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Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association.

Matsui T, Maeda M, Doi Y, Yonemura S, Amano M, Kaibuchi K, Tsukita S, Tsukita S - J. Cell Biol. (1998)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

ABSTRACT
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 approximately 30 and approximately 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.

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Production of mAb 297S that distinguishes T564-phosphorylated from nonphosphorylated radixin. Nonphosphorylated  C-rad (100 ng; C-rad), T564-phosphorylated C-rad (100 ng; P-C-rad),  and whole-cell lysate of Swiss 3T3 cells under conventional culture conditions (25 μg; Swiss 3T3 lysate) were immunoblotted  with pAb TK89 (pAb TK89) or mAb 297S (mAb 297S). The  former antibody recognized radixin as well as ezrin and moesin  irrespective of their phosphorylation state, whereas the latter distinguished T564-phosphorylated C-rad from the nonphosphorylated molecule. The mAb 297S recognized ezrin and moesin that  were phosphorylated at the corresponding threonine residues,  T567 and T558, respectively.
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Figure 3: Production of mAb 297S that distinguishes T564-phosphorylated from nonphosphorylated radixin. Nonphosphorylated C-rad (100 ng; C-rad), T564-phosphorylated C-rad (100 ng; P-C-rad), and whole-cell lysate of Swiss 3T3 cells under conventional culture conditions (25 μg; Swiss 3T3 lysate) were immunoblotted with pAb TK89 (pAb TK89) or mAb 297S (mAb 297S). The former antibody recognized radixin as well as ezrin and moesin irrespective of their phosphorylation state, whereas the latter distinguished T564-phosphorylated C-rad from the nonphosphorylated molecule. The mAb 297S recognized ezrin and moesin that were phosphorylated at the corresponding threonine residues, T567 and T558, respectively.

Mentions: Thus, to selectively detect T564 phosphorylation of radixin, we raised an mAb that can distinguish T564-phosphorylated radixin from the nonphosphorylated molecule. As an antigen, we synthesized a phosphopeptide corresponding to the COOH-terminal amino acids 559–569 of radixin in which T564 is phosphorylated. Since this amino acid sequence was completely conserved among ERM proteins, it was expected that mAb specific for this antigen would recognize not only T564-phosphorylated radixin but also T567-phosphorylated ezrin and T558-phosphorylated moesin. After intensive screening, one mAb, 297S, was obtained. As shown in Fig. 3, this mAb specifically recognized T564-phosphorylated C-rad, but not nonphosphorylated C-rad. When the whole cell lysate of a semiconfluent culture of Swiss 3T3 cells was immunoblotted with mAb 297S, ezrin and moesin as well as radixin were clearly detected. Since these cells were cultured in the presence of serum, we next examined the phosphorylation level of respective T567, T564, and T558 of ERM proteins in the serum-starved cells.


Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association.

Matsui T, Maeda M, Doi Y, Yonemura S, Amano M, Kaibuchi K, Tsukita S, Tsukita S - J. Cell Biol. (1998)

Production of mAb 297S that distinguishes T564-phosphorylated from nonphosphorylated radixin. Nonphosphorylated  C-rad (100 ng; C-rad), T564-phosphorylated C-rad (100 ng; P-C-rad),  and whole-cell lysate of Swiss 3T3 cells under conventional culture conditions (25 μg; Swiss 3T3 lysate) were immunoblotted  with pAb TK89 (pAb TK89) or mAb 297S (mAb 297S). The  former antibody recognized radixin as well as ezrin and moesin  irrespective of their phosphorylation state, whereas the latter distinguished T564-phosphorylated C-rad from the nonphosphorylated molecule. The mAb 297S recognized ezrin and moesin that  were phosphorylated at the corresponding threonine residues,  T567 and T558, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140160&req=5

Figure 3: Production of mAb 297S that distinguishes T564-phosphorylated from nonphosphorylated radixin. Nonphosphorylated C-rad (100 ng; C-rad), T564-phosphorylated C-rad (100 ng; P-C-rad), and whole-cell lysate of Swiss 3T3 cells under conventional culture conditions (25 μg; Swiss 3T3 lysate) were immunoblotted with pAb TK89 (pAb TK89) or mAb 297S (mAb 297S). The former antibody recognized radixin as well as ezrin and moesin irrespective of their phosphorylation state, whereas the latter distinguished T564-phosphorylated C-rad from the nonphosphorylated molecule. The mAb 297S recognized ezrin and moesin that were phosphorylated at the corresponding threonine residues, T567 and T558, respectively.
Mentions: Thus, to selectively detect T564 phosphorylation of radixin, we raised an mAb that can distinguish T564-phosphorylated radixin from the nonphosphorylated molecule. As an antigen, we synthesized a phosphopeptide corresponding to the COOH-terminal amino acids 559–569 of radixin in which T564 is phosphorylated. Since this amino acid sequence was completely conserved among ERM proteins, it was expected that mAb specific for this antigen would recognize not only T564-phosphorylated radixin but also T567-phosphorylated ezrin and T558-phosphorylated moesin. After intensive screening, one mAb, 297S, was obtained. As shown in Fig. 3, this mAb specifically recognized T564-phosphorylated C-rad, but not nonphosphorylated C-rad. When the whole cell lysate of a semiconfluent culture of Swiss 3T3 cells was immunoblotted with mAb 297S, ezrin and moesin as well as radixin were clearly detected. Since these cells were cultured in the presence of serum, we next examined the phosphorylation level of respective T567, T564, and T558 of ERM proteins in the serum-starved cells.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

ABSTRACT
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 approximately 30 and approximately 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.

Show MeSH
Related in: MedlinePlus