* Department of Cell Biology, First Department of Surgery, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan; § Division of Signal Transduction, Nara Institute of Science and Technology, Nara 630-01, Japan; and
College of Medical
Technology, Kyoto University, Sakyo-ku, Kyoto 606, Japan
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Abstract |
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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 ~30 and ~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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Introduction |
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THE ezrin/radixin/moesin (ERM)1 family consists of
three closely related proteins, ezrin, radixin, and
moesin (Bretscher et al., 1983; Pakkanen et al.,
1987; Lankes et al., 1988
; Tsukita et al., 1989
; Sato et al.,
1992
). Sequencing analyses have revealed that the three
ERM proteins are highly homologous; in the mouse, the
levels of identity are 75, 72, and 80% for ezrin/radixin, ezrin/moesin, and radixin/moesin, respectively (Gould et
al., 1989
; Turunen et al., 1989
; Funayama et al., 1991
; Lankes
and Furthmayr, 1991
; Sato et al., 1992
). The sequences of
their NH2-terminal halves are highly conserved (~85%
identity for all pairs). A tumor suppressor molecule responsible for neurofibromatosis type 2 named merlin or
schwannomin was identified and shown to have significant sequence similarity to ERM proteins; ~49% identity overall and ~65% identity in the NH2-terminal halves (Rouleau et al., 1993
; Trofatter et al., 1993
). This highly conserved
NH2-terminal sequence is also found in the NH2-terminal
ends of some membrane-associated proteins such as band
4.1 protein, talin, PTPH1, and PTPMEG, indicating the
existence of a band 4.1 superfamily (Conboy et al., 1986
; Rees et al., 1990
; Gu et al., 1991
; Yang and Tonks, 1991
;
Arpin et al., 1994
; Takeuchi et al., 1994a
).
ERM family proteins are thought to function as general
cross-linkers between the plasma membrane and actin filaments (Bretscher, 1983; Pakkanen et al., 1987
; Tsukita et
al., 1989
; Algrain et al., 1993
). The suppression of ERM
protein expression with antisense oligonucleotides destroys microvilli, cell-cell, and cell-matrix adhesion sites
(Takeuchi et al., 1994b
), and the introduction of dominant-negative constructs of radixin impairs cytokinesis (Henry et al., 1995
). The highly conserved NH2-terminal
halves of ERM proteins are responsible for their association with the plasma membrane. CD44 was identified as a
binding partner of ERM proteins on the plasma membrane (Tsukita et al., 1994
). Integral membrane proteins
such as CD43, intercellular adhesion molecule (ICAM)-2,
ICAM-3, and the H+/K+ ATPase pump have also been reported to be colocalized with ERM proteins (Hanzel et al.,
1991
; Yonemura et al., 1993
; Helander et al., 1996
; Serrador et al., 1997
). On the other hand, the COOH-terminal halves of ERM proteins, especially the COOH-terminal 34 amino acids, interacts with actin filaments (Turunen et al.,
1994
; Pestonjamasp et al., 1995
). The coexistence of
plasma membrane-binding and actin filament-binding domains in individual molecules allows ERM proteins to
function as plasma membrane/actin filament cross-linkers.
However, both the actin- and membrane-binding domains are thought to be masked in native full-length ERM
proteins. As mentioned above, the COOH-terminal half
of ezrin binds to actin filaments. However, the binding of
native full-length ERM proteins to actin filaments has not
been directly established under physiological conditions,
although one recent paper describes that purified ezrin
binds to nonmuscle -actin filaments with high affinity (Yao et al., 1996
). Similarly, at physiological ionic strength, full-length ERM proteins show very low affinity to the cytoplasmic domain of CD44 in vitro, whereas the NH2-terminal halves of ERM proteins lacking the COOH-terminal halves bind to CD44 with high affinity (Hirao et al.,
1996
). Furthermore, the NH2-terminal halves of ERM
proteins can be directly associated with their COOH-terminal halves in vitro (Gary and Bretscher, 1993
, 1995
; Andréoli et al., 1994
; Magendantz et al., 1995
). These findings
suggested an intramolecular and/or intermolecular head-to-tail association mechanism for ERM protein activation
and inactivation (Berryman et al., 1995
; Bretscher et al.,
1995
; Martin et al., 1995
). Through head-to-tail association, the NH2- and COOH-terminal halves of native ERM
proteins are thought to mutually suppress their functions, i.e., membrane and actin binding, respectively.
Some signal must release the mutual suppression in
ERM proteins within cells, so that they can function as
cross-linkers just beneath the plasma membrane. Previously we reported that Rho, a small GTP-binding protein,
regulates the formation of CD44/ERM protein complex
(Hirao et al., 1996). Takaishi et al. (1995)
and Kotani et al.
(1997)
also suggested that in MDCK cells Rho regulates the association of ERM proteins with plasma membranes.
Ridley and coworkers (Ridley and Hall, 1992
; Ridley et
al., 1992
) found using serum-starved Swiss 3T3 cells that
Rho plays a central role in the coordinated assembly of focal adhesions and stress fibers induced by growth factors
such as lysophosphatidic acid (LPA). More recently, this
group identified moesin as an essential factor for the Rho-dependent formation of stress fibers in serum-starved Swiss 3T3 cells (Mackay et al., 1997
). However, how the
Rho signaling pathway is involved in the regulation of the
cross-linking activity of ERM proteins at the molecular
level is still unclear.
Rho was reported to regulate the activities of some
serine/threonine kinases including Rho-associated kinase
(Rho-kinase)/ROK (Leung et al., 1995
; Matsui et al.,
1996
), p160 ROCK (Ishizaki et al., 1996
), protein kinase N
(Amano et al., 1996a
; Watanabe et al., 1996
), and protein
kinase C1 (Nonaka et al., 1995
). On the other hand, ERM
proteins are highly serine/threonine phosphorylated (Gould
et al., 1986
; Urushidani et al., 1989
; Nakamura et al., 1995
,
1996
), and their phosphorylation has been suggested to be involved in the regulation of the ERM protein/membrane
association (Chen et al., 1995
). In this study, we examined
whether ERM proteins are directly phosphorylated by
Rho-kinase and whether their functions are regulated by
Rho-kinase-dependent phosphorylation. First we incubated recombinant full-length radixin (F-rad) or COOH-terminal half radixin (C-rad) with the constitutively active
catalytic domain of Rho-kinase and found that ~100% of
C-rad molecules were phosphorylated mainly at the
COOH-terminal threonine residue (T564), which was
identical to the phosphorylation site of moesin in thrombin-stimulated platelets (Nakamura et al., 1995
, 1996
).
Furthermore, using mAb we found that this type of threonine phosphorylation was induced not only in radixin but
also in ezrin and moesin in vivo in a Rho-dependent manner. Finally, this type of phosphorylation affected the intramolecular and/or intermolecular head-to-tail association of ERM proteins. These findings will lead to a better
understanding of the Rho-dependent mechanism of regulation, not only ERM protein functions but also general
actin/plasma membrane interactions.
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Materials and Methods |
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Antibodies and Recombinant Proteins
The polyclonal antibody (pAb) TK89 was raised in rabbits against a synthesized peptide corresponding to mouse radixin sequence from amino acids 551-570. TK89 detected the COOH-terminal halves of all ERM proteins.
Recombinant F-rad, C-rad (311-583 amino acids), and glutathione-
S-transferase (GST) fusion proteins with the catalytic domain of Rho-kinase
(Rho-Kc; amino acids 6-553) were produced in Sf9 cells by recombinant
baculovirus infection and purified as described previously (Amano et al.,
1996b; Hirao et al., 1996
). The GST fusion protein with the NH2-terminal
half of radixin (1-310 amino acids; GST-N-rad) was expressed in Escherichia coli. Purified GST-N-rad was then cleaved with thrombin to remove
the GST according to the manufacturer's instructions. Purified recombinant proteins were concentrated with a Centricon-10 concentrator (Amicon, Beverly, MA).
In Vitro Kinase Reaction
The kinase reaction for Rho-Kc was carried out in 50 µl of reaction mixture containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 5 mM MgCl2, 250 µM -[32P]ATP (1-20 GBq/mmol), 2 pmol
Rho-Kc, and various amounts of F-rad, C-rad, or their mixture. 0.2 pmol
Rho-Kc was also used in some experiments, but to complete the kinase reaction as quickly as possible, 2 pmol Rho-Kc was used in most experiments. Initially, we produced F-rad and C-rad in E. coli. However, these
recombinant proteins were not phosphorylated efficiently by Rho-Kc, and
so we produced them in Sf9 cells by recombinant baculovirus infection. After 10 min of incubation at 30°C, the reaction mixture was boiled in
SDS-PAGE sample buffer and resolved by SDS-PAGE. The 32P signals
were analyzed by autoradiography (Fujix Bioimage Analyzer Bas 2000 System; Fuji Film Co. Ltd., Tokyo, Japan), and F-rad or C-rad was detected by silver staining.
Phosphopeptide Mapping
Two-dimensional mapping of N-1-tosylamide-2-phenylethylchloromethyl
ketone (TPCK)-treated trypsin peptides from F-rad and C-rad was performed as described previously with slight modification (Tanabe et al.,
1981). F-rad or C-rad (5 µg) was maximally phosphorylated as described
above and resolved by SDS-PAGE. Each band was cut out from the gel,
and the gel slices containing radioactive F-rad or C-rad were homogenized
in 500 µl of 50 mM NH4HCO3 containing 25 µg trypsin, pH 8.4. After 20 h
incubation at 37°C, the solution containing tryptic peptides was lyophilized and dissolved in 20 µl of TLE buffer (mixture of acetic acid, formic acid, and H2O at a 15:5:80 ratio). 10-µl aliquots of these solutions
were spotted on silica gel-coated thin layer chromatography plates, and
tryptic peptides were resolved by electrophoresis in the first dimension at
1,000 V for 1 h in TLE buffer using an AB Multiphor II (Pharmacia Biotech Sverige, Uppsala, Sweden) at 4°C, and by chromatography in the second dimension in thin layer chromatography buffer (mixture of butanol,
pyridine, acetic acid, and H2O at a 32.5:25:5:20 ratio) for 4 h. The plates
were then dried and the 32P signals were analyzed by autoradiography.
Determination of Phosphorylated Amino Acid Residues
C-rad (30 µg) was phosphorylated by Rho-Kc as described above, except
that the reaction was carried out for 60 min in the presence of cold ATP
instead of -[32P]ATP. As a control, nonphosphorylated C-rad was prepared in the same reaction buffer containing no ATP. Both phosphorylated and nonphosphorylated samples were separated by HPLC on a RESOURCETM RPC column (1 ml; Pharmacia Biotech Sverige) preequilibrated
with 0.1% trifluoroacetic acid (TFA). Elution was performed for 60 min
with a linear gradient of acetonitrile (0-90%) containing 0.1% TFA at a
flow rate of 1 ml/min. Purified proteins were evaporated and digested with
1 µg of lysyl endopeptidase in 260 µl of 100 mM Tris-HCl, pH 8.5, for 12 h
at 37°C. The digested peptides were separated by HPLC on a TSKgel
ODS-80Ts column (0.46 × 15 cm; Tosoh Co., Tokyo, Japan) preequilibrated with 0.1% TFA. Elution was performed for 75 min with a linear
gradient of acetonitrile (0-50%) containing 0.1% TFA. Elution profiles at
206 nm were compared between phosphorylated and nonphosphorylated
C-rad, and the phosphorylated amino acid residues were determined by
amino acid sequence analysis according to the method developed previously (see Results for details; Kato et al., 1994
; Fujita et al., 1996
).
mAb Production
mAbs were raised using BDF1 mice according to the method previously
described (Tsukita et al., 1994). A phosphopeptide (CRDKYKpTLRQIR) corresponding to amino acids 559-569 of radixin was synthesized and used as an antigen. One hybridoma clone producing an antibody (297S) that could distinguish between phosphorylated and nonphosphorylated C-rad was selected, expanded, and recloned.
Lysophosphatidic Acid Treatment of Serum-starved Swiss 3T3 Cells
Confluent serum-starved Swiss 3T3 cells were prepared according to the
method developed by Ridley and Hall (1992) with slight modifications
(Kumagai et al., 1993
). Cells were seeded and cultured at a density of 3 × 105 in 6-cm culture dishes in DME supplemented with 10% FCS for 7-10 d.
They were then transferred to FCS-free DME and culture was continued
for 12 h. In some experiments, before serum starvation, cells were treated
with 30 µg/ml C3 exoenzyme in the presence of 3 µl/ml lipofectamine
(GIBCO BRL, Gaithersburg, MD) for 24 h in DME containing FCS. The
serum-starved cells were stimulated with 1 µg/ml lysophosphatidic acid
(LPA), incubated for various periods, and then treated with 75 µl of SDS-PAGE sample buffer. After sonication, samples were then resolved by
SDS-PAGE and transfered to polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore Corp., Bedford, MA), followed by immunoblotting with TK89 pAb or 297S mAb.
Cosedimentation Experiments with Actin Filaments
Actin was purified from rabbit skeletal muscle as described previously
(Tsukita et al., 1988). Gel-filtered G-actin was stored in G-buffer (2 mM
Tris-HCl, pH 7.5, 0.2 mM ATP, 0.5 mM DTT, and 0.2% NaN3), and diluted at 20°C to 8 µM with F-buffer (20 mM Tris-HCl, pH 7.5, 75 mM KCl,
10 mM NaCl, 2 mM DTT, and 2.5 mM MgCl2) to initiate polymerization.
Phosphorylated and nonphosphorylated C-rad were prepared as described above, except that the reaction was carried out for 60 min in the
presence of cold ATP instead of
-[32P]ATP, and then dialyzed against
F-buffer, followed by centrifugation at 100,000 g for 30 min at 20°C. After
actin filaments were polymerized for 30 min, various amounts of phosphorylated or nonphosphorylated C-rad in F-buffer (20 µl) were added to 20 µl of actin filament solution (or the solution containing 0.72 mg/ml BSA)
and incubated for 30 min at 20°C. After centrifugation at 100,000 g for 30 min at 20°C in a Beckman TLA100 rotor (Beckman Instrs., Inc., Fullerton, CA) the supernatant and pellet were resolved by SDS-PAGE. Coomassie brilliant blue-stained gels were analyzed densitometrically using Adobe PhotoshopTM 3.0J.
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.
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Alkaline Phosphatase Treatment
Fully phosphorylated C-rad (10 µg) was dialyzed against AP buffer (50 mM Tris-HCl, pH 8.2, 50 mM NaCl, 1 mM MgCl2, 1 mM DTT, and 1 mM p-amidino PMSF), then incubated with 50 µl of glutathione-Sepharose 4B beads (Pharmacia Biotech Sverige) for 1 h at 4°C to absorb Rho-Kc (GST fusion protein). After the Rho-Kc-bound beads were removed by centrifugation, phosphorylated C-rad was concentrated with a Centricon-10 concentrator (Amicon Corp., Danvers, MA) up to 500 µg/ml. Calf intestine alkaline phosphatase (20 U/500 ng phosphorylated C-rad; Takara Shuzo Co., Ltd., Ohtsu, Japan) was then added and incubated for 1 h at 30°C.
Protein Iodination
Purified N-rad was iodinated with 125I using IODO-BEADS (Pierce Chemical Co., Rockford, IL). In brief, 100 µg of N-rad was incubated with IODO-BEADS for 4 min at 20°C in the presence of 0.5 mCi of [125I]NaI in 0.5 ml PBS, pH 7.5. The reaction was terminated by removal of solution from the IODO-BEADS, and labeled protein was separated from free 125I on a NAP-10 column (Pharmacia Biotech Sverige).
Gel Overlay Assay for Interdomain Interaction
Phosphorylated, nonphosphorylated C-rad, and alkaline phosphatase-treated phosphorylated C-rad were prepared as described above, except that the reaction was carried out for 60 min in the presence of cold ATP. The same amounts of each C-rad preparation (92 ng) was resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Nitrocellulose membranes were incubated for 60 min with 1% nonfat dried milk, 0.1% Tween 20, 25 mM Tris-HCl, pH 7.5, and 150 mM NaCl, and then with 5 µg/ml purified 125I-labeled N-rad (107 cpm/pmol) in 1% nonfat dried milk, 0.1% Tween 20, 25 mM Tris-HCl, pH 7.5, and 150 mM NaCl, for 1 h at 20°C. After washing with 10 mM Tris-HCl and 250 mM NaCl, pH 7.5, C-rad and bound 125I-labeled N-rad were detected by immunoblotting with pAb TK89 (or mAb 297S) and autoradiography, respectively.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting
One-dimensional SDS-PAGE (12.5%) was performed according to the
method of Laemmli (1970). After electrophoresis, proteins were electrophoretically transferred from gels onto nitrocellulose membranes that
were then incubated with the first antibody. Bound antibodies were visualized with alkaline phosphatase-conjugated goat anti-rabbit IgG and the
appropriate substrates as described by the manufacturer (Amersham International, Buckinghamshire, UK).
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Results |
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Phosphorylation of Radixin by Rho-Kinase In Vitro
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
-[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.
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Rho-Kinase-dependent Phosphorylation Sites of Radixin
We next compared the phosphorylation sites of F-rad and C-rad by phosphopeptide mapping. F-rad and C-rad were phosphorylated in vitro, digested completely with TPCK-treated trypsin, and subjected to two-dimensional peptide mapping followed by autoradiography. As shown in Fig. 1 C, phosphorylated F-rad and C-rad gave the identical phosphopeptide mapping pattern, indicating that the Rho-kinase-dependent phosphorylation sites of F-rad are located only in its COOH-terminal half.
The phosphorylated amino acid residues in C-rad were then determined. Phosphorylated and nonphosphorylated C-rad were purified to ~90% homogeneity by RESOURCETMRPC 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-dependent Phosphorylation of the COOH-terminal Threonine Residue of ERM Proteins In Vivo
We then attempted to determine whether T564 of radixin
was phosphorylated in a Rho-dependent manner in vivo.
Rho was reported to be activated when serum-starved
confluent Swiss 3T3 are stimulated with LPA, resulting in
the formation of focal contacts and stress fibers (Ridley
and Hall 1992; Ridley et al., 1992
). Using this system with
32P-labeled Swiss 3T3 cells, we first examined whether
ERM proteins were phosphorylated in a Rho-dependent
manner. However, even under the serum-starved condition, ERM proteins were fairly phosphorylated, and the
Rho-dependent increase of ERM phosphorylation was
failed to be detected (data not shown).
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.
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In serum-starved confluent Swiss 3T3 cells, we quantitatively determined the amount of total and T564-phosphorylated radixin/T558-phosphorylated moesin by scanning
densitometry of pAb TK89 and mAb 297S immunoblots, respectively, using purified phosphorylated C-rad to generate standard curves. The phosphorylation level of T567
in ezrin was difficult to be quantitatively analyzed due to
its low expression level in Swiss 3T3 cells. We found that
even under the serum-starved condition ~25% of radixin/
moesin were phosphorylated at their COOH-terminal
threonine residue. Immunoblotting with mAb 297S revealed that within 30 s after LPA stimulation, the COOH-terminal threonine residue of radixin/moesin was rapidly
phosphorylated (two- to threefold over the basal level)
followed by rapid dephosphorylation (Fig. 4, A and B).
This LPA-induced rapid phosphorylation of radixin/moesin was significantly suppressed in the presence of C3 toxin, a
potent inhibitor of Rho (Aktories et al., 1988; Kikuchi et
al., 1988
; Narumiya et al., 1988
; Braun et al., 1989
; Fig. 4 C).
Taking it into consideration that the phosphorylation level
of T567 in ezrin also appeared to rapidly increase then decrease within 2 min after LPA stimulation (Fig. 4 A), we
concluded that T567 (ezrin), T564 (radixin), and T558
(moesin) were phosphorylated in vivo in a Rho-dependent
manner. These findings suggest that the activation of Rho
causes the phosphorylation of ERM proteins through activation of Rho-kinase.
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Effects of T564 Phosphorylation on Actin-binding Ability of Radixin
T567 (ezrin), T564 (radixin), and T558 (moesin) are located in the putative actin-binding domain (KYKTL) of
ERM proteins (Turunen et al., 1994; Pestonjamasp et al.,
1995
). Thus, we compared the actin-binding ability of
T564-phosphorylated C-rad with that of the nonphosphorylated molecule in vitro by actin filament cosedimentation analysis. As shown in Fig. 5 A, when nonphosphorylated
and fully phosphorylated C-rad were incubated with skeletal muscle actin filaments followed by centrifugation, both
were sedimented in equal amounts. Quantitative analysis
indicated no significant difference in actin-binding ability
between non- and fully phosphorylated C-rad, indicating
that the phosphorylation of T564 does not affect the direct
binding of C-rad to actin filaments (Fig. 5 B).
We also attempted to examine the actin-binding ability of partially phosphorylated F-rad. However, as shown in Fig. 5 C, approximately half of the amount of F-rad, especially most of phosphorylated F-rad, was precipitated in the absence of actin filaments even at 10,000 g centrifugation, whereas phosphorylated C-rad was mostly recovered in the 100,000 g supernatant. This indicated that the actin filament cosedimentation analysis was not potent to assess the actin-binding ability of F-rad.
Effects of T564 Phosphorylation on Interdomain Interaction of Radixin
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.
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Discussion |
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C-rad was fully phosphorylated in vitro mainly at its
COOH-terminal threonine (T564) by the constitutively active of Rho-kinase. For in vivo analysis of this type of phosphorylation, we produced a mAb (297S) that distinguished
T564-phosphorylated from nonphosphorylated radixin.
Because the amino acid sequence around T564 of radixin is completely conserved among ERM proteins, this mAb
also recognized T567-phosphorylated ezrin and T558-phosphorylated moesin. Immunoblots of LPA-stimulated
serum-starved Swiss 3T3 cells with this mAb revealed that
not only T564 of radixin, but also T567 of ezrin and T558
of moesin, were phosphorylated in vivo in a Rho-dependent manner. Of course, these findings do not directly
prove that it is the Rho-kinase that phosphorylates ERM
proteins in vivo. However, considering that Rho-kinase is
one of the major targets for Rho in terms of the reorganization of actin-based cytoskeletons (Leung et al., 1996;
Amano et al., 1997
; Ishizaki et al., 1997
), we were led to
conclude that, when Rho is activated in vivo, radixin, as
well as ezrin and moesin, are similarly phosphorylated
through the activation of Rho-kinase. This conclusion favors the notion that ERM proteins are functionally redundant (Takeuchi et al., 1994b
; Hirao et al., 1996
; Tsukita et
al., 1997a
,b). These observations are also consistent with
the previous report by Nakamura et al. (1995
, 1996
) that in
platelets, thrombin stimulation induced the T558 phosphorylation of moesin. As moesin is predominant among ERM
proteins in platelets (Nakamura et al., 1995
) and Rho signaling is involved in the thrombin-induced activation of
platelets (Morii et al., 1992
), it is likely that T558 of moesin
in platelets is phosphorylated by Rho-kinase.
Two possible functions have been proposed for the
COOH-terminal highly conserved amino acid sequence
around ezrin T567, radixin T564, or moesin T558. First,
this domain is responsible for actin filament binding of
ERM proteins. The actin-binding site was narrowed down
in ezrin to the COOH-terminal 34 amino acids. The sequence around T567, KYKTL, in the COOH-terminal 34 amino acids was regarded as a consensus sequence for actin binding (KYKXL) that was also found in other actin-binding proteins such as the myosin heavy chain and subunit of Cap-Z (Turunen et al., 1994
), although there
has been no evidence showing the direct interaction of actin filaments with KYKTL in ERM proteins. It was thus
expected that phosphorylation of the KYKTL site would
affect actin binding of the COOH halves of ERM proteins,
although T is not necessarily required as an actin-binding
consensus sequence. As shown in Fig. 5, nonphosphorylated C-rad was cosedimented with actin filaments, but
T564-phosphorylated C-rad was also cosedimented to the
same extent, indicating that KYK-pT-L shows the same affinity to actin filaments as KYKTL.
Second, the COOH-terminal end domain is responsible
for the head-to-tail association of ERM proteins. As described in the introduction, the intra- or intermolecular
head-to-tail association is thought to be very important for
the regulation of ERM protein activity (Berryman et al.,
1995; Bretscher et al., 1995
). When the NH2- and COOH-terminal halves bind to each other to form closed forms
(and/or oligomers), ERM proteins are inactive as cross-linkers between actin filaments and plasma membranes.
When some signal interferes with this binding, ERM proteins are activated (opened). In this study, we found that
the T564 phosphorylation of radixin markedly suppressed
its head-to-tail association. This suggests that the T564-phosphorylation of radixin (and probably also the phosphorylation of ezrin T567 and moesin T558) keeps them
open and active. If the life time of the opened form of
ERM proteins is prolonged by this phosphorylation, the
actin filament/plasma membrane association is upregulated, which is consistent with the previous observation
that the CD44/ERM protein complex is stabilized by the
activation of Rho (Hirao et al., 1996
). We also found previously that Rho-GDI (GDP dissociation inhibitor; Araki et al., 1990
; Takai et al., 1995
) was coimmunoprecipitated
with the CD44/ERM protein complex (Hirao et al., 1996
).
Furthermore, it was recently shown that Rho-GDI carrying the GDP-bound form Rho (GDP-Rho) directly binds
to the NH2-terminal half of ERM proteins, and that this
binding dissociates the Rho-GDI/GDP-Rho complex to
release free GDP-Rho (Takahashi et al., 1997
). Thus we
speculate that GDP-Rho, which is recruited to ERM proteins, is converted to GTP-Rho and that GTP-Rho activates Rho-kinase to phosphorylate ERM proteins. The
phosphorylated ERM proteins with opened conformation may function as actin filament/plasma membrane cross-linkers.
Rho-kinase phosphorylated T564 in ~100% of C-rad
molecules but in at most ~30% of F-rad in vitro, indicating that Rho-kinase phosphorylates the opened form of
radixin more efficiently than the closed form at least in
vitro. Therefore, we speculate that the Rho-kinase-dependent phosphorylation of ERM proteins does not activate
(open) ERM proteins but stabilizes the activated (opened)
conformation. However, it is also possible that Rho-kinase can open ERM proteins in vivo in the presence of other
Rho-independent kinases, other targets for Rho and
ERM-binding proteins such as EBP50 (Zhang et al., 1995;
Chong et al., 1994
; Bowman et al., 1993
; Malcolm et al.,
1994
; Madaule et al., 1995
; Amano et al., 1996a
; Kimura et
al., 1996
; Reid et al., 1996
; Watanabe et al., 1996
, 1997
;
Reczek et al., 1997
). Actually, ezrin is effectively tyrosine
phosphorylated by EGF and HGF stimulation (Krieg et
al., 1992; Crepaldi et al., 1997
) and functions as a protein
kinase A-anchoring protein (Dransfield et al., 1997
). It is
also serine/threonine phosphorylated in vivo by protein kinase A probably at residues other than those phosphorylated by Rho-kinase (Urushidani et al., 1989
).
If the phosphorylation of respective T567, T564, and
T558 of ERM proteins keeps them open without affecting
the actin-binding ability of their COOH-terminal regions,
it was expected that Rho-kinase-dependent phosphorylation would upregulate the actin-binding ability of F-rad.
As shown in Fig. 5 C, however, even in the absence of actin filaments, F-rad, especially T564-phosphorylated F-rad,
was mostly recovered in the low speed pellet, making it
difficult to evaluate the actin-binding ability of F-rad in
vitro by the cosedimentation analysis. Most recently, another F-actin-binding domain was identified in the NH2-terminal domain of ezrin (Martin et al., 1997; Roy et al.,
1997
). Furthermore, this domain also bound to G-actin, which was consistent with our initial observation of the
barbed-end capping activity of purified native radixin
(Tsukita et al., 1989
). Therefore, to understand the regulatory mechanism of the physiological functions of ERM
proteins, detailed comparison of the F-actin- and/or G-actin-
binding ability between nonphosphorylated and phosphorylated full-length ERM proteins will be required in the next step.
The relationship between Rho-signaling and the actin-based cytoskeleton was first noted by Ridley and coworkers (Ridley and Hall, 1992; Ridley et al., 1992
). They showed
using serum-starved Swiss 3T3 cells that Rho plays a central role in the coordinated assembly of focal adhesions
and stress fibers induced by growth factors. However, its
molecular mechanism has still yet to be elucidated in detail. Constitutively active Rho-kinase induces the formation of focal adhesions and stress fibers (Leung et al., 1996
;
Amano et al., 1997
; Ishizaki et al., 1997
). Rho-kinase was reported to activate myosin ATPase, which is thought to
be important for stress fiber formation (Amano et al.,
1996a
; Kimura et al., 1996
). On the other hand, moesin
was identified as an essential factor for the Rho-dependent formation of stress fibers in serum-starved Swiss 3T3
cells (Mackay et al., 1997
). This study showed that the
Rho-dependent threonine phosphorylation of ERM proteins occurred very rapidly before the coordinated assembly of focal adhesions and stress fibers in serum-starved
Swiss 3T3 cells. Further analyses of the physiological relationship between the Rho-kinase-dependent activation of
myosin ATPase and ERM protein phosphorylation will lead to a better understanding of the Rho-dependent regulation of the actin-based cytoskeleton.
![]() |
Footnotes |
---|
Address correspondence to Shoichiro Tsukita, MD, PhD, Department of Cell Biology, Kyoto University Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan. Tel.: 81 75 753 4372. Fax: 81 75 753 4660. E-mail: htsukita{at}mfour.med.kyoto-u.ac.jp
Received for publication 7 July 1997 and in revised form 5 December 1997.
We would like to thank Professor Y. Takai (Department of Molecular Biology and Biochemistry, Osaka University, Japan) and all the members of our laboratory (Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan) for their helpful discussions throughout this study. Our thanks are also due to Dr. A. Hall for his generous gift of pGEX-C3. We are grateful to Miss M. Sato and Mr. T. Nakagawa for their excellent technical assistance.
This work was supported in part by a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science and Culture of Japan (to S. Tsukita).
![]() |
Abbreviations used in this paper |
---|
C-rad, COOH-terminal half recombinant radixin; ERM, ezrin/radixin/moesin; F-rad, full-length recombinant radixin; GST, glutathione-S-transferase; LPA, lysophosphatidic acid; N-rad, NH2-terminal half recombinant radixin; pAb, polyclonal antibody; Rho-Kc, recombinant Rho-kinase catalytic domain; Rho-kinase, Rho- associated kinase; TFA, trifluoroacetic acid.
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References |
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