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© The Rockefeller University Press, 0021-9525/1998//647 $5.00
The Journal of Cell Biology, Volume 140, Number 3, , 1998 647-657

Article

Rho-Kinase Phosphorylates COOH-terminal Threonines of Ezrin/Radixin/Moesin (ERM) Proteins and Regulates Their Head-to-Tail Association



Takeshi Matsui*, Masato Maeda*,{ddagger}, Yoshinori Doi*, Shigenobu Yonemura*, Mutsuki Amano§, Kozo Kaibuchi§, Sachiko Tsukita*,||, and Shoichiro Tsukita*

* Department of Cell Biology, {ddagger} First Department of Surgery, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan; § Division of Signal Transduction, Nara Institute of Science and Technology, Nara 630-01, Japan; and || College of Medical Technology, Kyoto University, Sakyo-ku, Kyoto 606, Japan

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho- associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and ~30 and ~100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase–dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase–dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.

Abbreviations used in this paper: C-rad, COOH-terminal half recombinant radixin; ERM, ezrin/radixin/moesin; F-rad, full-length recombinant radixin; GST, glutathione-S-transferase; LPA, lysophosphatidic acid; N-rad, NH2-terminal half recombinant radixin; pAb, polyclonal antibody; Rho-Kc, recombinant Rho-kinase catalytic domain; Rho-kinase, Rho- associated kinase; TFA, trifluoroacetic acid.


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