Phosphorylation of Vimentin by Rho-associated Kinase at a
Unique Amino-terminal Site That Is Specifically Phosphorylated
during Cytokinesis*
Hidemasa
Goto
§,
Hidetaka
Kosako
,
Kazushi
Tanabe¶,
Maki
Yanagida
,
Minoru
Sakurai§,
Mutsuki
Amano
,
Kozo
Kaibuchi
, and
Masaki
Inagaki
**
From the Laboratory of
Biochemistry, and
¶ Biophysics Unit, Aichi Cancer Center Research Institute,
Chikusa-ku, Nagoya, Aichi 464, the § Department of
Pediatrics, Mie University School of Medicine, Edobashi, Tsu 514, and
the
Division of Signal Transduction, Nara Institute of Science
and Technology, 8916-5 Takayama, Ikoma 630-01, Japan
 |
ABSTRACT |
We found that vimentin, the most widely
expressed intermediate filament protein, served as an excellent
substrate for Rho-associated kinase (Rho-kinase) and that vimentin
phosphorylated by Rho-kinase lost its ability to form filaments
in vitro. Two amino-terminal sites on vimentin,
Ser38 and Ser71, were identified as the major
phosphorylation sites for Rho-kinase, and Ser71 was the
most favored and unique phosphorylation site for Rho-kinase in
vitro. To analyze the vimentin phosphorylation by Rho-kinase in vivo, we prepared an antibody GK71 that specifically
recognizes the phosphorylation of vimentin-Ser71. Ectopic
expression of constitutively active Rho-kinase in COS-7 cells induced
phosphorylation of vimentin at Ser71, followed by the
reorganization of vimentin filament networks. During the cell cycle,
the phosphorylation of vimentin-Ser71 occurred only at the
cleavage furrow in late mitotic cells but not in interphase or early
mitotic cells. This cleavage furrow-specific phosphorylation of
vimentin-Ser71 was observed in the various types of cells
we examined. All these accumulating observations increase the
possibility that Rho-kinase may have a definite role in governing
regulatory processes in assembly-disassembly and turnover of vimentin
filaments at the cleavage furrow during cytokinesis.
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INTRODUCTION |
Intermediate filaments
(IFs)1 constitute one of the
three major cytoskeletal elements in eukaryotic cells. An important
feature of IF proteins is their tissue preferential expression. For
example, glial fibrillary acidic protein (GFAP) is expressed
specifically in astroglia. On the other hand, vimentin is the most
common IF protein and is expressed during development in a wide range
of cells, in mesenchymal cells and in a variety of cultured cells and
tumors. Previous studies have demonstrated that IFs can undergo dynamic
changes in their organization during different stages of the cell cycle
or during cell signaling (for review see Refs. 1-3). The
reorganization of IFs is thought to be regulated by site-specific
phosphorylation of IF proteins at serine and threonine residues, and
several protein kinases have been shown to act as IF kinases in
vivo (Ref. 4; for review see Ref. 5). Site- and phosphorylation
state-specific antibodies that can recognize a phosphorylated
serine/threonine residue and its flanking sequence are powerful tools
to visualize site-specific IF phosphorylation in cells and to identify
in vivo IF kinases (Refs. 6 and 7; for review see Ref. 8).
By using several types of such antibodies, we previously detected a
protein kinase activity that phosphorylates GFAP at Thr7,
Ser13, and Ser34 specifically at the cleavage
furrow during cytokinesis (6, 9). This kinase activity, tentatively
named cleavage furrow (CF) kinase activity, was observed not only in
astroglial cells but also in other cultured cells in which GFAP was
ectopically expressed (10). These findings indicate that the activation of CF kinase occurs in a wide range of cell types, suggesting its
important role in cytokinesis. Using a series of monoclonal antibodies
(MO6, YT33, TM50, 4A4, and MO82) which specifically recognize the
phosphorylation of vimentin at Ser6, Ser33,
Ser50, Ser55, and Ser82,
respectively, we visualized in vivo vimentin kinase
activities in cell signaling or mitosis (11-14), but we detected no CF
kinase-like activity that phosphorylates vimentin during
cytokinesis.
The small GTP-binding protein Rho is implicated in the formation of
stress fibers and focal adhesion complexes (15, 16) and in the
regulation of cell morphology (17), cell aggregation (18), cell
motility (19), smooth muscle contraction (20, 21), and cytokinesis
(Refs. 22-24; for review see Refs. 25 and 26). Upon stimulation with
certain signals, the GDP-bound inactive form of Rho may be converted to
GTP-bound active form, which presumably binds to specific targets and
thereby exerts its biological functions. The putative target proteins
for Rho include protein kinase N (27, 28), rhophilin (28), citron (29),
rhotekin (30), the myosin binding subunit of myosin phosphatase (31),
p140mDia (32), and Rho-kinase (33) (also called ROK (34) or ROCK (35))
(for review see Ref. 36). Recently, we have reported that Rho-kinase
phosphorylates GFAP at Thr7, Ser13, and
Ser34 in vitro, the same sites that are
phosphorylated by CF kinase in vivo (37). These observations
raise the possibility that Rho-kinase may act downstream of Rho in the
regulation of cleavage furrow-specific phosphorylation of GFAP during
cytokinesis. However, one could not rule out the possibility that other
unknown kinase(s) are responsible for the CF kinase activity because
these three phosphorylation sites are phosphorylated by several kinases
in vitro.
In this report, we showed that vimentin was phosphorylated by
Rho-kinase in a GTP·Rho-dependent manner and that
vimentin phosphorylation by Rho-kinase resulted in a nearly complete
inhibition of its filament formation in vitro. We then
identified Ser71 on vimentin as the most favored and unique
phosphorylation site for Rho-kinase in vitro. By producing a
site- and phosphorylation state-specific antibody for this site, we
demonstrated that vimentin-Ser71 was phosphorylated
specifically at the cleavage furrow during cytokinesis in various types
of cells.
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EXPERIMENTAL PROCEDURES |
Preparation of Proteins--
Recombinant mouse vimentin was
prepared from Escherichia coli as described previously (12).
Vimentin phosphorylated by the catalytic subunit of
cAMP-dependent protein kinase A,
Ca2+-calmodulin-dependent protein kinase II
(CaM kinase II), protein kinase C, and Cdc2 kinase were prepared as
described previously (11). Rho-kinase was purified from bovine brain
(33). GST-RhoA was purified and loaded with guanine nucleotides (38).
GST-Rho-kinase was purified from Sf9 cells as described
previously (39).
Phosphorylation of Vimentin--
The phosphorylation reaction
for Rho-kinase was performed for 30 min at 25 °C in 20 µl of 25 mM Tris-Cl (pH 7.5), 0.2% CHAPS, 4 mM
MgCl2, 3.6 mM EDTA, 100 µM
[
-32P]ATP, 0.1 µM calyculin A, 150 µg/ml vimentin, and 0.5 µg/ml purified Rho-kinase in the presence
of either GST, GDP·GST-RhoA, or GTP
S·GST-RhoA (each 1 µM). The phosphorylation reaction for GST-Rho-kinase was performed for 30 min at 25 °C in 20 µl of the reaction mixture (25 mM Tris-Cl (pH 7.5), 0.4 mM MgCl2,
100 µM ATP, 0.1 µM calyculin A, 150 µg/ml
vimentin, and 9.0 µg/ml GST-Rho-kinase). Reaction mixtures were
boiled in Laemmli's sample buffer and subjected to SDS-PAGE.
Fragmentation of Phosphorylated Vimentin--
Vimentin (150 µg) was phosphorylated by GST-Rho-kinase (4.5 µg) at 25 °C for
60 min to a stoichiometry of 2.0 mol of phosphate/mol of vimentin in 1 ml of the reaction mixture as described above with
[
-32P]ATP. The radioactive vimentin was precipitated
with 10% trichloroacetic acid, dissolved in 100 µl of 50 mM Tris-Cl (pH 8.0) containing 4 M urea, and
digested with 5 µg of lysyl-endopeptidase (Wako Pure Chemical, Osaka,
Japan) for 2 h at 30 °C. The digested sample was fractionated
by reverse-phase HPLC on a Zorbax C8 column (0.46 × 25 cm)
equilibrated with 5% (v/v) 2-propanol/acetonitrile (7:3) containing
0.1% trifluoroacetic acid. The peptides were eluted with a 60-min
linear gradient of 5-50% (v/v) 2-propanol/acetonitrile, followed by a
further 10-min linear gradient of 50-80% (v/v)
2-propanol/acetonitrile. All radioactivity loaded was recovered in a
single peptide (a 12-kDa fragment from the amino-terminal domain of
vimentin) with the retention time of 65-68 min. This phosphorylated
head domain was vacuum-dried, resuspended in 50 mM Tris-Cl
(pH 7.5), and treated with 1:50 (w/w)
L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma) at 37 °C for 8 h. The samples were retreated
identically for an additional 8 h. The obtained peptides were
fractionated by HPLC on the Zorbax C8 column as described above. All
the chromatographies were performed at room temperature with a flow
rate of 0.8 ml/min and a fraction size of 0.8 ml.
Amino Acid Sequence Analysis and Phosphoamino Acid Analysis of
Tryptic Peptides--
Amino acid sequences of each phosphopeptide were
analyzed using an ABI 476A gas-phase sequencer. To determine at which
position on vimentin each peptide exists, the sequences were then
compared with the published amino acid sequence predicted from mouse
vimentin cDNA (40). Two-dimensional phosphoamino acid analysis of
each peptide was performed as described previously (41).
Phosphoserine-containing peptides were treated with ethanethiol at
alkaline pH as described previously (42). The ethanethiol-modified
peptides were then sequenced as above.
Peptide Synthesis and Production of Anti-PV71 Antibody
(GK71)--
Vimentin peptides PV71
(Cys-Ala-Val-Arg-Leu-Arg-phospho-Ser71-Ser-Val-Pro-Gly-Val),
V71 (Cys-Ala-Val-Arg-Leu-Arg-Ser71-Ser-Val-Pro-Gly-Val),
PV6
(Cys-Ser-Thr-Arg-Ser-Val-phospho-Ser6-Ser-Ser-Ser-Tyr-Arg),
PV24
(Cys-Thr-Ser-Ser-Arg-Pro-phospho-Ser24-Ser-Asn-Arg-Ser-Tyr),
PV33
(Cys-Ser-Tyr-Val-Thr-Thr-phospho-Ser33-Thr-Arg-Thr-Tyr-Ser),
PV38
(Cys-Ser-Thr-Arg-Thr-Tyr-phospho-Ser38-Leu-Gly-Ser-Ala-Leu),
PV41
(Cys-Thr-Tyr-Ser-Leu-Gly-phospho-Ser41-Ala-Leu-Arg-Pro-Ser),
PV46
(Cys-Ser-Ala-Leu-Arg-Pro-phospho-Ser46-Thr-Ser-Arg-Ser-Leu),
PV50
(Cys-Pro-Ser-Thr-Ser-Arg-phospho-Ser50-Leu-Tyr-Ser-Ser-Ser),
PV55
(Ser-Leu-Tyr-Ser-Ser-phospho-Ser55-Pro-Gly-Gly-Ala-Tyr-Cys),
PV65
(Cys-Tyr-Val-Thr-Arg-Ser-phospho-Ser65-Ala-Val-Arg-Leu-Arg),
and PV82
(Cys-Arg-Leu-Leu-Gln-Asp-phospho-Ser82-Val-Asp-Phe-Ser-Leu)
were synthesized and purified as described previously (11). Antibodies
against PV71 were prepared by injecting four rabbits with PV71 coupled
to keyhole limpet hemocyanin. Monospecific antibodies against PV71 were
purified from the obtained serum by two-step chromatography: affinity
chromatography on PV71-coupled Cellulofine (Seikagaku Corp.) and then
absorption in V71-coupled Cellulofine. Protein concentrations were
determined by absorbance at 280 nm using rabbit IgG (Sigma) as a
standard. This anti-PV71 antibody (referred to as GK71) obtained from
one of the rabbits was used for the following experiments.
Immunoblotting was performed as described previously (6), using
horseradish peroxidase-conjugated secondary antibodies and the ECL
Western blotting detection system (Amersham Pharmacia Biotech).
Transfection--
The pEF-BOS-myc mammalian expression plasmids
encoding the catalytic domain of bovine Rho-kinase (CAT; amino acids
6-553) and the catalytic domain mutated at the ATP-binding site
(CAT-KD; Lys121
Gly) were constructed as described
previously (43). COS-7 cells (obtained from RIKEN Cell Bank) were
plated at a density of 1.5 × 105 cells per 35-mm
dish. After culturing for 1 day, cells were transfected with 2 µg of
the plasmid DNA by the application of SuperFect-DNA complexes (Qiagen).
At 2 h, Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum was added, and the cells were cultured for another 24 h. These cells were used for immunoblotting and immunofluorescence
studies.
Immunofluorescence Microscopy--
Cells growing on glass
coverslips were fixed with 3.7% formaldehyde in ice-cold PBS for 10 min and then treated with methanol at
20 °C for 10 min. For double
immunostaining with GK71 and DM1A, cells were fixed with 3.7%
formaldehyde in PBS for 10 min at 37 °C and then permeabilized with
0.1% Triton X-100 in PBS for 10 min at room temperature. Incubation
with primary antibodies diluted in PBS containing 1% sucrose and 1%
bovine serum albumin was for 2 h at 37 °C. After three washes
with PBS, cells were incubated for 1 h with appropriate secondary
antibodies diluted 1:100 and subsequently washed with PBS. Then DNAs
were stained with 0.5 µg/ml propidium iodide (Sigma) or 0.5 µg/ml
DAPI (Boehringer Mannheim) for 10 min at room temperature.
The following antibodies were used for indirect immunofluorescence
microscopy: GK71 (anti-phospho-Ser71 of vimentin) rabbit
polyclonal antibody diluted 1:100; 1B8 (anti-vimentin) mouse mAb (11)
diluted 1:2; 9E10 (anti-Myc, from Babco) mouse mAb diluted 1:200; DM1A
(anti-
-tubulin, from Sigma) mouse mAb diluted 1:500; fluorescein
isothiocyanate-conjugated goat anti-rabbit or anti-mouse
immunoglobulins (BioSource International, Camarillo, CA); and Texas
Red-conjugated sheep anti-mouse immunoglobulins (Amersham Pharmacia
Biotech).
Fluorescently labeled cells were examined either with an Olympus
BH2-RFCA microscope or an Olympus LSM-GB200 confocal microscope.
Preparation of Interphase, Early Mitotic, and Late Mitotic Cell
Lysates for Western Blotting--
Just before cells reached
confluence, the cells were arrested in early mitosis by the addition of
15 ng/ml TN-16 (Wako Pure Chemical, Osaka, Japan) for 4 h. Mitotic
cells were collected by mechanical shake off, and the adherent cells
were used as interphase cells. Mitotic cells were rinsed twice,
suspended in TN-16-free Dulbecco's modified Eagle's medium containing
10% fetal bovine serum, and plated onto the culture dish. Cells at 0 or 30 min after removal of TN-16 were used as early or late mitotic
cells, respectively. These cells were treated with 10% trichloroacetic acid and then collected. After centrifugation, cell pellets were dissolved in Laemmli's sample buffer, with brief sonication.
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RESULTS |
Phosphorylation of Vimentin by Rho-kinase--
We examined whether
Rho-kinase purified from bovine brain can phosphorylate vimentin in a
GTP·Rho-dependent manner. GTP
S·GST-RhoA enhanced the
phosphorylation of vimentin by Rho-kinase about 58-fold relative to
GST, about 26-fold relative to GDP·GST-RhoA (Fig. 1A). The recombinant
Rho-kinase (GST-Rho-kinase), which is constitutively active, also
phosphorylated vimentin (Fig. 1A). The pattern of tryptic
phosphopeptide mapping of vimentin phosphorylated by GST-Rho-kinase was
identical to that by purified Rho-kinase in the presence of GTP
S·GST-RhoA (Fig. 1B). This result suggests that
catalytic characteristics of GST-Rho-kinase are similar to those of
native Rho-kinase activated by GTP
S-bound RhoA.

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Fig. 1.
Phosphorylation of vimentin by Rho-kinase.
A, vimentin was phosphorylated by Rho-kinase (0.5 µg/ml)
in the presence of either GST (a), GDP·GST-RhoA
(b), or GTP S·GST-RhoA (c), or by
GST-Rho-kinase (1.0 µg/ml) (d). Radiolabeled bands were
visualized by autoradiography. After staining with Coomassie Brilliant
Blue, each band of vimentin was cut from the gel, and each
radioactivity was measured in 32P Beckman liquid
scintillation counter. The values in parentheses represent fold
stimulation relative to Rho-kinase in the presence of GST. The position
of vimentin is indicated (arrowhead). The positions of
molecular size standards (in kilodaltons) are indicated on the
left. B, tryptic phosphopeptide mapping of
vimentin phosphorylated by either Rho-kinase in the presence of
GTP S·GST-RhoA (c) or GST-Rho-kinase (d).
Each radioactive vimentin was eluted from the gel strip described above
(A-c or A-d), precipitated
with trichloroacetic acid, oxidized with performic acid, and digested
with trypsin as described previously (41). Each tryptic peptide was
loaded onto a thin layer cellulose gel plate (horizontal dimension,
electrophoresis with formic acid/acetic acid/H2O
(25:78:897) at 1500 V for 40 min; vertical dimension, ascending
chromatography in n-butanol/pyridine/acetic
acid/H2O (15:10:3:12)). Mixed map was generated by loading
equal counts of trypsin-digested vimentin phosphorylated by Rho-kinase
in the presence of GTP S·GST-RhoA and by GST-Rho-kinase
(c+d). Phosphopeptides were visualized by
autoradiography.
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The level of the vimentin phosphorylation by GST-Rho-kinase increased
in a time-dependent manner and was approximately 2.5 mol of
phosphate/mol of protein at 2 h (Fig.
2A). To investigate which
structural domain of vimentin is phosphorylated, the radiolabeled protein was digested with lysyl-endopeptidase. SDS-PAGE analysis revealed that almost all the radioactivity in the phosphorylated vimentin was recovered in the 12-kDa amino-terminal head domain of
vimentin (Fig. 2B), indicating that the phosphorylation
sites were restricted to only the head domain of vimentin.

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Fig. 2.
The effect of domain-specific phosphorylation
by GST-Rho-kinase on the formation of vimentin filament. A,
time course of phosphorylation of vimentin by GST-Rho-kinase.
B, vimentin (Vim.) phosphorylated by
GST-Rho-kinase was incubated in the presence (b) or absence
(a) of lysyl-endopeptidase and subjected to SDS-PAGE.
Radiolabeled bands were visualized by autoradiography. The positions of
vimentin and the head domain of vimentin are indicated
(arrowheads). The phosphorylation reaction was performed in
the absence (control) or the presence
(GST-Rho-kinase) of GST-Rho-kinase. The samples were then
incubated with 100 mM NaCl at 37 °C for further 60 min.
C, after the incubation, the samples were subjected to high
speed centrifugation (12,000 × g). The supernatant
(s) and the precipitate (p) were subjected to
SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue.
D, after the incubation, the samples were placed directly on
carbon film-coated specimen grids, stained with 2% uranyl acetate, and
subjected to the electron microscopy. Bar, 200 nm.
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We then examined the effect of vimentin phosphorylation by Rho-kinase
on its filament forming ability. Soluble vimentin was preincubated with
or without GST-Rho-kinase for 1 h, and the samples were incubated
under conditions of polymerization (100 mM NaCl at
37 °C) for a further 1 h. Analyses of these samples by
centrifugation (Fig. 2C) and by electron microscopy (Fig.
2D) revealed that the phosphorylation of vimentin by
Rho-kinase dramatically inhibited its filament formation. Furthermore,
the phosphorylation of preformed vimentin filaments by GST-Rho-kinase
also induced disassembly of filament structures in vitro
(data not shown). These results indicate that the vimentin
phosphorylation by Rho-kinase affects the state of polymerization of
vimentin in vitro.
Identification of Phosphorylation Sites on Vimentin by
Rho-kinase--
To identify the phosphorylation sites on vimentin by
Rho-kinase, vimentin (150 µg) was phosphorylated by GST-Rho-kinase in the presence of [
-32P]ATP to ~2.0 mol of
phosphate/mol of protein. Then the radioactive head domain of vimentin
was obtained by the treatment with lysyl-endopeptidase. This head
domain purified by reverse-phase HPLC was digested with trypsin, and
the resulting peptides were again separated by reverse-phase HPLC. As
shown in Fig. 3A, three major
radioactive peptides, RV1 to RV3, were obtained. All of these peptides
were phosphorylated at serine residues, as determined by
two-dimensional phosphoamino acid analysis (Fig. 3B). The
phosphoserine-containing peptides were then sequenced after ethanethiol
treatment which specifically converts phosphoserine into
S-ethylcysteine. The generation of S-ethylcysteine at a particular Edman degradation cycle
where serine is predicted provides a definitive way to locate the
phosphoserine residue(s) on each peptide. The lack of generation of
S-ethylcysteine indicates that phosphoserine is located in
the amino-terminal serine residue as there is no conversion of the
amino-terminal phosphoserine to S-ethylcysteine (42). Fig.
3C shows that phosphates of RV1, RV2, and RV3 peptides were
located on Ser71, Ser71, and Ser38,
respectively. As shown in Fig. 3C and Table
I, RV1 peptide was the complete digestion
product of RV2 peptide. Phosphates at Ser71 and
Ser38 accounted for about 41.7 and 23.3% of those on
vimentin phosphorylated by GST-Rho-kinase, respectively (Table I). As
shown in Fig. 4A and Table I,
vimentin-Ser71 was the most favored and unique
phosphorylation site for Rho-kinase.

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Fig. 3.
Tryptic phosphopeptides derived from vimentin
phosphorylated by GST-Rho-kinase. A, the radioactive
amino-terminal head domain of vimentin was digested with trypsin and
fractionated by reverse-phase HPLC. The radioactivity of each fraction
(0.8 ml) was measured in 32P Beckman liquid scintillation
counter. B, phosphopeptides indicated above
(RV1-RV3) was subjected to two-dimensional phosphoamino
acid analysis. The positions of phosphoserine (P-Ser),
phosphothreonine (P-Thr), and phosphotyrosine
(P-Tyr) are indicated. C, each
phosphoserine-containing peptide (RV1-RV3) was
incubated with ethanethiol at alkaline pH to convert phosphoserine into
S-ethylcysteine. Each modified peptide was then subjected to
amino acid sequence analysis. Relative amount of
S-ethylcysteine (closed circles) in the
phenylthiohydantoin chromatogram of each degradation step is shown.
Phosphoserine residues in each peptide are underlined (bold
letters).
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Fig. 4.
Specificity of the antibody GK71 analyzed by
Western blotting. A, a map of vimentin molecule showing
phosphorylation sites by Cdc2 kinase, protein kinase A, protein kinase
C, CaM kinase II, or Rho-kinase (5). The phosphorylation sites are
indicated by P within a circle. The amino acid
sequence corresponding to the synthetic peptide PV71 is
underlined. B, vimentin (Vim.) was
unphosphorylated (control) or phosphorylated at 2.0 mol of
phosphate/mol of protein by GST-Rho-kinase (Rho-kinase), at
4.0 mol of phosphate/mol of protein by the catalytic subunit of protein
kinase A, at 0.7 mol of phosphate/mol of protein by CaM kinase II, at
1.5 mol of phosphate/mol of protein by Cdc2 kinase, or at 2.2 mol of
phosphate/mol of protein by protein kinase C, respectively. After
SDS-PAGE (100 ng in each lane), samples were transferred onto a
poly(vinylidene difluoride) membrane. The membrane was immunoblotted
with the antibody GK71 (4 µg/ml) and then stained with Coomassie
Brilliant Blue (C.B.B.). C, specificity of GK71
determined by a competition assay. Vimentin (Vim.)
phosphorylated by GST-Rho-kinase was immunoblotted with GK71 (2 µg/ml) after preincubation with synthetic peptides (50 µg/ml V71,
PV71, PV6, PV24, PV33, PV38, PV41, PV46, PV50, PV55, PV65, or PV82). As
a control, the phosphorylated vimentin was immunoblotted with GK71 (2 µg/ml) after preincubation with TBS-T.
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Production and Characterization of the Site- and Phosphorylation
State-specific Antibody for Vimentin-Ser71--
Since
vimentin-Ser71 is the phosphorylation site specific to
Rho-kinase among known vimentin kinases in vitro (Fig.
4A), this residue can serve as a pertinent indicator to
study in vivo vimentin phosphorylation by Rho-kinase. Thus
we prepared a rabbit polyclonal antibody (referred to as GK71), raised
against the synthetic phosphopeptide PV71
(phosphovimentin-Ser-71;
Cys-Ala-Val-Arg-Leu-Arg-phospho-Ser71-Ser-Val-Pro-Gly-Val)
(Fig. 4A). In Fig. 4, B and C, the
specificity of GK71 was examined by Western blotting. GK71 reacted with
vimentin phosphorylated by Rho-kinase but not with nonphosphorylated
vimentin or vimentin phosphorylated by protein kinase A, protein kinase C, Cdc2 kinase, or CaM kinase II (Fig. 4B). As shown in Fig.
4C, the immunoreactivity of GK71 for vimentin phosphorylated
by Rho-kinase was neutralized by preincubation with the phosphopeptide
PV71 but not with the nonphosphopeptide V71
(Cys-Ala-Val-Arg-Leu-Arg-Ser71-Ser-Val-Pro-Gly-Val) or
other phosphopeptides such as PV6, PV24, PV33, PV38, PV41, PV46, PV50,
PV55, PV65, and PV82 (which were designed to represent vimentin
phosphorylated at other sites, Ser6, Ser24,
Ser33, Ser38, Ser41,
Ser46, Ser50, Ser55,
Ser65, and Ser82, respectively). These results
indicate that GK71 specifically recognizes the phosphorylation of
vimentin at Ser71 by Rho-kinase.
Phosphorylation of Vimentin-Ser71 by Ectopic Expression
of Active Rho-kinase in COS-7 Cells--
To examine whether Rho-kinase
can phosphorylate vimentin at Ser71 in cells, we next
analyzed the phosphorylation of vimentin-Ser71 in monkey
kidney epithelial (COS-7) cells which ectopically express the active
form of Rho-kinase. Cells were transfected with the pEF-BOS vector, or
the pEF-BOS-myc plasmid carrying the catalytic domain of Rho-kinase
(CAT), or the catalytic domain mutated at the ATP-binding site
(CAT-KD). Expression levels of the Myc epitope-tagged CAT and CAT-KD
were almost the same, and endogenous vimentin was equally expressed in
the three types of transfected cells (Fig. 5A). However, immunoblot
analysis using GK71 revealed that the phosphorylation of
vimentin-Ser71 occurred only in lysates from CAT-expressing
cells (Fig. 5A). Immunofluorescence analysis of the
transfected cells is shown in Fig. 5B. The phosphorylation
of vimentin-Ser71 was observed in cells expressing
Myc-tagged CAT but not in cells expressing Myc-tagged CAT-KD. These
results demonstrate that constitutively active Rho-kinase can
phosphorylate vimentin at Ser71 in intact cells.

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Fig. 5.
Phosphorylation of vimentin-Ser71
in COS-7 cells overexpressing Rho-kinase mutants. A, COS-7
cells were transfected with pEF-BOS (vector),
pEF-BOS-myc-CAT (CAT), or pEF-BOS-myc-CAT-KD
(CAT-KD) and were lysed in Laemmli's sample buffer after
26 h. Each lysate was immunoblotted with anti-Myc
(9E10), anti-vimentin (1B8), and
anti-phospho-Ser71 of vimentin (Vim.)
(GK71). B, indirect double label
immunofluorescence of COS-7 cells transfected with pEF-BOS-myc-CAT or
pEF-BOS-myc-CAT-KD. Cells were double-stained with 9E10 and GK71. DNAs
were simultaneously stained with DAPI. C, effects of
overexpressed constitutively active Rho-kinase on vimentin filament
networks in COS-7 cells. An untransfected cell (control) and
a cell transfected with pEF-BOS-myc-CAT (CAT) were
double-stained with 1B8 and GK71. Bars, 20 µm.
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We often observed abnormal vimentin filament networks in COS-7 cells
which ectopically expressed CAT (Fig. 5C). In some cases, fiber bundles or granulates of phospho-vimentin were observed in these
cells. Together with the data shown in Fig. 2, C and D, these results may suggest that the phosphorylation by
Rho-kinase induces dynamic changes in vimentin-IF organization.
Specific Phosphorylation of Vimentin-Ser71 at the
Cleavage Furrow during Cytokinesis--
Fig.
6 shows U251 glioma cells immunostained
with 1B8 or GK71. 1B8, which reacts with both the phosphorylated and
unphosphorylated forms of vimentin (11), stained filamentous structures
in both mitotic and interphase cells (Fig. 6A). In contrast,
the immunoreactivity of GK71 was observed specifically at the cleavage
furrow (Fig. 6B). The immunoreactivity of GK71 appeared at
the onset of anaphase, was maintained until telophase, and decreased at
the exit of mitosis (Fig. 6C). We also analyzed late mitotic
cells doubly labeled with 1B8 and GK71. As shown in Fig.
7, GK71 indeed immunoreacted with
vimentin at the cleavage furrow.

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Fig. 6.
Indirect immunofluorescence analysis of U251
cells stained with anti-vimentin (1B8) or
anti-phospho-Ser71 of vimentin (GK71). Confocal
microscopic images of U251 cells stained with 1B8 (A) or
GK71 (B and C) (green). DNAs were
stained with propidium iodide (red). Images represent
horizontal optical sections (A and B) or
projections of Z series scans (C). Bars, 20 µm
(A and B); 10 µm (C).
|
|

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Fig. 7.
Indirect double label immunofluorescence of
late mitotic U251 cells stained with 1B8 and GK71. DNAs were
stained with DAPI. Bar, 10 µm.
|
|
To confirm that vimentin-Ser71 is specifically
phosphorylated during cytokinesis, Western blot analysis of U251 cell
lysates was carried out. As shown in Fig.
8, GK71-immunoreactive band at 57 kDa
corresponding to the position of vimentin was detected only in the late
mitotic cell lysate. No GK71-immunoreactive band was detected in the
lysate of interphase or metaphase cells. These results strongly suggest
that the immunostaining with the antibody GK71 during late mitotic
phase represents the presence of phospho-Ser71 of vimentin
specifically at the cleavage furrow.

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Fig. 8.
Western blot analysis of phosphorylation of
vimentin-Ser71 in U251 cell lysates. Lysates of 4 × 104 cells were loaded on lanes and resolved by SDS-PAGE.
The gel was stained with Coomassie Brilliant Blue (C.B.B.)
or transferred onto a poly(vinylidene difluoride) membrane. The
membrane was immunoblotted with GK71 (5 µg/ml). Lanes a
and d, interphase cells; lanes b and
e, early mitotic cells (metaphase-rich cells); lanes
c and f, late mitotic cells (anaphase- and
telophase-rich cells). Vim., vimentin.
|
|
We then examined the spatial distribution of vimentin-Ser71
phosphorylation at the cleavage furrow. Immunocytochemical analysis with GK71 using confocal laser scanning microscopy revealed that vimentin phosphorylated at Ser71 was associated with the
cleavage furrow to form a ring-like structure (Fig.
9A) and was localized at the
outside of spindle microtubules in the cleavage furrow (Fig.
9B).

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Fig. 9.
Confocal micrographs of U251 cells in
anaphase. A, images on six serial focal planes of an
anaphase cell stained with GK71 (green) and propidium iodine
(red). Serial optical sections were obtained by confocal
microscopy at 1.5-µm intervals. B, an optical section of
an anaphase cell double-stained with GK71 (green) and DM1A
(anti- -tubulin, red). Bars, 10 µm.
|
|
To examine whether or not phosphorylation of vimentin-Ser71
is generally observed at the cleavage furrow during cytokinesis, various cell lines were stained with the antibody GK71, as shown in
Fig. 10 (A,
Ltk
mouse fibroblastic cell; B, Swiss 3T3
mouse fibroblastic cell; C, Madin-Darby canine kidney
epithelial cell; D, COS-7 monkey kidney epithelial cell).
GK71 reacted with vimentin only at the cleavage furrow during
cytokinesis in these cells. Therefore, we believe that
vimentin-Ser71 phosphorylation at the cleavage furrow
during cytokinesis is a general feature in vimentin-expressing
cells.

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Fig. 10.
Indirect immunofluorescence staining with
GK71 in Ltk mouse fibroblastic cells (A),
Swiss 3T3 mouse fibroblastic cells (B), Madin-Darby canine
kidney epithelial cells (MDCK) (C), and COS-7
monkey kidney epithelial cells (D). Confocal
microscopic images of anaphase/telophase cells stained with GK71
(green) and propidium iodide (red). All images
represent projections of Z series scans. Bars, 10 µm.
|
|
 |
DISCUSSION |
In the present study, we obtained evidence that Rho-kinase
phosphorylates vimentin in a GTP·Rho-dependent manner and
that the phosphorylation of vimentin by Rho-kinase prevents its
filament formation in vitro. Vimentin-Ser71,
which was identified here as the phosphorylation site specific to
Rho-kinase in vitro, was shown to be specifically
phosphorylated at the cleavage furrow during cytokinesis.
One of the dynamic changes in cellular morphology during mitosis is the
reorganization of three major cytoskeletal structures, microfilaments
(actin filaments), microtubules, and intermediate filaments (IFs). Two
distinct cytoskeletal structures, a bipolar mitotic spindle and a
contractile ring, appear transiently and play active roles in the
mitotic phase of animal cells (for review, see Refs. 44 and 45). A
bipolar mitotic spindle is composed of microtubules and their
associated proteins and divides the replicated chromosomes for each
daughter cell. A contractile ring is composed of actin filaments and
myosin just beneath the plasma membrane and divides the cell into two
by pulling the membrane inward (for review, see Refs. 46 and 47).
Unlike microtubules and actin filaments which are largely reorganized
for the specific mitotic functions described above, the behavior of IFs
during mitosis differs depending on cell types. Rosevear et
al. (48) reported changes of IF network in baby hamster kidney
(BHK-21) cells during different stages of mitosis. During
prometaphase/metaphase, the typical network of long 10-nm diameter IFs
characteric of interphase cells disassembled into aggregates containing
short 4-6-nm filaments. During anaphase/telophase, arrays of short IFs reappeared throughout cytoplasm, and in cytokinesis, the majority of
IFs was longer and concentrated mainly in a juxtanuclear cap. Franke
et al. (49, 50) observed punctate or granular structures of
IFs even during cytokinesis in some types of cells. However, IFs of
many types of cells appeared to be interrupted as intact bundles in the
plane of the cleavage furrow during cytokinesis (51-54). There seems
to be a mechanism that accounts for the locally controlled breakdown of
the filaments before the final separation of daughter cells. In
vitro studies revealed that the site-specific phosphorylation of
IF proteins by several kinases induced disassembly of the filament
structure (for review, see Ref. 5). Protein kinase A, protein kinase C,
CaM kinase II, and Cdc2 kinase have been known as such kinases. In a
previous study (37) and in the present study, we demonstrated that
Rho-kinase also acts as an in vitro IF kinase which induces
alterations in the filament structure.
Identifying protein kinases responsible for in vivo IF
phosphorylation is of great importance in order to understand how
cellular IF reorganization is regulated. As a method for the
identification of in vivo IF kinases, we have utilized site-
and phosphorylation state-specific antibodies (for review, see Ref. 8).
Among the in vitro phosphorylation sites, there are sites
specifically phosphorylated by a single kinase. For example,
Ser33, Ser55, and Ser82 on vimentin
are site-specific for protein kinase C, Cdc2 kinase, and CaM kinase II,
respectively (Fig. 4A). These specific sites can serve as a
pertinent indicator for the detection of in vivo IF
phosphorylation by the kinase. To determine whether Cdc2 kinase phosphorylates vimentin in vivo, a monoclonal antibody 4A4
that recognizes the phosphorylation of Ser55 on vimentin
was produced (11). Ser55 was phosphorylated in various
types of cells only during early mitotic phase, and the chromatographic
analysis of mitotic cell lysates revealed a single peak of
Ser55 kinase activity that is identical to Cdc2 kinase.
These data together with data obtained by tryptic phosphopeptide
analysis (55) strongly suggest that Cdc2 kinase directly phosphorylates vimentin in mitotic cells. By using antibodies recognizing the phosphorylation of the distinct specific sites on vimentin, we further
identified protein kinase C and CaM kinase II as in vivo vimentin kinases that act during cell cycle and cell signaling, respectively (12-14). Here, we have identified Ser71 on
vimentin as a unique site for Rho-kinase in vitro. By
producing and utilizing the site- and phosphorylation state-specific
antibody GK71 which recognizes the phosphorylation of
Ser71, we have demonstrated that vimentin-Ser71
is specifically phosphorylated at the cleavage furrow during cytokinesis. This observation suggests the possibility that Rho-kinase may be responsible for the cleavage furrow-specific phosphorylation of
vimentin.
Rho was reported to be translocated from the cytosol to the cleavage
furrow (56) and to play a critical role in inducing and maintaining the
contractile ring during cytokinesis (22-24). We recently found that
Rho-kinase is also translocated to the cleavage
furrow.2 These accumulating
observations allow us to speculate on the possible mechanism regulating
cytokinesis. The GTP-bound active form of Rho concentrated at the
cleavage furrow may bind to and activate its specific targets around
the cleavage furrow. Rho-kinase may phosphorylate several proteins
including vimentin specifically at the cleavage furrow. Since
phosphorylation of GFAP at Thr7, Ser13, and
Ser34 was also observed at the cleavage furrow (9, 10) and
these three sites were phosphorylated by Rho-kinase in vitro
(37), Rho-kinase might also phosphorylate GFAP during cytokinesis. The cleavage furrow-specific phosphorylation of vimentin and GFAP might
contribute to the efficient separation of these IF structures and allow
the cleavage furrow to contract unencumbered by continuous filaments.
So far, myosin binding subunit of myosin phosphatase (31) and myosin
light chain (39) have been identified as other putative substrates for
Rho-kinase. Phosphorylation of these proteins by Rho-kinase resulted in
activation of myosin ATPase by actin (31, 39). Therefore, there is a
possibility that Rho-kinase may also play an important role in the
contraction and the formation of the contractile ring through the
phosphorylation of these proteins.
Rho is known to regulate the assembly of focal adhesions and actin
stress fibers in response to extracellular signals, such as
lysophosphatidic acid (15). Rho-kinase was recently reported to act
downstream of Rho in the regulation of the formation of stress fibers
and focal adhesion complexes (43, 57, 58). These studies suggested that
Rho may activate Rho-kinase to control actin filament reorganization in
response to extracellular signals during interphase. However, vimentin
phosphorylation at Ser71 did not occur in interphase cells
cultured in the presence of the serum, which contains lysophosphatidic
acid (Fig. 7). Furthermore, the phosphorylation of
vimentin-Ser71 was not observed when quiescent
serum-starved Swiss 3T3 cells were stimulated by lysophosphatidic
acid.3 Why was vimentin
phosphorylated by constitutively active Rho-kinase in COS-7 cells but
not by endogenous activated Rho-kinase in interphase cells? One
possible explanation is the compartmentalized distribution of activated
Rho-kinase in the cell. Since Rho-kinase in the cytoplasm is thought to
be translocated to membranes by forming a complex with GTP-bound Rho
(33, 34), endogenous activated Rho-kinase might be kept apart from
cytoplasmic vimentin-IFs and unable to phosphorylate vimentin in
interphase cells. Definitive mechanism governing the cleavage
furrow-specific phosphorylation of vimentin at Ser71
remains unclear, but the contractile force might partly contribute to
the interaction between cytoplasmic vimentin-IFs and membrane-bound active Rho-kinase at the cleavage furrow.
Since Rho-kinase belongs to a family of related serine/threonine
kinases including myotonic dystrophy kinase, these kinases may
phosphorylate the similar sites on vimentin and GFAP. Further investigations are necessary to elucidate the relationship between IF
phosphorylation at the cleavage furrow and Rho-kinase or its family
members during cytokinesis.
 |
ACKNOWLEDGEMENTS |
We are grateful to K. Matsuzawa and K. Hara
(our laboratory) for technical assistance. We also thank Dr. Y. Nishi
(our laboratory) for help in the electron microscopy, Drs. K. Okawa and
A. Iwamatsu (Kirin Brewery Co. Ltd.) for comments on the Rho-kinase
phosphorylation sites of vimentin, and Drs. N. Inagaki (our laboratory)
and M. Ohara for critique of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid for
scientific research and cancer research from the Ministry of Education, Science, Sports, and Culture of Japan, and special coordination funds
from the Science and Technology Agency of the Government of Japan, and
Japan Society of the Promotion of Science Research for the Future.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Laboratory of
Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464, Japan. Tel.: 81-52-762-6111 (ext. 8824); Fax: 81-52-763-5233.
1
The abbreviations used are: IF, intermediate
filament; GFAP, glial fibrillary acidic protein; CF kinase, cleavage
furrow kinase; protein kinase A, cAMP-dependent protein
kinase; CaM kinase II, Ca2+-calmodulin-dependent protein kinase II;
GST, glutathione S-transferase; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
GTP
S, guanosine 5'-(3-O-thio)-triphosphate; PAGE,
polyacrylamide gel electrophoresis; HPLC, high performance liquid
chromatography; DAPI, 4',6-diamidine-2-phenylindole-dihydrochloride;
mAb, monoclonal antibody; TN-16,
3-(1-anillinoethylidene)-5-benzylpyrrolidine-2,4-dione; PBS,
phosphate-buffered saline.
2
H. Kosako and M. Inagaki, unpublished
observations.
3
H. Goto, H. Kosako, and M. Inagaki, unpublished
observations.
 |
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