Phosphorylation of Moesin by Rho-associated Kinase (Rho-kinase) Plays a Crucial Role in the Formation of Microvilli-like Structures

doi:10.1074/jbc.273.52.34663

Phosphorylation of Moesin by Rho-associated Kinase (Rho-kinase) Plays a Crucial Role in the Formation of Microvilli-like Structures*

From the Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan

Abstract

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates moesin at Thr⁵⁵⁸ in vitro. Here, using a site- and phosphorylation state-specific antibody, we found that the expression of dominant active RhoA in COS7 cells induced moesin phosphorylation and the formation of microvilli-like structures at apical membranes where the Thr⁵⁵⁸-phosphorylated moesin accumulated, whereas the expression of dominant negative Rho-kinase inhibited both of these processes. The expression of dominant active Rho-kinase also induced moesin phosphorylation. When COS7 cells expressing moesin or moesin^T558A (substitution of Thr by Ala) were cultured under serum-depleted conditions, there were few microvilli-like structures, whereas microvilli-like structures remained in the cells expressing moesin^T558D (substitution of Thr by Asp). The expression of moesin^T558A inhibited the dominant active RhoA-induced formation of microvilli-like structures. These results indicate that Rho-kinase regulates moesin phosphorylation downstream of Rho in vivo and that the phosphorylation of moesin by Rho-kinase plays a crucial role in the formation of microvilli-like structures.

Footnotes

↵* This work was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, and Culture, Japan (1998), by Japan Society of the Promotion of Science Research for the Future, by the Human Frontier Science Program, and by the grant from Kirin Brewery Company Limited.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. Tel.: 81-743-72-5440; Fax: 81-743-72-5449; E-mail: kaibuchi{at}bs.aist-nara.ac.jp.
↵2 M. Amano, K. Chihara, N. Nakamura, Y. Marsuura, and K. Kaibuchi, manuscript in preparation.
↵3 N. Nakamura, M. Amano, Y. Fukata, N. Oshiro, T. Leung, L. Lim, and K. Kaibuchi, manuscript in preparation.
Abbreviations:

Rho-kinase

Rho-associated kinase

MBS

myosin-binding subunit

MLC

myosin light chain

aa

amino acids

HA

hemagglutinin

Ab

antibody

TRITC

tetramethylrhodamine isothiocyanate

LPA

lysophosphatidic acid

CAT

catalytic domain

DMEM

Dulbecco’s modified Eagle medium

FBS

fetal bovine serum

PBS

phosphate-buffered saline.
- Received August 11, 1998.
- Revision received October 16, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.