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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: We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho.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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In vitro phosphorylation of the full-length radixin (F-rad) and the COOH-terminal half radixin (C-rad) by the constitutively active catalytic domain of Rho-kinase (Rho-Kc). (A)  SDS-PAGE banding pattern of the reaction mixture (Silver  Staining) and its accompanying autoradiogram (Autoradiogram).  Purified 3 pmol F-rad (F-rad + Rho-Kc), C-rad (C-rad + Rho-Kc), or their mixture (F-rad + C-rad + Rho-Kc) was incubated  with 2 pmol Rho-Kc for 10 min at 30°C. Phosphorylated proteins  were resolved by SDS-PAGE followed by autoradiography.  C-rad (C-rad) was phosphorylated more heavily than F-rad  (F-rad). Rho-Kc (Rho-Kc) was autophosphorylated. (B) Time  course and stoichiometry of the phosphorylation reaction. After  30, 60, and 120 min of incubation, the phosphorylation levels of  F-rad (filled circle) and C-rad (open circle) were quantified. 2  pmol of Rho-Kc and 500 pmol of γ-[32P]ATP were added to the  reaction mixture after 60 min of incubation (arrows). (C) Phosphopeptide mapping. Phosphorylated F-rad and C-rad were digested completely with TPCK-trypsin, subjected to two-dimensional peptide mapping (first dimension, Electrophoresis; second  dimension, Chromatography) as described in Materials and  Methods, and then analyzed by autoradiography. Two radioactive spots in F-rad and C-rad may correspond to the T564- and  T573-containing tryptic fragments (see Fig. 2). To visualize the  weak spot from F-rad the autoradiogram of C-rad was overexposed, but the intensity ratio of two spots from C-rad was the  same as that from F-rad. Asterisks represent the origins of spotted samples.
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Figure 1: In vitro phosphorylation of the full-length radixin (F-rad) and the COOH-terminal half radixin (C-rad) by the constitutively active catalytic domain of Rho-kinase (Rho-Kc). (A) SDS-PAGE banding pattern of the reaction mixture (Silver Staining) and its accompanying autoradiogram (Autoradiogram). Purified 3 pmol F-rad (F-rad + Rho-Kc), C-rad (C-rad + Rho-Kc), or their mixture (F-rad + C-rad + Rho-Kc) was incubated with 2 pmol Rho-Kc for 10 min at 30°C. Phosphorylated proteins were resolved by SDS-PAGE followed by autoradiography. C-rad (C-rad) was phosphorylated more heavily than F-rad (F-rad). Rho-Kc (Rho-Kc) was autophosphorylated. (B) Time course and stoichiometry of the phosphorylation reaction. After 30, 60, and 120 min of incubation, the phosphorylation levels of F-rad (filled circle) and C-rad (open circle) were quantified. 2 pmol of Rho-Kc and 500 pmol of γ-[32P]ATP were added to the reaction mixture after 60 min of incubation (arrows). (C) Phosphopeptide mapping. Phosphorylated F-rad and C-rad were digested completely with TPCK-trypsin, subjected to two-dimensional peptide mapping (first dimension, Electrophoresis; second dimension, Chromatography) as described in Materials and Methods, and then analyzed by autoradiography. Two radioactive spots in F-rad and C-rad may correspond to the T564- and T573-containing tryptic fragments (see Fig. 2). To visualize the weak spot from F-rad the autoradiogram of C-rad was overexposed, but the intensity ratio of two spots from C-rad was the same as that from F-rad. Asterisks represent the origins of spotted samples.

Mentions: To examine whether ERM proteins are phosphorylated by Rho-kinase in a cell-free system, F-rad and C-rad (amino acids 311–583) were produced in Sf9 cells by recombinant baculovirus infection. Purified F-rad, C-rad, and the mixture of F- and C-rad were then incubated with the Rho-kinase catalytic domain (Rho-Kc) in the presence of γ-[32P]ATP. Rho-Kc, which was also produced by recombinant baculovirus infection, was previously shown to be constitutively active (Amano et al., 1996b, 1997). Autoradiography revealed that both F-rad and C-rad were phosphorylated, and that the latter was phosphorylated more efficiently than the former (Fig. 1 A). As shown in Fig. 1 B, ∼1.3 mol of phosphate was maximally incorporated into 1 mol of C-rad in a time-dependent manner, whereas at most ∼0.3 mol of phosphate was detected per mole of F-rad.


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)

In vitro phosphorylation of the full-length radixin (F-rad) and the COOH-terminal half radixin (C-rad) by the constitutively active catalytic domain of Rho-kinase (Rho-Kc). (A)  SDS-PAGE banding pattern of the reaction mixture (Silver  Staining) and its accompanying autoradiogram (Autoradiogram).  Purified 3 pmol F-rad (F-rad + Rho-Kc), C-rad (C-rad + Rho-Kc), or their mixture (F-rad + C-rad + Rho-Kc) was incubated  with 2 pmol Rho-Kc for 10 min at 30°C. Phosphorylated proteins  were resolved by SDS-PAGE followed by autoradiography.  C-rad (C-rad) was phosphorylated more heavily than F-rad  (F-rad). Rho-Kc (Rho-Kc) was autophosphorylated. (B) Time  course and stoichiometry of the phosphorylation reaction. After  30, 60, and 120 min of incubation, the phosphorylation levels of  F-rad (filled circle) and C-rad (open circle) were quantified. 2  pmol of Rho-Kc and 500 pmol of γ-[32P]ATP were added to the  reaction mixture after 60 min of incubation (arrows). (C) Phosphopeptide mapping. Phosphorylated F-rad and C-rad were digested completely with TPCK-trypsin, subjected to two-dimensional peptide mapping (first dimension, Electrophoresis; second  dimension, Chromatography) as described in Materials and  Methods, and then analyzed by autoradiography. Two radioactive spots in F-rad and C-rad may correspond to the T564- and  T573-containing tryptic fragments (see Fig. 2). To visualize the  weak spot from F-rad the autoradiogram of C-rad was overexposed, but the intensity ratio of two spots from C-rad was the  same as that from F-rad. Asterisks represent the origins of spotted samples.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: In vitro phosphorylation of the full-length radixin (F-rad) and the COOH-terminal half radixin (C-rad) by the constitutively active catalytic domain of Rho-kinase (Rho-Kc). (A) SDS-PAGE banding pattern of the reaction mixture (Silver Staining) and its accompanying autoradiogram (Autoradiogram). Purified 3 pmol F-rad (F-rad + Rho-Kc), C-rad (C-rad + Rho-Kc), or their mixture (F-rad + C-rad + Rho-Kc) was incubated with 2 pmol Rho-Kc for 10 min at 30°C. Phosphorylated proteins were resolved by SDS-PAGE followed by autoradiography. C-rad (C-rad) was phosphorylated more heavily than F-rad (F-rad). Rho-Kc (Rho-Kc) was autophosphorylated. (B) Time course and stoichiometry of the phosphorylation reaction. After 30, 60, and 120 min of incubation, the phosphorylation levels of F-rad (filled circle) and C-rad (open circle) were quantified. 2 pmol of Rho-Kc and 500 pmol of γ-[32P]ATP were added to the reaction mixture after 60 min of incubation (arrows). (C) Phosphopeptide mapping. Phosphorylated F-rad and C-rad were digested completely with TPCK-trypsin, subjected to two-dimensional peptide mapping (first dimension, Electrophoresis; second dimension, Chromatography) as described in Materials and Methods, and then analyzed by autoradiography. Two radioactive spots in F-rad and C-rad may correspond to the T564- and T573-containing tryptic fragments (see Fig. 2). To visualize the weak spot from F-rad the autoradiogram of C-rad was overexposed, but the intensity ratio of two spots from C-rad was the same as that from F-rad. Asterisks represent the origins of spotted samples.
Mentions: To examine whether ERM proteins are phosphorylated by Rho-kinase in a cell-free system, F-rad and C-rad (amino acids 311–583) were produced in Sf9 cells by recombinant baculovirus infection. Purified F-rad, C-rad, and the mixture of F- and C-rad were then incubated with the Rho-kinase catalytic domain (Rho-Kc) in the presence of γ-[32P]ATP. Rho-Kc, which was also produced by recombinant baculovirus infection, was previously shown to be constitutively active (Amano et al., 1996b, 1997). Autoradiography revealed that both F-rad and C-rad were phosphorylated, and that the latter was phosphorylated more efficiently than the former (Fig. 1 A). As shown in Fig. 1 B, ∼1.3 mol of phosphate was maximally incorporated into 1 mol of C-rad in a time-dependent manner, whereas at most ∼0.3 mol of phosphate was detected per mole of F-rad.

Bottom Line: We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho.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