* Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan; Howard Hughes Medical
Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710;
and § Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
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Abstract |
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Adducin is a membrane skeletal protein that
binds to actin filaments (F-actin) and thereby promotes
the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such
as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates -adducin and thereby
enhances the F-actin-binding activity of
-adducin in
vitro. Here we identified the sites of phosphorylation of
-adducin by Rho-kinase as Thr445 and Thr480. We
prepared antibody that specifically recognized
-adducin phosphorylated at Thr445, and found by use of this
antibody that Rho-kinase phosphorylated
-adducin at
Thr445 in COS7 cells in a Rho-dependent manner.
Phosphorylated
-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney
(MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF).
The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or
-adducinT445A,T480A (substitution of Thr445 and
Thr480 by Ala) inhibited the TPA-induced membrane
ruffling in MDCK cells and wound-induced migra-
tion in NRK49F cells.
-AdducinT445D,T480D (substi-
tution of Thr445 and Thr480 by Asp), but not
-adducinT445A,T480A, counteracted the inhibitory
effect of the dominant negative Rho-kinase on the
TPA-induced membrane ruffling in MDCK cells. Taken
together, these results indicate that Rho-kinase phosphorylates
-adducin downstream of Rho in vivo, and
that the phosphorylation of adducin by Rho-kinase
plays a crucial role in the regulation of membrane ruffling and cell motility.
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Introduction |
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MEMBRANE ruffling, a dynamically fluctuating movement of protrusion consisting of filopodia and
lamellipodia, is rapidly induced in response to
certain extracellular signals and is also induced in the leading edges of motile cells (Stossel, 1993; Lauffenburger and
Horwitz, 1996
; Mitchison and Cramer, 1996
; Welch et al.,
1997
). Membrane ruffling is thought to be important for
cell motility (Mitchison and Cramer, 1996
). The small GTPase Rho regulates the formation of actin stress fibers and
focal adhesions (Ridley and Hall, 1992
, 1994
; Hall, 1998
),
membrane ruffling (Nishiyama et al., 1994
; Takaishi et al.,
1995
) and cell motility (Takaishi et al., 1993
, 1994
) in certain types of cells, cell morphology (Paterson et al., 1990
),
cell aggregation (Tominaga et al., 1993
), smooth muscle
contraction (Hirata et al., 1992
; Gong et al., 1996
), neurite
retraction in neuronal cells (Nishiki et al., 1990
; Jalink et al.,
1994
), the formation of microvilli (Shaw et al., 1998
), and
cytokinesis (Kishi et al., 1993
; Mabuchi et al., 1993
). Rho exerts its biological functions through interaction with specific targets (Van Aelst and D'Souza-Schorey, 1997
). Several targets of Rho have been identified, including protein
kinase N (PKN)1 (Amano et al., 1996b
; Watanabe et al.,
1996
), Rho-kinase/ROK/ROCK (Leung et al., 1995
; Ishizaki et al., 1996
; Matsui et al., 1996
), the myosin-binding
subunit (MBS) of myosin phosphatase (Kimura et al.,
1996
), p140mDia (Watanabe et al., 1997
), citron (Madaule et al., 1995
), citron-kinase (Madaule et al., 1998
), rhophilin, rhotekin (Reid et al., 1996
), and kv1.2 (Cachero et al.,
1998
). Rho-kinase, which is activated by Rho, regulates
the phosphorylation of myosin light chain (MLC) by the
direct phosphorylation of MLC (Amano et al., 1996a
) and
by the inactivation of myosin phosphatase through the
phosphorylation of MBS (Kimura et al., 1996
). Rho-kinase regulates the formation of stress fibers and focal adhesions
(Leung et al., 1996
; Amano et al., 1997
; Ishizaki et al.,
1997
), smooth muscle contraction (Kureishi et al., 1997
),
and neurite retraction (Amano et al., 1998
; Hirose et al.,
1998
; Katoh et al., 1998
) through the phosphorylation of
MLC. Rho-kinase also phosphorylates the ezrin/radixin/
moesin (ERM) family proteins (Matsui et al., 1998
) and
then regulates the formation of microvilli (Oshiro et al.,
1998
). However, it remains to be clarified how Rho regulates membrane ruffling and cell motility.
Recently, we identified adducin as an MBS-binding protein and a substrate of Rho-kinase in vitro (Kimura et al.,
1998). Adducin is a membrane skeletal protein that binds
to the fast growing ends (Kuhlman et al., 1996
; Li et al.,
1998
) and the sides (Mische et al., 1987
) of actin filaments
(F-actin), and thereby promotes the association of spectrin
with F-actin to form a spectrin-actin meshwork beneath
plasma membranes (Gardner and Bennett, 1987
; Bennett
et al., 1988
). Adducin is composed of either
and
, or
and
subunits which are closely related in amino acid
sequence and domain organization (Gardner and Bennett, 1986
; Bennett et al., 1988
; Dong et al., 1995
). Each
adducin subunit has three distinct domains: an amino-terminal head domain, a neck domain, and a carboxy-terminal tail domain (Joshi and Bennett, 1990
; Joshi et al.,
1991
; Dong et al., 1995
). The tail domain has a domain called the myristoylated alanine-rich C kinase substrate
(MARCKS)-related domain, which contains a highly basic
stretch and binds to calmodulin (Joshi et al., 1991
; Dong et
al., 1995
; Matsuoka et al., 1996
). Adducin is also a substrate for PKC and PKA (Palfrey and Waseem, 1985
; Kaiser
et al., 1989
; Dong et al., 1995
). The phosphorylation of adducin by PKA reduces the activity of adducin to associate with F-actin and spectrin-F-actin complexes and to recruit
spectrin to F-actin (Matsuoka et al., 1996
). The phosphorylation of adducin by PKC also inhibits activity of adducin
in binding to F-actin and recruiting spectrin to F-actin
(Matsuoka et al., 1998
). On the other hand, the phosphorylation of adducin by Rho-kinase results in the interaction
of adducin with F-actin in vitro (Kimura et al., 1998
).
In this study, we identified the sites of phosphorylation
of -adducin by Rho-kinase as Thr445 and Thr480, and
prepared antibody that specifically recognized
-adducin
phosphorylated at Thr445 to elucidate the functions of
-adducin phosphorylated by Rho-kinase. We found that
-adducin was phosphorylated at Thr445 via the Rho/ Rho-kinase pathway in intact cells, and that the phosphorylation of
-adducin by Rho-kinase plays a crucial
role in membrane ruffling and cell motility.
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Materials and Methods |
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Materials and Chemicals
MDCK cells were kindly provided by Dr. S. Tsukita (Kyoto University,
Kyoto, Japan). NRK49F cells were purchased from American Type Culture Collection. pEF-BOS-HA-RhoAV14, RhoAN19, Rac1V12, Cdc42V12,
pEF-BOS-myc-Rho-kinase (full-length, 6-1388 amino acids [aa]), Rho-binding domain (RB; 941-1075 aa), RB/PH (TT) (941-1388 aa), and CAT
(the catalytic domain of Rho-kinase, 6-553 aa) were constructed as described (Kuroda et al., 1996; Matsui et al., 1996
; Amano et al., 1997
, 1998
).
pcDSR
-PKC
lacking C1 domain at positions 6-159 aa was constructed
as described (Kaibuchi et al., 1989
). pQE-rat
-adducin fragment (319-671
aa; 35H fragment-4D) was kindly provided by Dr. S. Jaken (University of
Vermont, Burlington, VT), and 6 X His-tagged
-adducin fragment was
produced and purified as described (Dong et al., 1995
). The expression
plasmid of C3 transferase (pGEX-C3) was kindly provided by Dr. A. Hall
(University College London, London, UK), and C3 was produced and
purified as described (Fukata et al., 1998
). Glutathione-S-transferase (GST)-RB was produced and purified from Escherichia coli as described (Amano et al., 1997
). GST-CAT was produced and purified from Sf9 cells
as described previously (Matsuura et al., 1987
; Amano et al., 1996a
).
Rac1N17 was produced and purified from E. coli as described (Kuroda et al., 1996
). Anti-hemagglutinin (HA) monoclonal Ab (12CA5) was purchased from Boehringer Mannheim. [
-32P]ATP was purchased from Amersham Corp. All materials used in the nucleic acid study were purchased
from Takara Shuzo Corp. Other materials and chemicals were obtained
from commercial sources.
Plasmid Constructs
The cDNA encoding human -adducin (1-737 aa; Joshi et al., 1991
) was
inserted into the KpnI site of pEF-BOS-HA, into the KpnI site of
pAcYM1-HA to obtain recombinant
-adducin by the use of a baculovirus system, and into the BamHI site of pGEX-2T. The cDNA encoding
human
-adducin (1-726 aa; Joshi et al., 1991
) was also inserted into the
KpnI site of pAcYM1-HA. The cDNAs of
-adducinT445A,T480A (AA) and
-adducinT445D,T480D (DD), which were substituted for Thr at both 445 and
480 amino acid residues by Ala or Asp, were generated with the use of a site-directed mutagenesis kit (Stratagene), and subcloned into pEF-BOS-HA, pAcYM1-HA, and pGEX-2T. The cDNAs of
-adducinT445A and
-adducinT480A were also generated as above and subcloned into pGEX-2T.
Protein Purification
RB/PH (TT) (Amano et al., 1998) was expressed as a maltose-binding
protein (MBP) fusion protein in E. coli and purified. GST-
-adducin, GST-
-adducin-AA, GST-
-adducinT445A, and GST-
-adducinT480A were
produced and purified from E. coli. HA-
-adducin, HA-
-adducin, HA-
-adducin-AA, and HA-
-adducin-DD were produced in Sf9 cells. The
cells expressing HA-
-adducin were suspended in homogenizing buffer
(20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 10 µM A-PMSF, 10 µg/ml leupeptin). The suspension was sonicated and centrifuged at
100,000 g for 1 h at 4°C. The supernatant was applied onto a Mono Q column (Pharmacia LKB Biotechnology) which had been equilibrated with
buffer A (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT). The column was then washed, and the proteins were eluted with a linear concentration gradient of NaCl (0-600 mM) in buffer A. HA-
-adducin was
eluted with ~200 mM NaCl and purified to near homogeneity. HA-
-adducin, HA-
-adducin-AA, and HA-
-adducin-DD were prepared as
HA-
-adducin.
Determination of Phosphorylation Sites of -Adducin
by Rho-Kinase
HA--adducin (70 µg of protein) was phosphorylated with GST-CAT
(120 ng of protein) in 2 ml of kinase buffer (50 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) containing 100 µM
[
-32P]ATP for 1 h at 30°C, and the reaction product was digested with Achromobacter protease-1 (AP-1) at 37°C for 20 h. The obtained peptides were applied onto a C18 reverse-phase column (4.6 × 250 mm;
Shiseido, Japan) and eluted with a linear gradient of 0-48% acetonitrile
for 100 min at a flow rate of 1.0 ml/min by HPLC (System Gold, Beckman). The radioactive peaks were separated and phosphoamino acid sequencing was carried out with a peptide sequencer (PPSQ-10; Shimadzu,
Japan) as described (Bodwell et al., 1991
). The fractions obtained from
each Edoman degradation cycle were measured for 32P in a Beckman liquid scintillation counter.
Production of Site- and Phosphorylation State-specific
Antibody for -Adducin
The phosphopeptide Cys-Gln440-Gln-Arg-Glu-Lys-phospho-Thr-Arg-Trp-
Leu-Asn-Ser450 was chemically synthesized as an antigen by Peptide Institute Inc. Rabbit polyclonal antibody (anti-pT445) was raised against the
phosphopeptide and affinity purified as described (Inagaki et al., 1997).
To examine the specificity of anti-pT445, equal amounts of HA-
-adducin
(40 fmol) with various ratios between phosphorylated and nonphosphorylated proteins were subjected to SDS-PAGE. HA-
-adducin-AA (40 fmol) was incubated in kinase buffer containing GST-CAT and ATP, and
subjected to SDS-PAGE. The amount of phosphates incorporated into
HA-
-adducin was simultaneously determined by [
-32P]ATP. Immunoblot analysis was then performed with anti-pT445, anti-pT445 which was
preincubated with a 100-fold amount of antigen-phosphopeptide, and
anti-HA Ab. For some experiments, HA-
-adducin, HA-
-adducin, or 6 X
His-
-adducin fragment was separately phosphorylated with GST-CAT,
and 40 fmol (as 32P-incorporated amount) of phosphorylated proteins was
subjected to immunoblot analysis with anti-pT445.
Cell Culture
MDCK cells and NIH3T3 cells were maintained in DMEM containing
10% calf serum, streptomycin, and penicillin. COS7 cells were maintained
in DMEM containing 10% FBS, streptomycin, and penicillin. NRK49F
cells were maintained in DMEM containing 5% calf serum and 1% nonessential amino acids. To obtain MDCK cells stably expressing HA--adducin, MDCK cells were transfected with pEF-BOS-HA-
-adducin along
with pSVIISR
vector containing the neomycin resistance gene using Lipofectamine (GIBCO BRL) as described (Kuroda et al., 1998
), and neomycin-resistant clones were selected.
Detection of Thr445-phosphorylated -Adducin in
Intact Cells
The transfection of plasmids into COS7 cells was carried out by the standard DEAE-dextran method. COS7 cells were cotransfected with pEF-BOS-HA--adducin and pEF-BOS-vector, pEF-BOS encoding HA-Rac1V12, HA-Cdc42V12, HA-RhoAV14, HA-RhoAN19, myc-Rho-kinase
(full-length), HA-RhoAV14 and myc-Rho-kinase (full-length), HA-RhoAV14 and myc-RB, HA-RhoAV14 and myc-RB/PH (TT), myc-CAT, or
pcDSR
-PKC
. Separately, COS7 cells were cotransfected with pEF-BOS-HA-
-adducin-AA and myc-CAT. After a 24-h incubation, the
transfected cells were incubated in serum-free medium for 24 h. The cells
were treated with 10% (wt/vol) trichloroacetic acid. The resulting precipitates were subjected to immunoblot analysis using anti-HA Ab and anti-pT445. MDCK cells stably expressing HA-
-adducin were incubated in
the absence or the presence of tetradecanoylphorbol-13-acetate (TPA) for 15 min followed by incubation in the presence of 0.1 µM calyculin A
for 10 min. The cells were then treated as COS7 cells and subjected to immunoblot analysis.
Immunofluorescence Analysis
MDCK cells were deprived of serum for 24 h and incubated in DMEM
containing 200 nM TPA or 50 pM recombinant human hepatocyte growth
factor (HGF; Calbiochem) for 15 min at 37°C. For cell scattering, the cells
were not deprived of serum and incubated in DMEM containing 5% calf
serum and 200 nM TPA for 2 h. The cells were fixed with 3.0% formaldehyde in PBS for 10 min and treated with PBS containing 0.2% Triton
X-100 for 10 min. Phosphorylated adducin and F-actin were doubly
stained with anti-pT445 and TRITC-phalloidin. For anti-pT445, the cells
were then labeled with FITC-conjugated anti-rabbit Ig Ab. For a double
immunofluorescence study, the clone stably expressing HA--adducin
was stimulated as described above. Phosphorylated adducin and HA-
-adducin were doubly stained with anti-pT445 and anti-HA Ab. The
cells were then labeled with FITC-conjugated anti-rabbit Ig Ab and Texas
red-conjugated anti-mouse Ig Ab. The cells were examined using a Zeiss
Axiophot microscope or a confocal microscope (Carl Zeiss).
Microinjection
MDCK cells were seeded at a density of 2.5 × 103 cells per 13-mm cover
glass in 60-mm tissue culture dishes and incubated for 8 h. Then, the cells
were deprived of serum for 24 h. For microinjection, proteins were concentrated and during the concentration the buffer was replaced by microinjection buffer (20 mM Tris-HCl, pH 7.4, 20 mM NaCl, 1 mM MgCl2,
0.1 mM EDTA, 5 mM 2-mercaptoethanol). The indicated proteins were
microinjected along with a marker protein (rabbit or mouse IgG at 0.5 mg/ml) into the cytoplasm of cells. Using microcapillaries (tip diameter is
0.5 µm), ~3 × 1014 liter of each protein was microinjected by one injection. When C3 was microinjected at 0.1 mg/ml, the injected amount per
cell was ~3 × 10
15 g (the intracellular concentration was estimated to be
~0.15 µM). When HA-
-adducin mutants were microinjected at 5.0 mg/ml, the injected amount per cell was ~150 × 10
15 g (the intracellular concentration was estimated to be ~2.0 µM, and it seemed to be much higher
than the level of endogenous
-adducin). 30 min after the microinjection,
the cells were incubated in DMEM containing 200 nM TPA for 15 min at
37°C. The cells were then fixed and stained with anti-pT445 or TRITC-phalloidin as described above.
Wound Healing Assay
Cell migration assay using a wound healing model was performed as described previously (Santos et al., 1997). The confluent monolayer of the
cells was scraped with a white chip. Soon after wounding, the medium was
changed and indicated recombinant proteins were microinjected along
with a marker protein (rabbit IgG, 1.0 mg/ml) into the cytoplasm of only
the cells along the wound edge as described above. The original wound
edge was determined by taking photographs soon after the cells were
wounded and microinjected. After 6 h, the cells were then stained with
TRITC-phalloidin, and the cells that moved (>20 µm) from the original
wound edge were counted by taking photographs of the same fields as the
original photograph.
Other Procedures
SDS-PAGE was performed as described (Laemmli, 1970). Protein concentrations were determined with BSA as the reference protein as described (Bradford, 1976
).
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Results |
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Determination of the Sites of Phosphorylation of
-Adducin by Rho-Kinase
We first determined the sites of phosphorylation of human
-adducin by Rho-kinase as follows. HA-tagged
-adducin, which was phosphorylated by Rho-kinase in the presence of [
-32P]ATP, was digested with AP-I and subjected
to reverse-phase HPLC as described (Shirataki et al.,
1993
). Two major radioactive peaks (named peak 1 and 2, respectively) were obtained (Fig. 1 A) and their amino
acid sequences were determined. The sequence obtained from peak 1 was TRWLNSGRGDEASEEGQNGSSPK,
corresponding to residues 445-467 of human
-adducin,
and Thr445 turned out to be the phosphorylation site for
Rho-kinase (Fig. 1 B, panel a). The sequence obtained from peak 2 was EDGHRTSTSAVPNLFVPLNTNPK,
corresponding to residues 475-497 of human
-adducin,
and Thr480 turned out to be the phosphorylation site for
Rho-kinase (Fig. 1 B, panel b). Both phosphorylation sites
were in the neck domain of
-adducin, and were different from previously identified sites of phosphorylation by
PKA (Ser408, Ser436, Ser481, and Ser726) and by PKC
(Ser726) (Matsuoka et al., 1996
) (Fig. 1 C). When GST-
-adducinT445A or GST-
-adducinT480A (substitution of
Thr445 or Thr480 by Ala) was phosphorylated by Rho-kinase, GST-
-adducinT480A was more efficiently phosphorylated by Rho-kinase than was GST-
-adducinT445A (Fig. 1
D). This result is apparently inconsistent with the observation that the radioactivity of peak 1 containing Thr445 is
lower than that of peak 2 containing Thr480 in the HPLC
analysis (Fig. 1 A). This may be due to the different recovery of each peptide during separation by HPLC. Taken together, these results suggest that Thr445 is a preferential
site of phosphorylation by Rho-kinase in vitro.
|
Production and Characterization of the Site- and
Phosphorylation State-specific Antibody for -Adducin
An antibody that recognizes the phosphorylated state of a
substrate at a specific site is a useful tool with which to
evaluate site-specific phosphorylation in vivo and to visualize the cellular distribution of the protein phosphorylated at a specific site (Inagaki et al., 1997). We prepared
and affinity purified the polyclonal antibody that recognized the Thr445-phosphorylated
-adducin (anti-pT445),
against the synthetic phosphopeptide Cys-Gln-Gln-Arg-Glu-Lys-phospho-Thr445-Arg-Trp-Leu-Asn-Ser. An immunoblot analysis revealed that anti-pT445 specifically bound
to HA-
-adducin phosphorylated by Rho-kinase in a
dose-dependent manner, but not to nonphosphorylated
HA-
-adducin or HA-
-adducinT445A,T480A (substitution of
Thr445 and Thr480 by Ala; HA-
-adducin-AA) incubated with Rho-kinase and ATP (Fig. 2 A). HA-
-adducin,
which was phosphorylated by PKC or PKN at levels similar to that by Rho-kinase, was not recognized by anti-pT445 (data not shown). The binding of anti-pT445 to
-adducin phosphorylated by Rho-kinase was neutralized
by preincubation of the antibody with the antigen phosphopeptide. Rho-kinase phosphorylated
- and
-adducin
in vitro as well (Fig. 2 B). Neither
- nor
-adducin phosphorylated by Rho-kinase was recognized by anti-pT445
(Fig. 2 B). These results indicate that anti-pT445 specifically recognizes only
-adducin phosphorylated at Thr445.
|
Rho/Rho-Kinase-dependent Phosphorylation of
-Adducin in COS7 Cells
We examined whether -adducin was phosphorylated via
the Rho/Rho-kinase pathway in COS7 cells by immunoblot analysis with anti-pT445. cDNA of HA-
-adducin
was cotransfected with plasmids carrying the cDNA of
dominant active Rho family members, or dominant active
or negative Rho-kinase (Fig. 3). HA-
-adducin was almost equally expressed in all the HA-
-adducin-transfected
cells.
-Adducin phosphorylated at Thr445 was undetectable in serum-starved COS7 cells expressing HA-
-adducin alone. The expression of dominant active RhoA
(RhoAV14), a RhoA mutant that is defective in GTPase activity and thought to be constitutively the GTP-bound
form in the cells, induced a small increment of the
-adducin phosphorylation at Thr445, whereas the expression of
dominant negative RhoA (RhoAN19), a RhoA mutant that
preferentially binds GDP rather than GTP and is thought
to be constitutively the GDP-bound form in the cells, had
minimal effects. Although the exact reason for the minimal increase in phosphorylated
-adducin in COS7 cells
expressing RhoAN19 is unknown, it is likely that the overexpression of GDP-bound RhoAN19 induced the phosphorylation of
-adducin to a minimal extent because
GDP · Rho also activates Rho-kinase in vitro to a small extent as described (Matsui et al., 1996
). Dominant active
Rac1 (Rac1V12) or dominant active Cdc42 (Cdc42V12) had
no effects. Under the conditions, RhoAV14, RhoAN19,
Rac1V12, and Cdc42V12 were almost equally expressed
(data not shown). The expression of Rho-kinase induced
the
-adducin phosphorylation to some extent. By the coexpression of RhoAV14 and Rho-kinase, the
-adducin
phosphorylation was further enhanced. Rho-kinase is
composed of catalytic (CAT), coiled-coil (COIL), Rho-binding (RB), and pleckstrin-homology (PH) domains
(Matsui et al., 1996
). RB (941-1075 aa) binds to Rho and
inhibits the Rho-dependent Rho-kinase activity in vitro
and in vivo (Amano et al., 1997
). RB/PH (TT) (941-1388
aa), which is the carboxy-terminal portion of Rho-kinase
and encompasses the RB and PH domains, has point mutations in the RB domain and does not bind to Rho
(Fujisawa et al., 1996
; Leung et al., 1996
; Amano et al.,
1998
). RB/PH (TT) inhibits the lysophosphatidic acid
(LPA)-induced stress fiber formation in fibroblasts and
neurite retraction in neuroblastoma cells (Amano et al.,
1998
), suggesting that RB/PH (TT) serves as the dominant
negative form of Rho-kinase. Recently, we found that RB/
PH (TT) interacted with the catalytic domain of Rho-kinase and thereby inhibited the Rho-kinase activity in
vitro without titrating out Rho (Amano, M., K. Chihara,
N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi,
manuscript submitted for publication). We further found
that RB/PH (TT) had no effect on catalytic activity of
PKN or myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), which is identified as a target of
Cdc42 and is homologous to Rho-kinase within the kinase
domain (Leung et al., 1998
), in vitro (Amano, M., K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi, manuscript submitted for publication). RB/PH (TT) inhibited the Rho-kinase-induced stress fiber, but
not MRCK-induced stress fiber (Leung et al., 1998
) in fibroblasts (Amano, M., K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi, manuscript submitted for publication). These observations indicate that
RB/PH (TT) more specifically inhibits the Rho-kinase activity in intact cells.
-Adducin phosphorylation at Thr445
by RhoAV14 was suppressed by RB and RB/PH (TT) (Fig.
3). Constitutively active Rho-kinase (CAT) greatly induced
the
-adducin phosphorylation (Fig. 3), whereas constitutively active MRCK did not (data not shown). When the
dominant active form of PKC, which activates the TPA-responsive element linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester
(Kaibuchi et al., 1989
), was expressed, the phosphorylation
of
-adducin was not detected by anti-pT445 (Fig. 3). HA-
-adducin-AA coexpressed with CAT was not recognized
by anti-pT445 (Fig. 3). These results indicate that
-adducin was specifically phosphorylated at Thr445 via the Rho/
Rho-kinase pathway in intact cells.
|
Specific Phosphorylation of -Adducin at Thr445 in the
TPA-induced Membrane Ruffling Areas
Both Rho and Rac are thought to be necessary for the
HGF- and TPA-induced membrane ruffling and HGF-induced cell motility in MDCK epithelial cells, although
neither RhoAV14 nor Rac1V12 by itself induces membrane
ruffling or cell motility (Takaishi et al., 1993, 1994
, 1995
;
Nishiyama et al., 1994
; Ridley et al., 1995
). We examined
the distribution of Thr445-phosphorylated
-adducin in
the TPA- or HGF-stimulated MDCK cells. We have found
by immunoblotting with isoform-specific antibodies that
120-kD
-adducin and 90-kD
-adducin (alternatively
spliced form of 80-kD
-adducin) were expressed almost
equally in MDCK cells. We have also found that 110-kD
-adducin was expressed to a lesser extent in MDCK cells. Therefore, it is likely that anti-pT445 recognizes only
phospho-Thr445 in
-adducin in MDCK cells. The addition of TPA induced membrane ruffling at the outer edge
of cell colonies (Fig. 4 A) as described (Nishiyama et al.,
1994
; Takaishi et al., 1995
). The immunoreactivity of anti-pT445 accumulated in the membrane ruffling areas, where F-actin (Fig. 4 A) and spectrin (data not shown) also accumulated. Phosphorylated
-adducin was also detected in
the perinuclear region. Similar results were obtained when
the cells were stimulated with HGF instead of TPA (Fig. 4
A). The immunoreactivity with anti-pT445 was abolished
by preincubation of the antibody with the antigen
phosphopeptide (data not shown). In the nonstimulated MDCK cells, phosphorylated
-adducin was diffusely
present in the cytoplasm, but not in the free ends of the
plasma membrane. Between 2 and 16 h after the addition
of TPA, the cells dissociated from each other and scattered, with polarized morphology and membrane ruffling
in the leading edge as described (Takaishi et al., 1994
; Ridley et al., 1995
). Phosphorylated
-adducin also accumulated in the leading edge of TPA-induced scattering cells
(Fig. 4 A). To compare the localization of phosphorylated
-adducin and total
-adducin, a double-label immunofluorescence study was performed in MDCK cells stably expressing HA-
-adducin. Immunoreactivity showing the
distribution of HA-
-adducin (red) was detected strongly in the cell-cell contact sites (Fig. 4 B, asterisk), weakly in the membrane ruffling areas (Fig. 4 B, arrow), and diffusely in the cytoplasm, as described (Kaiser et al., 1989
;
Marrs et al., 1993
). Phosphorylated
-adducin (green) was
enriched in the membrane ruffling areas in the TPA-stimulated MDCK cells (Fig. 4 B, arrow). The merged image
of red and green immunofluorescence revealed the enrichment of phosphorylated
-adducin in the membrane ruffling areas and a small increment of phosphorylated
-adducin in the cytoplasm in the TPA-stimulated cells
(Fig. 4 B, panel f, greenish image). Phosphorylated
-adducin did not accumulate at the cell-cell contact sites
(Fig. 4 B, panels c and f, reddish images indicated by the
asterisks). Similar results were obtained when the cells were stimulated with HGF. To confirm that
-adducin is
specifically phosphorylated at Thr445 during the action of
TPA, immunoblot analysis with anti-pT445 was performed. The addition of TPA to the MDCK cells stably
expressing HA-
-adducin increased the phosphorylation level of HA-
-adducin at Thr445 (Fig. 4 C).
|
The Inhibition of the TPA-induced Membrane
Ruffling by the Dominant Negative Rho-Kinase and
-Adducin Mutant
We next examined whether the Rho/Rho-kinase pathway
was required for the TPA-induced membrane ruffling.
The microinjection of C3, which is thought to interfere
with endogenous Rho functions, RB, or RB/PH (TT) inhibited the TPA-induced membrane ruffling and the accumulation of phosphorylated adducin in the free end of
the plasma membrane (Fig. 5, A and B); membrane ruffling was induced in 64% of TPA-stimulated cells in the
outer edges of cell colonies, but in only 14% of cells injected with C3, 15% of cells injected with RB, and 22% of
cells injected with RB/PH (TT). Dominant negative Rac
(Rac1N17) also inhibited the TPA-induced membrane ruffling to a similar extent (Ridley et al., 1995). These results
indicate that the Rho-kinase function is required for the
TPA-induced membrane ruffling in MDCK cells.
|
To determine whether the phosphorylation of adducin
by Rho-kinase is necessary or sufficient for the membrane
ruffling, we produced and used cDNAs encoding -adducin
mutants; HA-
-adducinT445A,T480A (HA-
-adducin-AA,
substitution of Thr residues by Ala), which was not phosphorylated by Rho-kinase and expected to serve as the
dominant negative form, and HA-
-adducinT445D,T480D
(HA-
-adducin-DD, substitution of Thr residues by Asp),
which was expected to mimic phosphorylated
-adducin
and to serve as the constitutively active form (Sweeney et al.,
1994
). The microinjection of HA-
-adducin, HA-
-adducin-AA, or HA-
-adducin-DD by itself did not induce the
membrane ruffling in MDCK cells (data not shown). This
observation is consistent with the notion that RhoAV14
alone does not induce the membrane ruffling, and that a
lot of processes are required for the induction of membrane ruffling. HA-
-adducin or HA-
-adducin-DD was
translocated to the membrane ruffling area (data not
shown) and did not inhibit the TPA-induced membrane
ruffling, whereas HA-
-adducin-AA inhibited the ruffling; membrane ruffling was induced in ~60% of TPA-stimulated cells injected with HA-
-adducin or HA-
-adducin-DD, but in only 24% of cells injected with
HA-
-adducin-AA (Fig. 5, A and B). Although the inhibitory mechanism of HA-
-adducin-AA is not known,
HA-
-adducin-AA may substitute for endogenous
-adducin to oligomerize with endogenous
- or
-adducin, and thereby stay as an inactive complex to inhibit functions of endogenous adducins. Essentially similar results as to the effects of RB/PH (TT) or HA-adducin-AA
on membrane ruffling were obtained when MDCK cells
were stimulated with HGF instead of TPA (data not
shown). To confirm that HA-
-adducin-AA specifically inhibits the function of endogenous
-adducin but not
those of other substrates of Rho-kinase such as MLC or
the ERM family proteins, we examined whether the inhibitory effect of HA-
-adducin-AA on the membrane ruffling in MDCK cells was reversed by coinjection of HA-
-adducin or HA-
-adducin-DD. As shown in Fig. 5 C,
HA-
-adducin reversed the inhibitory effect of HA-
-adducin-AA in a dose-dependent manner. HA-
-adducin-DD
more efficiently reversed the inhibitory effect. These results indicate that HA-
-adducin-AA specifically inhibits
the function of endogenous
-adducin but not those of other substrates. When HA-
-adducin-AA was microinjected into NIH3T3 cells, HA-
-adducin-AA did not affect
the LPA-induced formation of stress fibers, which is mediated by MLC phosphorylation through the Rho/Rho-kinase
pathway (Chihara et al., 1997
; Amano et al., 1998
), whereas
RB/PH (TT) inhibited the LPA-induced formation of stress
fibers as described (Amano et al., 1998
; Fig. 5 D). HA-
-adducin-AA did not affect the LPA-induced formation of microvilli, which is mediated by ERM phosphorylation
through the Rho/Rho-kinase pathway, under the conditions
in which RB/PH (TT) inhibited the LPA-induced formation
of microvilli (Oshiro et al., 1998
; data not shown). Taken together, these results suggest that HA-
-adducin-AA specifically interferes with the pathway mediated through the
-adducin phosphorylated by Rho-kinase in intact cells.
When HA--adducin-DD, but not HA-
-adducin-AA,
was comicroinjected with RB or RB/PH (TT), the inhibitory effect of RB or RB/PH (TT) on the TPA-induced
membrane ruffling was counteracted (Fig. 6, A and B).
Under these conditions, HA-
-adducin also counteracted the inhibitory effect of RB/PH (TT), but not that of RB
(Fig. 6 B). This difference is likely due to the distinct mode
of action between RB and RB/PH (TT). RB/PH (TT) interacts with the catalytic domain of Rho-kinase and inhibits the activity of Rho-kinase in a manner competitive with
peptide substrates of Rho-kinase such as RRRLSSLRA
without titrating out Rho (Amano, M., K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi,
manuscript submitted for publication). The excess of HA-
-adducin over RB/PH (TT) in the cells may neutralize
a competitive inhibition of Rho-kinase by RB/PH (TT),
and then HA-
-adducin can be phosphorylated by the
activated Rho-kinase. Consistently, when RB/PH (TT)
was microinjected at higher concentrations, HA-
-adducin could not counteract the inhibitory effect of RB/PH
(TT), whereas HA-
-adducin-DD was still capable of
counteracting (data not shown). In contrast to RB/PH (TT), RB binds to the effector domain of Rho, and is expected to inhibit the interaction of Rho-kinase with Rho
and thereby the activation of Rho-kinase by Rho (Amano
et al., 1997
). Thus, HA-
-adducin may not overcome the inhibitory effect of RB in MDCK cells. On the other hand,
because HA-
-adducin-DD can mimic
-adducin phosphorylated at Thr445 and Thr480 in vivo, HA-
-adducin-DD may bypass Rho-kinase to induce membrane
ruffling during the action of TPA irrespective of the Rho-kinase activity. HA-
-adducin-DD counteracted neither
the inhibitory effect of C3 nor that of Rac1N17 (Fig. 6, A
and B). GTP
S-bound RhoAI41, which is insensitive to C3,
could completely overcome the inhibitory effect of C3 as
described (Nishiyama et al., 1994
; data not shown). Taken
together, these results indicate that HA-
-adducin-DD
serves as the constitutively active form, and that
-adducin
is a crucial substrate of Rho-kinase downstream of Rho
for membrane ruffling.
|
Adducin Phosphorylation via the Rho/Rho-Kinase Pathway and Cell Motility
Membrane ruffling is thought to be an essential event in
cell motility (Mitchison and Cramer, 1996). We then investigated whether the
-adducin phosphorylation by Rho-kinase is involved in cell motility. C3 has been shown to inhibit the wound-induced migration of IEC-6 cells (Santos
et al., 1997
). We performed a wound healing assay by the
use of NRK49F cells to investigate roles of Rho-kinase
and adducin in cell motility, since it was difficult to estimate the precise distance moved in MDCK cells and to
evaluate cell motility induced by TPA or HGF, and
NRK49F cells were more suitable for a wound healing assay than were MDCK cells. Linear wounds were made in a
confluent monolayer of NRK49F cells and ~200 cells
along the wound edge were microinjected with C3, RB/PH
(TT), or the
-adducin mutants. 6 h after wounding, the
cells that moved (>20 µm) from the original wound edge
were counted. We also evaluated the wound-induced migration by a time-lapse recording. When MBP was injected as a control protein, 63% of the total injected cells
showed wound-oriented migration, and ~60% of the total
injected cells had membrane rufflings (Fig. 7, B and C).
About 90% of the migrating injected cells had membrane rufflings in their leading edges (Fig. 7 B). We found that
-adducin phosphorylated at Thr445 accumulated at the
membrane ruffling areas in the leading edge and the perinuclear region in migrating NRK49F cells (Fig. 7 A), as
shown in MDCK cells. C3 and RB/PH (TT) inhibited the
migration to the wound; only 24% of cells injected with C3
and 17% of cells injected with RB/PH (TT) migrated to the wound (Fig. 7, B and C). The microinjection of HA-
-adducin-AA also inhibited the migration; only 21% of
cells injected with HA-
-adducin-AA migrated to the wound
(Fig. 7, B and C). When C3, RB/PH (TT), or
-adducin-AA was injected, the percentages of the cells with membrane ruffling in the total injected cells (~20%) were
roughly the same as those of the migrating cells in the total
injected cells (17-24%), and the stationary injected cells
had few membrane rufflings (Fig. 7 B). Whatever proteins were injected, the majority (~90%) of migrating injected
cells had membrane ruffling in the leading edge. These
results suggest that the inhibition of cell migration by HA-
-adducin-AA, C3, or RB/PH (TT) correlates with the
inhibition of membrane ruffling. In the HA-
-adducin-
injected cells, the wound-oriented migration and membrane
ruffling were not inhibited (Fig. 7, B and C). HA-
-adducin-DD also inhibited the migration; 32% of cells injected
with HA-
-adducin-DD migrated to the wound (Fig. 7, B
and C). However, the percentage of the cells with membrane ruffling in the total injected cells (~60%) was much
bigger than that of the migrating cells in the total injected
cells (32%). The majority of migrating cells injected with
-adducin-DD had membrane rufflings in the leading
edge, and about one-half of the stationary cells injected
with
-adducin-DD had membrane rufflings (Fig. 7 B,
panel k). These results indicate that
-adducin-DD did not
inhibit the membrane ruffling but did inhibit cell migration, suggesting that cycling between the phosphorylated and dephosphorylated states of
-adducin appears to be
necessary for cell migration, but not for membrane ruffling. Consistently, RhoAV14 or CAT as well as Rac1N17 inhibited the wound-induced migration (data not shown).
|
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Discussion |
---|
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---|
Phosphorylation Sites of -Adducin by Rho-Kinase and
Site- and Phosphorylation State-specific Antibodies
In this study, we identified the sites of phosphorylation of
-adducin by Rho-kinase as Thr445 and Thr480 in the
neck domain.
-Adducin is phosphorylated at Ser408,
Ser436, and Ser481 in the neck domain by PKA, and at
Ser726 in the MARCKS-related domain by PKC and PKA
(Matsuoka et al., 1996
). Among the in vitro phosphorylation sites of
-adducin, Thr445 and Thr480 are unique
for Rho-kinase. We raised and used a site- and phosphorylation state-specific antibody for
-adducin. Anti-pT445
recognized HA-
-adducin phosphorylated by Rho-kinase, but not by PKC or PKN. Anti-pT445 recognized neither nonphosphorylated HA-
-adducin nor HA-
-adducinT445A,T480A incubated with Rho-kinase and ATP
(Fig. 2 A). These results indicate that anti-pT445 is a useful tool with which to detect the specific phosphorylation
of
-adducin by Rho-kinase both in vitro and in vivo, distinguishing from the phosphorylation of
-adducin by PKC. Although Rho-kinase phosphorylated
- and
-adducin in vitro (Fig. 2 B), the phosphorylation sites have not
been determined. Since the surrounding amino acid sequences of Thr437 in rat
-adducin (QQREKTRWLNS)
are completely identical to those of Thr445 in
-adducin (selected as antigen phosphopeptide for anti-pT445), the
result that anti-pT445 did not cross-react with rat
-adducin phosphorylated by Rho-kinase (Fig. 2 B) revealed that
Rho-kinase does not primarily phosphorylate
-adducin at
Thr437. The surrounding amino acid sequences of Thr431
in
-adducin (QQKEKTRWLNT) are a little bit different
from those of Thr445 in
-adducin. At this stage, we do
not know whether Rho-kinase phosphorylates
-adducin
at Thr431. Adducin is thought to be comprised of
- and
-, or
- and
-adducin in vivo (Bennett et al., 1988
; Dong
et al., 1995
). We found that in MDCK and NRK49F cells a
heterodimer of adducin which consists of
- and
-adducin
was mainly expressed (data not shown). Therefore, it is
likely that anti-pT445 recognizes only phospho-Thr445 in
-adducin but not in
- and
-adducin in both cells (see
below). Further analysis is necessary to understand how
the phosphorylation of
- or
-adducin is regulated through
Rho-kinase in vitro and in vivo.
In Vivo Phosphorylation of -Adducin via the
Rho/Rho-Kinase Pathway
Using anti-pT445, we found that Thr445 phosphorylation
of -adducin occurred in a Rho/Rho-kinase-dependent
manner in COS7 cells (Fig. 3). We further found that
-adducin was phosphorylated at Thr445 in the TPA-stimulated MDCK cells (Fig. 4 C). TPA also drives the activation of PKC. Indeed, TPA induces the phosphorylation of
-adducin at Ser726 in renal proximal tubule epithelial
cells and MDCK cells (Fowler et al., 1998
; Matsuoka et al.,
1998
). However, it is unlikely that PKC directly phosphorylates
-adducin at Thr445 in the TPA-stimulated
cells, because anti-pT445 did not recognize
-adducin
which was phosphorylated by PKC in vitro and was coexpressed with the dominant active PKC in COS7 cells. Rac
and Cdc42 are also involved in TPA-induced phenomena
such as membrane ruffling (Ridley et al., 1995
). It is possible that the protein kinases activated by Rac or Cdc42
phosphorylate
-adducin. However, we found that neither
Rac1V12 nor Cdc42V12 induced the Thr445 phosphorylation
of
-adducin in COS7 cells, and that the dominant active
MRCK, which is identified as the target of Cdc42 (Leung
et al., 1998
) and Rac1 (Nakamura, N., M. Amano, Y. Fukata, N. Oshiro, T. Leung, L. Lim, and K. Kaibuchi, manuscript in preparation), induced the phosphorylation
of MLC but not of
-adducin at Thr445. Taken together,
these results indicate that in intact cells
-adducin was specifically phosphorylated at Thr445 via the Rho/Rho-kinase
pathway during the action of TPA. Although an in
vitro experiment using GST-
-adducinT445A and GST-
-adducinT480A (Fig. 1 D) suggests that Thr445 is a preferential site of phosphorylation by Rho-kinase in vitro, at
this stage it is unknown which of Thr445 or Thr480 is a
preferential site in intact cells.
Involvement of the Phosphorylation of -Adducin via
the Rho/Rho-Kinase Pathway in Membrane Ruffling
and Cell Motility
Membrane ruffling, which is induced by several growth
factors and is observed in the leading edges of motile cells,
is thought to play an essential role in cell motility. The regulatory mechanism of the membrane ruffling is less well
characterized. Not only Rac but also Rho is thought to
regulate the membrane ruffling downstream of extracellular signals in certain types of cells, because C3 inhibits the
TPA- or HGF-induced membrane ruffling in epithelial
cells such as MDCK and KB cells (Nishiyama et al., 1994;
Takaishi et al., 1995
). Here, we found that Thr445-phosphorylated
-adducin accumulated at the TPA-induced
membrane ruffling areas and the leading edges of migrating cells (Figs. 4 and 7 A). We furthermore found that RB
and RB/PH (TT) as well as C3 inhibited the TPA-induced
membrane ruffling, that
-adducin-AA also inhibited the
membrane ruffling as the dominant negative
-adducin
(Fig. 5, A and B), and that
-adducin-DD specifically
counteracted the inhibitory effect of RB and RB/PH (TT)
on the membrane ruffling (Fig. 6). These results indicate
that
-adducin is a crucial substrate of Rho-kinase downstream of Rho for membrane ruffling. The fact that
-adducin-DD counteracted the inhibitory effect of RB
but not that of C3 (Fig. 6 B) is probably due to difference
between the inhibitory mechanisms by RB and C3. RB is
the fragment of Rho-kinase which consists of the RB domain of Rho-kinase. RB binds to the effector domain of
Rho, and then inhibits the interaction of Rho with Rho-kinase
(Amano et al., 1997
). Recent studies revealed that the various effector mutants of RhoAV14, in which interaction
with some effectors is maintained but with others is lost,
make it possible to dissect the diverged pathways downstream of Rho. For example, RhoAY42C abolishes interaction with PKN but maintains interaction with ROCK-I (an
isoform of Rho-kinase) and RhoAF39L,C20R maintains interaction with PKN but abolishes interaction with ROCK-I (Sahai et al., 1998
). RhoAY42C induces stress fiber formation, whereas RhoAF39L,C20R does not induce stress fiber
formation, suggesting that ROCK-I but not PKN is required for stress fiber formation. These observations indicate that there are distinct interfaces in the effector domain of Rho for the different classes of effector proteins.
Although at this time we cannot completely rule out the
possibility that RB inhibits interaction of Rho with the
other Rho targets such as PKN and p140mDia, RB may inhibit preferentially the interaction of Rho with Rho-kinase
rather than with other Rho targets. On the other hand, C3
interferes with the whole functions of Rho. In fact, C3 inhibits the activity of PKN as well as Rho-kinase in Swiss 3T3
cells (Amano et al., 1996) and the Rho-dependent recruitment
of p140mDia to the dynamic membrane structures in Swiss
3T3 cells (Watanabe et al., 1997
). C3 may inhibit the membrane ruffling by impairing the whole functions of Rho,
whereas RB may inhibit the membrane ruffling by mainly
(or specifically) impairing the function of Rho-kinase.
We also found that not only C3 and RB/PH (TT) but
also -adducin-AA inhibited cell motility as well as membrane ruffling in NRK fibroblasts (Fig. 7, B and C). This
suggests that
-adducin is one of the substrates for Rho-kinase involved in cell motility, probably regulating the
membrane ruffling in the leading edges of motile cells. As
for the substrates of Rho-kinase, MLC is another major
substrate which is thought to play an important role in cell
motility. Indeed, injection of anti-MLC kinase antibody
diminishes the cell motility of macrophages (Wilson et al.,
1991
). Moreover, phosphorylated MLC is enriched in both
the leading edges and rear ends of motile fibroblasts and
epithelial cells (Matsumura et al., 1998
), suggesting that a
force derived from myosin-actin filament driven by the
MLC phosphorylation contributes to cell motility. Thus,
Rho, acting through Rho-kinase, appears to regulate cell
motility through the spatial and temporal regulation of
phosphorylation of certain substrates including adducin
and MLC.
It should be noted that in Swiss 3T3 or NIH3T3 fibroblasts, in which the dominant active Rac by itself is sufficient to induce the membrane ruffling (Hall, 1998),
-adducin phosphorylated at Thr445 was not observed in the
dominant active Rac-induced ruffling areas, and neither
C3, RB/PH (TT), nor HA-
-adducin-AA inhibited the
dominant active Rac-induced ruffling (data not shown).
On the other hand, we found that in KB epithelial cells as
well as MDCK epithelial cells,
-adducin phosphorylated
at Thr445 accumulated in the TPA-induced membrane
ruffling areas, and that C3 and RB/PH (TT) inhibited the
TPA-induced membrane ruffling (data not shown). When
RB/PH (TT) or
-adducin-AA was injected to MDCK
cells, the HGF-induced membrane ruffling was also inhibited as the TPA-induced membrane ruffling (data not
shown). The regulatory system of the membrane ruffling
may vary among cell types. It is conceivable that the membrane ruffling in a certain type of cell such as MDCK epithelial cells may require not only the Rac-mediated pathway but also the phosphorylation of adducin by the Rho/
Rho-kinase pathway, whereas that in other types of cells
such as Swiss 3T3 fibroblasts may require only the Rac-mediated pathway. Further studies are necessary to determine what causes the differences between the two types of
cell lines.
Regulation of -Adducin Function through
Phosphorylation by Rho-Kinase and PKC
These results indicate that the phosphorylation of -adducin by the Rho/Rho-kinase pathway plays a crucial role in
the regulation of membrane ruffling and cell motility.
What is the function of the
-adducin phosphorylation in
membrane ruffling and cell motility? Membrane ruffles
contain complicated cytoskeletal structures composed of
F-actin and other F-actin-associated proteins (Mitchison
and Cramer, 1996
). Spectrin, which binds to F-actin beneath plasma membrane and forms a membrane cortical
meshwork, also accumulates at the leading edges of motile
cells (Burridge et al., 1982
; Sormunen and Lehto, 1995
).
This lattice-like meshwork is dynamically reconstructed and required for the formation of membrane ruffling
(Lauffenburger and Horwitz, 1996
). Since the phosphorylation of
-adducin by Rho-kinase is known to promote
the binding of
-adducin to F-actin (Kimura et al., 1998
)
and this in turn induces the recruitment of spectrin to
F-actin (Gardner and Bennett, 1987
; Bennett et al., 1988
),
it is possible that the formation of a spectrin-F-actin meshwork is dynamically regulated in the active membrane ruffling by the Rho/Rho-kinase pathway (Fig. 8). It is also
possible that Rho-kinase modulates the F-actin-capping
activity (Li et al., 1998
) or cross-linking activity (Mische et al.,
1987
) of adducin and then regulates actin dynamics in the
membrane ruffling areas. Adducin is also a substrate of
PKC, which is activated in response to TPA or HGF, and
the phosphorylation of
-adducin by PKC inhibits the activity of adducin in promoting the formation of a spectrin- actin complex in vitro (Matsuoka et al., 1998
). TPA induces the phosphorylation of
-adducin at Ser726 and the
redistribution of
-adducin phosphorylated at Ser726 from
the cell membrane to cytoplasm accompanied by that of
spectrin in renal proximal tubule epithelial and MDCK
cells (Fowler et al., 1998
; Matsuoka et al., 1998
). Interestingly,
-adducin phosphorylated at Ser726 was also observed in the membrane ruffling areas (Matsuoka, Y., and
V. Bennett, manuscript in preparation). It is conceivable
that
-adducin phosphorylated by PKC dissociates from
a spectrin-F-actin meshwork during membrane ruffling.
Thus, it is tempting to speculate that the adducin function
may be cyclically regulated through the phosphorylation by Rho-kinase and PKC during the action of TPA or HGF,
and the alternative phosphorylation contributes to the
turnover of a spectrin-F-actin meshwork for membrane ruffling. In this regard, it is interesting that
-adducin-DD
inhibits the wound-induced migration of NRK49F cells
but not their membrane ruffling. This suggests that the "on" and "off" states of
-adducin are necessary for cell migration (Fig. 8).
|
![]() |
Footnotes |
---|
Address correspondence to Kozo Kaibuchi, M.D., Ph.D., Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. Tel.: 81-743-72-5440. Fax: 81-743-72-5449. E-mail: kaibuchi{at}bs.aist-nara.ac.jp
Received for publication 30 December 1998 and in revised form 8 March 1999.
We thank Dr. S. Jaken for providing pQE-35H fragment (4D) and antiserum against -adducin, Dr. A. Hall for providing pGEX-C3, and Dr. S. Tsukita for providing MDCK cells. We are also grateful to A. Takemura
for secretarial assistance.
This work was supported by a grant from Research for the Future of Japan Society of the Promotion of Science, by the Human Frontier Science Program, and by a grant from Kirin Brewery Company Limited.
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Note Added in Proof: |
---|
After we submitted this paper, it was reported that the GTPase cycle of Rho was important for cell migration in a wound healing assay (Nobes, C.D., and A. Hall. 1999. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J. Cell Biol. 144: 1235-1244).
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Abbreviations used in this paper |
---|
-adducin-AA,
-adducin with substitution of Thr445 and Thr480 by Ala;
-adducin-DD,
-adducin with substitution of Thr445 and Thr480 by Asp;
CAT, catalytic domain;
ERM, ezrin/radixin/moesin;
GST, glutathione-S-transferase;
HA, hemagglutinin;
HGF, hepatocyte growth factor;
LPA, lysophosphatidic acid;
MBP, maltose-binding protein;
MBS, myosin-binding subunit;
MLC, myosin light
chain;
PH, pleckstrin-homology domain;
PK, protein kinase;
RB, Rho-binding domain;
TPA, tetradecanoylphorbol-13-acetate.
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