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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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Determination of  amino acid residues of radixin that are phosphorylated by Rho-kinase. (A)  Comparison of elution profiles of lysyl endopeptidase-digested peptides of non- and  fully phosphorylated C-rad  from reverse-phase HPLC  (C-rad and P-C-rad, respectively). Rho-kinase–dependent phosphorylation divided  and shifted the peak 31 from  nonphosphorylated C-rad into  two peaks, peaks 30 and 29,  from phosphorylated C-rad.  For details see the text. (B)  Amino acid sequences of peaks  29, 30, and 31. The PTH-threonine/PTH-dehydroaminobutyric acid ratio (see C) revealed that T564/T573 in peak  29 and T564 in peak 30 were  phosphorylated (Kato et al.,  1994; Fujita et al., 1996). (C)  The PTH-threonine/PTH-dehydroaminobutyric acid ratio of T564 and T573 in peak 30. The amounts of PTH-threonine (PTH-T) and PTH-dehydroaminobutyric  acid (PTH-DABA) were determined from the absorbance at 269 and 322 nm, respectively. When the threonine residue (T564 in this  case) was phosphorylated, the PTH-T/PTH-DABA ratio fell to below 1.0.
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Figure 2: Determination of amino acid residues of radixin that are phosphorylated by Rho-kinase. (A) Comparison of elution profiles of lysyl endopeptidase-digested peptides of non- and fully phosphorylated C-rad from reverse-phase HPLC (C-rad and P-C-rad, respectively). Rho-kinase–dependent phosphorylation divided and shifted the peak 31 from nonphosphorylated C-rad into two peaks, peaks 30 and 29, from phosphorylated C-rad. For details see the text. (B) Amino acid sequences of peaks 29, 30, and 31. The PTH-threonine/PTH-dehydroaminobutyric acid ratio (see C) revealed that T564/T573 in peak 29 and T564 in peak 30 were phosphorylated (Kato et al., 1994; Fujita et al., 1996). (C) The PTH-threonine/PTH-dehydroaminobutyric acid ratio of T564 and T573 in peak 30. The amounts of PTH-threonine (PTH-T) and PTH-dehydroaminobutyric acid (PTH-DABA) were determined from the absorbance at 269 and 322 nm, respectively. When the threonine residue (T564 in this case) was phosphorylated, the PTH-T/PTH-DABA ratio fell to below 1.0.

Mentions: The phosphorylated amino acid residues in C-rad were then determined. Phosphorylated and nonphosphorylated C-rad were purified to ∼90% homogeneity by RESOURCE™RPC column chromatography. Purified proteins were completely digested with lysyl endopeptidase and applied to the TSKgel ODS-80Ts column. As shown in Fig. 2 A, peptides from phosphorylated as well as nonphosphorylated C-rad were separated into >20 peaks. In this chromatography procedure, phosphorylated peptides are eluted faster than nonphosphorylated peptides because of their hydrophilic modification. Peak 31 from nonphosphorylated C-rad was not detected in phosphorylated C-rad, and two peaks (30 and 29) that migrated slightly faster were detected only from phosphorylated C-rad. Sequencing showed that these three peaks had identical amino acid sequences and corresponded to the COOH-terminal 11 amino acid residues of radixin (amino acids 564–574). Of the two threonine residues in this sequence (T564 and T573), no threonine, only T564, and both T564 and T573 were phosphorylated in peaks 31, 30, and 29, respectively (Fig. 2 B and C). A peak of the phosphopeptide, in which only T573 was phosphorylated, was not detected. Quantitative analyses revealed that ∼100% of T564, but at most ∼40% of T573, was phosphorylated when C-rad was incubated with Rho-Kc for 1 h. Then we concluded that the major and primary phosphorylation site of radixin by Rho-kinase was T564 and referred to the Rho-Kc–phosphorylated C-rad as T564-phosphorylated C-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)

Determination of  amino acid residues of radixin that are phosphorylated by Rho-kinase. (A)  Comparison of elution profiles of lysyl endopeptidase-digested peptides of non- and  fully phosphorylated C-rad  from reverse-phase HPLC  (C-rad and P-C-rad, respectively). Rho-kinase–dependent phosphorylation divided  and shifted the peak 31 from  nonphosphorylated C-rad into  two peaks, peaks 30 and 29,  from phosphorylated C-rad.  For details see the text. (B)  Amino acid sequences of peaks  29, 30, and 31. The PTH-threonine/PTH-dehydroaminobutyric acid ratio (see C) revealed that T564/T573 in peak  29 and T564 in peak 30 were  phosphorylated (Kato et al.,  1994; Fujita et al., 1996). (C)  The PTH-threonine/PTH-dehydroaminobutyric acid ratio of T564 and T573 in peak 30. The amounts of PTH-threonine (PTH-T) and PTH-dehydroaminobutyric  acid (PTH-DABA) were determined from the absorbance at 269 and 322 nm, respectively. When the threonine residue (T564 in this  case) was phosphorylated, the PTH-T/PTH-DABA ratio fell to below 1.0.
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Related In: Results  -  Collection

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Figure 2: Determination of amino acid residues of radixin that are phosphorylated by Rho-kinase. (A) Comparison of elution profiles of lysyl endopeptidase-digested peptides of non- and fully phosphorylated C-rad from reverse-phase HPLC (C-rad and P-C-rad, respectively). Rho-kinase–dependent phosphorylation divided and shifted the peak 31 from nonphosphorylated C-rad into two peaks, peaks 30 and 29, from phosphorylated C-rad. For details see the text. (B) Amino acid sequences of peaks 29, 30, and 31. The PTH-threonine/PTH-dehydroaminobutyric acid ratio (see C) revealed that T564/T573 in peak 29 and T564 in peak 30 were phosphorylated (Kato et al., 1994; Fujita et al., 1996). (C) The PTH-threonine/PTH-dehydroaminobutyric acid ratio of T564 and T573 in peak 30. The amounts of PTH-threonine (PTH-T) and PTH-dehydroaminobutyric acid (PTH-DABA) were determined from the absorbance at 269 and 322 nm, respectively. When the threonine residue (T564 in this case) was phosphorylated, the PTH-T/PTH-DABA ratio fell to below 1.0.
Mentions: The phosphorylated amino acid residues in C-rad were then determined. Phosphorylated and nonphosphorylated C-rad were purified to ∼90% homogeneity by RESOURCE™RPC column chromatography. Purified proteins were completely digested with lysyl endopeptidase and applied to the TSKgel ODS-80Ts column. As shown in Fig. 2 A, peptides from phosphorylated as well as nonphosphorylated C-rad were separated into >20 peaks. In this chromatography procedure, phosphorylated peptides are eluted faster than nonphosphorylated peptides because of their hydrophilic modification. Peak 31 from nonphosphorylated C-rad was not detected in phosphorylated C-rad, and two peaks (30 and 29) that migrated slightly faster were detected only from phosphorylated C-rad. Sequencing showed that these three peaks had identical amino acid sequences and corresponded to the COOH-terminal 11 amino acid residues of radixin (amino acids 564–574). Of the two threonine residues in this sequence (T564 and T573), no threonine, only T564, and both T564 and T573 were phosphorylated in peaks 31, 30, and 29, respectively (Fig. 2 B and C). A peak of the phosphopeptide, in which only T573 was phosphorylated, was not detected. Quantitative analyses revealed that ∼100% of T564, but at most ∼40% of T573, was phosphorylated when C-rad was incubated with Rho-Kc for 1 h. Then we concluded that the major and primary phosphorylation site of radixin by Rho-kinase was T564 and referred to the Rho-Kc–phosphorylated C-rad as T564-phosphorylated C-rad.

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