Limits...
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.

Show MeSH
Effects of T564 phosphorylation in radixin on the direct  interaction between its NH2- and COOH-terminal halves. (A)  The same amounts (92 ng) of nonphosphorylated C-rad (C-rad),  T564-phosphorylated C-rad (P-C-rad), and alkaline phosphatase-treated T564-phosphorylated C-rad (P-C-rad/AP) were electrophoresed and then transferred onto nitrocellulose membranes.  They were immunoblotted with pAb TK89 (TK89) or mAb 297S  (297S) to check the amounts and the phosphorylation level of  each sample, respectively (Immunoblot). They were then incubated with the iodinated NH2-terminal half of radixin (125I-N-rad)  that was purified from GST fusion protein (Overlay). Bound 125I-N-rad was detected by autoradiography. 125I-N-rad specifically  bound to nonphosphorylated C-rad but not to T564-phosphorylated C-rad. (B) Various amounts (7.5–120 ng) of non- (C-rad)  and T564-phosphorylated C-rad (P-C-rad) were electrophoresed,  transferred onto nitrocellulose membranes, and incubated with  125I-N-rad. The amount of bound 125I-N-rad and electrophoresed  C-rad/P-C-rad was detected by autoradiography (125I-N-rad  Overlay) and Coomassie brilliant blue staining (Coomassie), respectively. 125I-N-rad specifically bound to nonphosphorylated  C-rad in a dose-dependent manner.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140160&req=5

Figure 6: Effects of T564 phosphorylation in radixin on the direct interaction between its NH2- and COOH-terminal halves. (A) The same amounts (92 ng) of nonphosphorylated C-rad (C-rad), T564-phosphorylated C-rad (P-C-rad), and alkaline phosphatase-treated T564-phosphorylated C-rad (P-C-rad/AP) were electrophoresed and then transferred onto nitrocellulose membranes. They were immunoblotted with pAb TK89 (TK89) or mAb 297S (297S) to check the amounts and the phosphorylation level of each sample, respectively (Immunoblot). They were then incubated with the iodinated NH2-terminal half of radixin (125I-N-rad) that was purified from GST fusion protein (Overlay). Bound 125I-N-rad was detected by autoradiography. 125I-N-rad specifically bound to nonphosphorylated C-rad but not to T564-phosphorylated C-rad. (B) Various amounts (7.5–120 ng) of non- (C-rad) and T564-phosphorylated C-rad (P-C-rad) were electrophoresed, transferred onto nitrocellulose membranes, and incubated with 125I-N-rad. The amount of bound 125I-N-rad and electrophoresed C-rad/P-C-rad was detected by autoradiography (125I-N-rad Overlay) and Coomassie brilliant blue staining (Coomassie), respectively. 125I-N-rad specifically bound to nonphosphorylated C-rad in a dose-dependent manner.

Mentions: The NH2-terminal halves of ERM proteins directly bind to their COOH-terminal halves, and this interdomain interaction has been reported to be important in the regulation of cross-linking activity of ERM proteins (Berryman et al., 1995; Bretscher et al., 1995). Using the gel overlay assay, domains responsible for the interdomain interaction were narrowed down in ezrin to the NH2-terminal amino acids 1–296 and the COOH-terminal amino acids 479–585 (Gary et al., 1995). T567 is located in the latter domain. Thus, we examined whether the T564 phosphorylation affects the interdomain interaction in radixin. The same amounts of non- and fully phosphorylated C-rad were electrophoresed and transferred onto nitrocellulose membranes and then incubated with the iodinated NH2-terminal half of radixin (125I-N-rad) that was purified from recombinant GST fusion protein produced in E. coli. As shown in Fig. 6 A, 125I-N-rad bound specifically to nonphosphorylated C-rad but not to phosphorylated C-rad. When phosphorylated C-rad was pretreated with alkaline phosphatase, it was dephosphorylated, which restored its binding ability to 125I-N-rad. The specific interaction between nonphosphorylated C-rad and 125I-N-rad was further confirmed by a dose–response experiment (Fig. 6 B). We thus concluded that the phosphorylation of T564 affected the direct binding between the NH2- and COOH-terminal halves of ERM proteins.


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 direct  interaction between its NH2- and COOH-terminal halves. (A)  The same amounts (92 ng) of nonphosphorylated C-rad (C-rad),  T564-phosphorylated C-rad (P-C-rad), and alkaline phosphatase-treated T564-phosphorylated C-rad (P-C-rad/AP) were electrophoresed and then transferred onto nitrocellulose membranes.  They were immunoblotted with pAb TK89 (TK89) or mAb 297S  (297S) to check the amounts and the phosphorylation level of  each sample, respectively (Immunoblot). They were then incubated with the iodinated NH2-terminal half of radixin (125I-N-rad)  that was purified from GST fusion protein (Overlay). Bound 125I-N-rad was detected by autoradiography. 125I-N-rad specifically  bound to nonphosphorylated C-rad but not to T564-phosphorylated C-rad. (B) Various amounts (7.5–120 ng) of non- (C-rad)  and T564-phosphorylated C-rad (P-C-rad) were electrophoresed,  transferred onto nitrocellulose membranes, and incubated with  125I-N-rad. The amount of bound 125I-N-rad and electrophoresed  C-rad/P-C-rad was detected by autoradiography (125I-N-rad  Overlay) and Coomassie brilliant blue staining (Coomassie), respectively. 125I-N-rad specifically bound to nonphosphorylated  C-rad in a dose-dependent manner.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Effects of T564 phosphorylation in radixin on the direct interaction between its NH2- and COOH-terminal halves. (A) The same amounts (92 ng) of nonphosphorylated C-rad (C-rad), T564-phosphorylated C-rad (P-C-rad), and alkaline phosphatase-treated T564-phosphorylated C-rad (P-C-rad/AP) were electrophoresed and then transferred onto nitrocellulose membranes. They were immunoblotted with pAb TK89 (TK89) or mAb 297S (297S) to check the amounts and the phosphorylation level of each sample, respectively (Immunoblot). They were then incubated with the iodinated NH2-terminal half of radixin (125I-N-rad) that was purified from GST fusion protein (Overlay). Bound 125I-N-rad was detected by autoradiography. 125I-N-rad specifically bound to nonphosphorylated C-rad but not to T564-phosphorylated C-rad. (B) Various amounts (7.5–120 ng) of non- (C-rad) and T564-phosphorylated C-rad (P-C-rad) were electrophoresed, transferred onto nitrocellulose membranes, and incubated with 125I-N-rad. The amount of bound 125I-N-rad and electrophoresed C-rad/P-C-rad was detected by autoradiography (125I-N-rad Overlay) and Coomassie brilliant blue staining (Coomassie), respectively. 125I-N-rad specifically bound to nonphosphorylated C-rad in a dose-dependent manner.
Mentions: The NH2-terminal halves of ERM proteins directly bind to their COOH-terminal halves, and this interdomain interaction has been reported to be important in the regulation of cross-linking activity of ERM proteins (Berryman et al., 1995; Bretscher et al., 1995). Using the gel overlay assay, domains responsible for the interdomain interaction were narrowed down in ezrin to the NH2-terminal amino acids 1–296 and the COOH-terminal amino acids 479–585 (Gary et al., 1995). T567 is located in the latter domain. Thus, we examined whether the T564 phosphorylation affects the interdomain interaction in radixin. The same amounts of non- and fully phosphorylated C-rad were electrophoresed and transferred onto nitrocellulose membranes and then incubated with the iodinated NH2-terminal half of radixin (125I-N-rad) that was purified from recombinant GST fusion protein produced in E. coli. As shown in Fig. 6 A, 125I-N-rad bound specifically to nonphosphorylated C-rad but not to phosphorylated C-rad. When phosphorylated C-rad was pretreated with alkaline phosphatase, it was dephosphorylated, which restored its binding ability to 125I-N-rad. The specific interaction between nonphosphorylated C-rad and 125I-N-rad was further confirmed by a dose–response experiment (Fig. 6 B). We thus concluded that the phosphorylation of T564 affected the direct binding between the NH2- and COOH-terminal halves of ERM proteins.

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