and spread to assume flat pancake-like shapes, and moesin is localized in filopodia and cell body. In resting platelets, approximately 25% of moesin molecules are phosphorylated as shown by metabolic labeling with P and by isoelectric focusing. Within seconds after exposure to thrombin, phosphorylation increases, reaching a maximum of 35% labeled molecules by 1 min, followed by a decrease to a new basal level within 5 min. This modification affects a single residue, Thr, which is located within or close to a binding site for F-actin. Rapid shifts (0-100%) in the number of phosphorylated molecules are observed in the presence of inhibitors of serine/threonine kinases and phosphatases. Inhibitors affecting tyrosine phosphorylation also modulate phosphorylation at this site suggesting that the enzymes involved in the modification of Thr are regulated by tyrosine phosphorylation. Platelets respond to both extremes of modification by forming extremely long filopodia and the absence of spreading on glass. Completely phosphorylated moesin is concentrated together with F-actin in the center of the cell. The rapid modification of moesin at or near its actin-binding domain suggests a model for regulated membrane-cytoskeleton interaction during cell activation.

Footnotes

↵^§ Recipient of partial support from the Japan Society for the Promotion of Science for Japanese Junior Scientists.
↵^¶ Supported by United States Public Health Service Training Grants CA09302 and 2T32GM07365.
↵* This work was supported in part by United States Public Health Service Grant AR41045 and California Tobacco-Related Disease Research Program Grant 4RT-0316. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽¹⁾ The abbreviations used are:

EGF

epidermal growth factor

pAs

polyclonal antiserum

pAb

polyclonal, affinity-purified antibody

BSA

bovine serum albumin

PGI²

prostacyclin

PGE¹

prostaglandin E¹

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

TEMED

N, N, N',N'-tetramethylethylenediamine

RIPA

radioimmunoprecipitation assay

PAO

phenylarsine oxide

GST

glutathione S-transferase

DIC

differential interference contrast.
↵⁽²⁾ H. Furthmayr, unpublished data.
- Received May 10, 1995.
- Revision received September 29, 1995.