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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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Effects of T564 phosphorylation in  radixin on the actin filament binding ability of  C-rad. (A) Cosedimentation of non- or T564-phosphorylated C-rad with actin filaments.  Non- (C-rad) or T564-phosphorylated C-rad  (P-C-rad) was incubated with BSA (BSA) or  with actin filaments (F-actin), then centrifuged at 100,000 g. Supernatant (S) and pellet  (P) were resolved by SDS-PAGE followed  by Coomassie brilliant blue staining. Both  non- and T564-phosphorylated C-rad (C-rad/ P-C-rad) were cosedimented with actin filaments (Actin) to the same extent, but not with  BSA (BSA). (B) Quantitative analysis. 2 μM  F-actin was incubated with various amounts  of non- or T564-phosphorylated C-rad and  centrifuged, then the amounts of cosedimented  non- (C-rad; filled circles) and T564-phosphorylated C-rad (P-C-rad; open squares) were  quantified by densitometric scanning of  Coomassie brilliant blue–stained gels. (C)  Sedimentation of F-rad. Partially [32P]-phosphorylated F-rad (P-F-rad) and fully [32P]- phosphorylated C-rad (P-C-rad) were centrifuged in the presence of BSA at 100,000 or  10,000 g for 30 min. Supernatant (S) and pellet (P) were resolved by SDS-PAGE followed by Coomassie brilliant blue staining  (Coomassie), immunoblot with TK89 (TK89  Immunoblot), or autoradiography (Autoradiogram). In the absence of actin filaments,  F-rad, especially P-F-rad, were mostly recovered in pellet even at 10,000 g, whereas P-C-rad  was mainly recovered in supernatant at  100,000 g as well as 10,000 g.
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Figure 5: Effects of T564 phosphorylation in radixin on the actin filament binding ability of C-rad. (A) Cosedimentation of non- or T564-phosphorylated C-rad with actin filaments. Non- (C-rad) or T564-phosphorylated C-rad (P-C-rad) was incubated with BSA (BSA) or with actin filaments (F-actin), then centrifuged at 100,000 g. Supernatant (S) and pellet (P) were resolved by SDS-PAGE followed by Coomassie brilliant blue staining. Both non- and T564-phosphorylated C-rad (C-rad/ P-C-rad) were cosedimented with actin filaments (Actin) to the same extent, but not with BSA (BSA). (B) Quantitative analysis. 2 μM F-actin was incubated with various amounts of non- or T564-phosphorylated C-rad and centrifuged, then the amounts of cosedimented non- (C-rad; filled circles) and T564-phosphorylated C-rad (P-C-rad; open squares) were quantified by densitometric scanning of Coomassie brilliant blue–stained gels. (C) Sedimentation of F-rad. Partially [32P]-phosphorylated F-rad (P-F-rad) and fully [32P]- phosphorylated C-rad (P-C-rad) were centrifuged in the presence of BSA at 100,000 or 10,000 g for 30 min. Supernatant (S) and pellet (P) were resolved by SDS-PAGE followed by Coomassie brilliant blue staining (Coomassie), immunoblot with TK89 (TK89 Immunoblot), or autoradiography (Autoradiogram). In the absence of actin filaments, F-rad, especially P-F-rad, were mostly recovered in pellet even at 10,000 g, whereas P-C-rad was mainly recovered in supernatant at 100,000 g as well as 10,000 g.

Mentions: In some experiments (see Fig. 5 C), 3 pmol of partially [32P]phosphorylated F-rad and fully [32P]phosphorylated C-rad, which had been dialyzed against F-buffer, were centrifuged in the presence of 0.36 mg/ml BSA at 100,000 g or 10,000 g for 30 min at 20°C using siliconized tubes. Resultant supernatant and pellet were resolved in SDS-PAGE followed by Coomassie brilliant blue staining, immunoblotting with TK89, or autoradiography.


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)

Effects of T564 phosphorylation in  radixin on the actin filament binding ability of  C-rad. (A) Cosedimentation of non- or T564-phosphorylated C-rad with actin filaments.  Non- (C-rad) or T564-phosphorylated C-rad  (P-C-rad) was incubated with BSA (BSA) or  with actin filaments (F-actin), then centrifuged at 100,000 g. Supernatant (S) and pellet  (P) were resolved by SDS-PAGE followed  by Coomassie brilliant blue staining. Both  non- and T564-phosphorylated C-rad (C-rad/ P-C-rad) were cosedimented with actin filaments (Actin) to the same extent, but not with  BSA (BSA). (B) Quantitative analysis. 2 μM  F-actin was incubated with various amounts  of non- or T564-phosphorylated C-rad and  centrifuged, then the amounts of cosedimented  non- (C-rad; filled circles) and T564-phosphorylated C-rad (P-C-rad; open squares) were  quantified by densitometric scanning of  Coomassie brilliant blue–stained gels. (C)  Sedimentation of F-rad. Partially [32P]-phosphorylated F-rad (P-F-rad) and fully [32P]- phosphorylated C-rad (P-C-rad) were centrifuged in the presence of BSA at 100,000 or  10,000 g for 30 min. Supernatant (S) and pellet (P) were resolved by SDS-PAGE followed by Coomassie brilliant blue staining  (Coomassie), immunoblot with TK89 (TK89  Immunoblot), or autoradiography (Autoradiogram). In the absence of actin filaments,  F-rad, especially P-F-rad, were mostly recovered in pellet even at 10,000 g, whereas P-C-rad  was mainly recovered in supernatant at  100,000 g as well as 10,000 g.
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Related In: Results  -  Collection

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Figure 5: Effects of T564 phosphorylation in radixin on the actin filament binding ability of C-rad. (A) Cosedimentation of non- or T564-phosphorylated C-rad with actin filaments. Non- (C-rad) or T564-phosphorylated C-rad (P-C-rad) was incubated with BSA (BSA) or with actin filaments (F-actin), then centrifuged at 100,000 g. Supernatant (S) and pellet (P) were resolved by SDS-PAGE followed by Coomassie brilliant blue staining. Both non- and T564-phosphorylated C-rad (C-rad/ P-C-rad) were cosedimented with actin filaments (Actin) to the same extent, but not with BSA (BSA). (B) Quantitative analysis. 2 μM F-actin was incubated with various amounts of non- or T564-phosphorylated C-rad and centrifuged, then the amounts of cosedimented non- (C-rad; filled circles) and T564-phosphorylated C-rad (P-C-rad; open squares) were quantified by densitometric scanning of Coomassie brilliant blue–stained gels. (C) Sedimentation of F-rad. Partially [32P]-phosphorylated F-rad (P-F-rad) and fully [32P]- phosphorylated C-rad (P-C-rad) were centrifuged in the presence of BSA at 100,000 or 10,000 g for 30 min. Supernatant (S) and pellet (P) were resolved by SDS-PAGE followed by Coomassie brilliant blue staining (Coomassie), immunoblot with TK89 (TK89 Immunoblot), or autoradiography (Autoradiogram). In the absence of actin filaments, F-rad, especially P-F-rad, were mostly recovered in pellet even at 10,000 g, whereas P-C-rad was mainly recovered in supernatant at 100,000 g as well as 10,000 g.
Mentions: In some experiments (see Fig. 5 C), 3 pmol of partially [32P]phosphorylated F-rad and fully [32P]phosphorylated C-rad, which had been dialyzed against F-buffer, were centrifuged in the presence of 0.36 mg/ml BSA at 100,000 g or 10,000 g for 30 min at 20°C using siliconized tubes. Resultant supernatant and pellet were resolved in SDS-PAGE followed by Coomassie brilliant blue staining, immunoblotting with TK89, or autoradiography.

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