From the Department of Anatomy and Neurobiology,
Graduate School of Medicine, Kyoto University, Konoe-Yoshida, Sakyo-ku,
Kyoto 606, Japan, the § Division of Signal Transduction,
Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma 630-01, Japan, the ¶ Howard Hughes
Medical Institute and Departments of Cell Biology and Biochemistry,
Duke University Medical Center, Durham, North Carolina 27710, the
Department of Virology II, National Institute of Infectious
Diseases, 1-23-1 Toyama, Shinjyuku-ku, Tokyo 162, Japan, and the
** Central Laboratories for Key Technology, Kirin Brewery Company
Limited, 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The small GTPase Rho is believed to regulate the
actin cytoskeleton and cell adhesion through its specific targets. We
previously identified the Rho targets: protein kinase N, Rho-associated
kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin
phosphatase. Here we purified MBS-interacting proteins, identified them
as adducin, and found that MBS specifically interacted with adducin in vitro and in vivo. Adducin is a
membrane-skeletal protein that promotes the binding of spectrin to
actin filaments and is concentrated at the cell-cell contact sites in
epithelial cells. We also found that Rho-kinase phosphorylated
-adducin in vitro and in vivo and that the
phosphorylation of
-adducin by Rho-kinase enhanced the interaction
of
-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed
phosphatase activity toward
-adducin, which was phosphorylated by
Rho-kinase. This phosphatase activity was inhibited by the
phosphorylation of MBS by Rho-kinase. These results suggest that
Rho-kinase and myosin phosphatase regulate the phosphorylation state of
adducin downstream of Rho and that the increased phosphorylation of
adducin by Rho-kinase causes the interaction of adducin with actin
filaments.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rho is a small GTPase that exhibits both GDP/GTP binding and
GTPase activities. Rho has GDP-bound inactive (GDP·Rho) and GTP-bound active (GTP·Rho) forms, which are interconvertible by GDP/GTP exchange and GTPase reactions (for reviews, see Refs. 1 and 2). When
cells are stimulated with certain extracellular signals such as
lysophosphatidic acid, GDP·Rho is thought to be converted to
GTP·Rho, which binds to specific targets and then exerts its biological functions. Rho participates in signaling pathways that regulate actin cytoskeletons such as stress fibers and in
cell-substratum adhesions such as focal adhesions in fibroblasts (3).
Rho is also involved in the regulation of cell morphology (4), cell aggregation (5), cadherin-mediated cell-cell adhesion (6), cell
motility (7), cytokinesis (8, 9), membrane ruffling (10), smooth muscle
contraction (11, 12), c-fos gene expression (13), the
synthesis of phosphatidylinositol 4,5-diphosphate via
phosphatidylinositol 5-kinase (14), and endocytosis (15). In budding
yeast, RHO1 (a homologue of RhoA) is implicated in the regulation of
cell morphology and budding (16). We identified the following three
targets of Rho: protein kinase N (17, 18), Rho-kinase1
(19), which is also known as ROK (20), and the
MBS of myosin phosphatase (21), which has
ankyrin-like repeats in the amino-terminal domain and a poly basic
region followed by a leucine zipper-like motif in the carboxyl-terminal
domain (22). p160 ROCK is an isoform of Rho-kinase (23). We showed that
Rho-kinase phosphorylates MBS and consequently inactivates myosin
phosphatase (21). We demonstrated that Rho-kinase phosphorylates MLC
and thereby activates myosin ATPase (24). Another group of
investigators has identified different Rho targets: Rhophilin,
Rhotekin, and Citron (18, 25). Phosphatidylinositol 5-kinase is shown
to be activated by GTP·Rho (14). Among these targets, Rho-kinase
appears to be involved in the formation of stress fibers and focal
adhesions downstream of Rho (26-28), smooth muscle contraction through
myosin phosphorylation (29), and c-fos gene expression
(30).
We recently showed that MBS is accumulated at cell-cell contact sites
apart from myosin fibers in polarized MDCK epithelial cells, whereas
MBS is colocalized with myosin fibers in REF52 fibroblasts (31). To
understand the function of MBS at cell-cell contact sites, we attempted
to identify MBS-interacting molecules other than Rho and myosin. We
have purified MBS-interacting proteins with molecular masses of about
85, 110, 115, 120, and 125 kDa and identified them as -,
-, and
-adducin.
Adducin is a membrane skeletal protein that was first purified
from human erythrocytes based on calmodulin binding activity (32).
Adducin associates with F-actin and spectrin-F-actin complexes to
promote the association of spectrin with F-actin (33). Adducin also
caps the fast growing end of actin filaments (34). Adducin is localized
at cell-cell contact sites in some epithelial cells (35). It is likely
that adducin participates in the assembly of the spectrin-actin network
of erythrocytes and epithelial cells. Adducin is composed of and
or
and
subunits closely related in amino acid sequence and
domain organization (36-38). Each adducin subunit has three distinct
domains as follows: an amino-terminal head domain, connected by a neck
domain to a carboxyl-terminal tail domain (36-38).
-Adducin and
-adducin form heterodimers and tetramers through the head domains
and tail domains (39). The tail domain of
-adducin binds mainly to
Ca2+/calmodulin (40), which inhibits both the ability of
adducin to recruit additional spectrin to adducin-spectrin-F-actin
complexes (33) and the ability of adducin to cap actin filaments (34). The tail domains are responsible for binding to spectrin-F-actin complexes (39).
Adducin is also a substrate for PKC and PKA (35, 41, 42). The
phosphorylation of -adducin by PKC or PKA inhibits the calmodulin
binding of
-adducin. The phosphorylation of adducin by PKA reduces
the activity of adducin to associate with F-actin and spectrin-F-actin
complexes and to promote the binding of spectrin to F-actin.
Phosphorylation by PKC has little effect on these activities (40).
In the present study, we found that MBS interacts with adducin both in vitro and in vivo and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of adducin. We also found that the phosphorylation of adducin by Rho-kinase results in the interaction of adducin with F-actin.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials and Chemicals--
cDNA of rat Notch1 was kindly
provided by Dr. M. Nakafuku (Tokyo University, Tokyo, Japan) (43).
Native Rho-kinase was purified from bovine brain as described (19).
GST-CAT (the catalytic domain of Rho-kinase (6-553 aa)) was produced
and purified as described (24). pEF-BOS-myc-CAT was constructed as
described (27). GST-RhoA was purified from Escherichia coli
and loaded guanine nucleotides as described (17). F-actin was purified from an acetone powder prepared from rabbit skeletal muscle as described (44). Chicken myosin phosphatase holoenzyme was kindly provided by Dr. M. Ito (Mie University, Japan) (22).
[-32P]ATP and [32P]orthophosphate were
purchased from Amersham Corp. (Buckinghamshire, UK). All materials used
in the nucleic acid study were purchased from Takara Shuzo Corp.
(Kyoto, Japan). Other materials and chemicals were obtained from
commercial sources.
Production and Purification of Recombinant MBS and
-Adducin--
The cDNAs encoding the NH2-terminal
domain (1-707 aa), the COOH-terminal domain (699-976 aa), the ankyrin
repeat domain (39-295 aa) of rat MBS, human
-adducin (1-642 aa)
(37), and the ankyrin repeat domain of rat Notch1 (1847-2099 aa) were
subcloned into pGEX to produce GST-MBS-N, GST-MBS-C, GST-MBS-ANK,
GST-
-adducin, and GST-Notch-ANK, respectively. GST-MBS-N, GST-MBS-C,
GST-MBS-ANK, and GST-
-adducin were produced and purified from
E. coli.
GST-MBS-ANK Affinity Column Chromatography-- The membrane extract of bovine brain gray matter, 190 g, was prepared (17). The membrane extract (16 ml) was passed through a 2.5-ml glutathione-Sepharose 4B column (Pharmacia) to remove endogenous GST (17). One-tenth of the pass-through fraction was loaded onto a 0.25-ml glutathione-Sepharose 4B column containing GST-MBS-ANK, GST-Notch-ANK, or GST. After washing the columns with 0.825 ml of Buffer A containing 50 mM NaCl three times, the bound proteins were coeluted with the respective GST fusion proteins by the addition of 0.825 ml of Buffer A containing 10 mM glutathione three times. To prepare affinity purified MBS-ANK interacting proteins for peptide sequencing, the pass-through fraction (16 ml) was loaded onto a 1-ml glutathione-Sepharose 4B column containing 24 nmol of GST-MBS-ANK. The proteins were eluted by the addition of 10 ml of Buffer A containing 10 mM glutathione, and fractions of 1 ml each were collected. The same procedures were repeated three times.
Peptide Sequence Analysis-- The affinity purified p85, p110, p115, p120, and p125 were dialyzed three times against distilled water and concentrated by freeze-drying. The concentrated samples were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (46). The immobilized p85, p110, p115, p120, and p125 were digested, fractionated, and subjected to amino acid sequencing as described (46).
In Vitro Binding Assay--
GST-MBS-ANK, GST-MBS-C, and GST (1 nmol each) were separately immobilized onto 35 µl of
glutathione-Sepharose 4B beads. The immobilized beads were incubated
with 8 µg of HA--adducin in 300 µl of Buffer A containing 1 mg/ml bovine serum albumin for 1 h at 4 °C. The beads were
washed six times with 116 µl (3.3 volumes) of Buffer A, and the bound
proteins were eluted with GST-MBS-ANK, GST-MBS-C, and GST by the
addition of 116 µl (3.3 volumes) of Buffer A containing 10 mM glutathione three times. The second eluates were
subjected to SDS-PAGE, and the proteins were detected by silver
staining.
Cell Culture-- MDCK cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum, streptomycin, and penicillin. COS7 cells were maintained in DMEM containing 10% fetal bovine serum, streptomycin, and penicillin. For the transfection of DNA, COS7 cells were seeded at the density of 1.7 × 105 cells in 35-mm tissue culture dishes and cultured overnight.
Immunoprecipitation Assay--
Rabbit anti-rat MBS pAb and
rabbit anti-human -adducin pAb were generated by use of GST-MBS-N
and GST-
-adducin. MDCK cells were grown in 100-mm tissue culture
dishes. After being washed with PBS, the cells were lysed with 1 ml of
extraction Buffer A (20 mM Tris/HCl at pH 8.0, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1%
Nonidet P-40, 10 µM A-PMSF, 10 µg/ml leupeptin). The
lysate was removed from the dishes with a rubber policeman, incubated
in a 1.5-ml tube for 15 min, and then clarified by centrifugation at
12,000 × g for 10 min. The soluble supernatant was
incubated with 2 µg of anti-
-adducin Ab or 2 µg of control rabbit IgG. The immunocomplex was then precipitated with protein A-Sepharose CL 4B (Pharmacia). The immunocomplex was washed five times
with the extraction Buffer A containing 0.5% Nonidet P-40, then eluted
by boiling in sample buffer for SDS-PAGE and subjected to immunoblot
analysis using the anti-MBS Ab as described (47).
Immunofluorescence Analysis--
The coiled-coil domain of
Rho-kinase (421-701 aa) was produced and purified as GST fusion
protein (GST-COIL). Rabbit anti-Rho-kinase pAb was generated by use of
GST-COIL. For anti-MBS Ab, MDCK cells were fixed with 3.7%
formaldehyde in PBS for 10 min and treated with ice-cold methanol for
10 min. For anti--adducin Ab and anti-Rho-kinase Ab, MDCK cells were
fixed with ice-cold methanol for 10 min. After being washed with PBS
three times, the cells were incubated with anti-MBS Ab, anti-Rho-kinase
Ab, or anti-
-adducin Ab overnight at room temperature. Then MDCK
cells were incubated with fluorescein isothiocyanate-conjugated
anti-rabbit Ig Ab. After being washed with PBS three times, the cells
were examined using a Zeiss axiophoto microscope or a confocal
microscope (Carl Zeiss, Oberkochen, Germany).
Phosphorylation Assay--
The kinase reaction for Rho-kinase
was carried out in 50 µl of the kinase buffer (50 mM
Tris/HCl at pH 7.5, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT)
containing 100 µM [-32P]ATP (1-20
GBq/mmol), purified enzyme, and 2.4 µg of HA-
-adducin with 1 µM GTP
S·GST-RhoA, GDP·GST-RhoA, or GST. After an
incubation for 10 min at 30 °C, the reaction mixtures were boiled in
SDS sample buffer and subjected to SDS-PAGE. The radiolabeled bands were visualized and estimated by an image analyzer (BAS-2000, Fuji,
Tokyo).
Cosedimentation Assay--
HA--adducin (7 µg of
protein) was phosphorylated with GST-CAT (3 µg of protein) in 200 µl of kinase buffer containing 0.1 µM calyculin A with
or without ATP for 1 h at 30 °C. F-actin was mixed with
HA-
-adducin phosphorylated as above in Buffer B (30 mM
Hepes at pH 7.4, 0.5 mM DTT, 2 mM
MgCl2, 50 mM KCl, 1 mM EGTA, 10%
(w/v) sucrose, 0.5 mM ATP) for 2 h at 4 °C. After
the incubation, 50 µl of each reaction mixture was layered onto a
100-µl sucrose barrier (20% (w/v) sucrose in Buffer B) and
centrifuged at 200,000 × g for 1 h at 4 °C.
The supernatants and pellets were separated and subjected to immunoblot
analysis using anti-
-adducin Ab.
Protein Phosphatase Assay--
HA--adducin (300 ng of
protein) was phosphorylated with GST-CAT (120 ng of protein) in 20 µl
of kinase buffer containing 100 µM
[
-32P]ATP for 1 h at 30 °C, and the reaction
was stopped by the addition of 200 nM staurosporine. Native
myosin phosphatase (5-75 ng) was preincubated in 30 µl of reaction
mixture (30 mM Tris/HCl at pH 7.5, 3 mM
MgCl2, 0.4 mM EDTA, 0.55 mM EGTA,
0.1 mg/ml bovine serum albumin, 0.3 mM CoCl2,
5-75 ng of myosin phosphatase) with or without 100 µM
ATP
S and GST-CAT (80 ng of protein) for 15 min at 30 °C, and the
reaction was stopped by the addition of 200 nM
staurosporine. The phosphatase reaction was then performed in 50 µl
of the reaction mixture containing 300 ng of 32P-labeled
HA-
-adducin for 15 min at 30 °C. The reaction mixture was then
boiled in sample buffer for SDS-PAGE and resolved by SDS-PAGE. The
32P-labeled band corresponding to HA-
-adducin was
visualized and estimated with an image analyzer. As a control
experiment, 32P-labeled MLC was used as the substrate of
myosin phosphatase.
In Vivo Phosphorylation of -Adducin by Rho-kinase--
To
express HA epitope-tagged
-adducin, the cDNA encoding human
-adducin (1-642 aa) was inserted into the KpnI site of
pEF-BOS-HA. The transfection of plasmids into COS7 cells was carried
out by the standard DEAE-dextran method (17, 21). The plasmid
pEF-BOS-HA-
-adducin was transfected with or without pEF-BOS-myc-CAT.
The transfected cells were cultured in DMEM containing 10% fetal
bovine serum for 2 days. The cells were then labeled with 18.5 MBq of
[32P]orthophosphate for 3 h and lysed with 0.3 ml of
extraction Buffer B (20 mM Tris/HCl at pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1%
Nonidet P-40, 2 mM Na3VO4, 50 mM NaF, 0.1 µM calyculin A, 10 µM A-PMSF, 10 µg/ml leupeptin). The lysates were
clarified by centrifugation at 12,000 × g for 15 min.
HA-
-adducin was then immunoprecipitated from the soluble
supernatant. The washed immunocomplexes were subjected to SDS-PAGE for
peptide mapping.
Peptide Mapping--
HA--adducin was phosphorylated by
Rho-kinase in vitro as described above. HA-
-adducins
phosphorylated in vitro and in vivo were isolated
by SDS-PAGE and digested with trypsin. Two-dimensional peptide mappings
were performed with silica gel thin layer plates as described (48).
Phosphorylated peptides were visualized by autoradiography.
Other Procedures-- SDS-PAGE was performed as described (49). Protein concentrations were determined with bovine serum albumin as the reference protein as described (50).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of MBS-interacting Molecule-- To detect MBS- interacting molecules, bovine brain membrane extract was loaded onto a glutathione-Sepharose affinity column on which GST, GST-MBS-ANK, or GST-Notch-ANK was immobilized. The proteins bound to the affinity columns were then coeluted with GST or GST fusion proteins by the addition of glutathione. Proteins with molecular masses of about 85 kDa (p85), 110 kDa (p110), 115 kDa (p115), 120 kDa (p120), and 125 kDa (p125) were detected in the glutathione eluate from the GST-MBS-ANK affinity column but not detected from the GST or the GST-Notch-ANK affinity column (Fig. 1A).
|
Interaction of MBS with -Adducin in Vitro and in Vivo--
We
examined whether recombinant adducin interacts with MBS in a cell-free
system. The purified recombinant HA-
-adducin was incubated with
GST-MBS-ANK, GST-MBS-C, or GST immobilized beads. After washing the
beads, the GST fusion proteins were eluted by the addition of
glutathione. HA-
-adducin was coeluted with GST-MBS-ANK but not with
GST-MBS-C or GST (Fig. 2A).
This result indicates that recombinant
-adducin binds directly to
the ankyrin repeat domain of MBS.
|
Similar Localization of MBS and -Adducin at Cell-Cell Contact
Sites--
We recently showed that MBS is accumulated at cell-cell
contact sites apart from myosin fibers in polarized MDCK epithelial cells, whereas MBS is colocalized with myosin fibers in REF52 fibroblasts (31). Adducin is also localized at the cell-cell contact
sites of MDCK epithelial cells (35). We then compared the localization
of MBS with that of
-adducin in confluent MDCK cells, which show
characteristics of polarized epithelial cells and form the junctional
complexes (including the tight junctions, adherens junctions, and
desmosomes) at cell-cell contact sites. Immunofluorescence analysis
revealed that MBS showed a distribution similar to that of
-adducin
at the cell-cell contact sites (Fig. 3,
A and B). Because Rho-kinase phosphorylates MBS
(21), we then examined the localization of Rho-kinase in confluent MDCK cells. Rho-kinase was partly accumulated at the cell-cell contact sites
(Fig. 3C).
|
In Vitro Phosphorylation of -Adducin by Rho-kinase--
We next
examined whether Rho-kinase phosphorylates adducin in a cell-free
system. Native Rho-kinase purified from bovine brain phosphorylated
HA-
-adducin, and this phosphorylation was markedly enhanced by the
addition of GTP
S·GST-RhoA but not of GDP·GST-RhoA (Fig.
4A). We found that GST-CAT
(the catalytic domain of Rho-kinase) phosphorylated recombinant
HA-
-adducin (Fig. 4A). We previously showed that CAT
serves as a constitutively active form in vitro and in
vivo (27). About 0.8 mol of phosphate could be maximally incorporated into 1 mol of HA-
-adducin by GST-CAT (Fig.
4B). It is reported that PKC and PKA phosphorylate
-adducin (35, 41, 42).
-Adducin is primarily phosphorylated at
Ser-408, Ser-436, Ser-481, and Ser-726 by PKA and at Ser-726 by PKC
(40). We performed a phosphoamino acid analysis and found that
phosphorylation by Rho-kinase occurred mainly on the threonine residue.
It is thus likely that the phosphorylation sites by Rho-kinase are
different from those by PKC and PKA. (The identification of
phosphorylation sites by Rho-kinase is currently under investigation
and will be described elsewhere.)
|
The Effect of the Phosphorylation of -Adducin by Rho-kinase on
Its F-Actin Binding Activity--
Adducin binds to F-actin and
spectrin-F-actin complex to promote the binding of spectrin to F-actin
(33). We speculated that phosphorylation by Rho-kinase might regulate
the F-actin binding activity of adducin. To examine whether the
phosphorylation of adducin by Rho-kinase modulates its F-actin binding
activity, a cosedimentation assay of recombinant
-adducin with
F-actin was performed. HA-
-adducin phosphorylated by GST-CAT was
cosedimentated with F-actin more efficiently than non-phosphorylated
HA-
-adducin (Fig. 5). A similar result
was obtained in the presence of spectrin (data not shown). These
findings suggest that the phosphorylation of adducin by Rho-kinase
enhances the F-actin binding activity of adducin.
|
Phosphatase Activity of Myosin Phosphatase toward -Adducin
Phosphorylated with Rho-kinase--
Rho-kinase phosphorylated
adducin stoichiometrically, as described above. We speculated that
myosin phosphatase might regulate the phosphorylation states of adducin
downstream of Rho as described for MLC (21). We next examined whether
myosin phosphatase dephosphorylates adducin which was phosphorylated by
Rho-kinase. The myosin phosphatase showed phosphatase activity toward
HA-
-adducin phosphorylated by GST-CAT (Fig.
6). We previously observed that the MBS
of the native myosin phosphatase was thiophosphorylated with Rho-kinase in the presence of ATP
S and that this thiophosphorylation of MBS was
associated with a decrease of phosphatase activity toward MLC (21).
Here, we examined whether Rho-kinase also modulates the phosphatase
activity of myosin phosphatase toward adducin through the
thiophosphorylation of MBS. We found that the thiophosphorylation of
MBS was associated with a decrease of phosphatase activity toward
HA-
-adducin (Fig. 6).
|
Phosphorylation of -Adducin by Rho-kinase in COS7 Cells--
We
performed a two-dimensional peptide map analysis of the phosphorylated
HA-
-adducin. HA-
-adducin phosphorylated by GST-CAT in
vitro was digested with trypsin and was subjected to thin layer chromatography using a silica gel plate, followed by autoradiography. Two major radioactive spots (named spots a and b)
and several minor radioactive spots were detected (Fig.
7A). To examine whether Rho-kinase can induce the phosphorylation of adducin in
vivo, the plasmid pEF-BOS-HA-
-adducin was transfected with or
without pEF-BOS-CAT into COS7 cells, and the cells were labeled with
[32P]orthophosphate. HA-
-adducin was then
immunoprecipitated from the cell lysates and subjected to
two-dimensional peptide mapping. When HA-
-adducin was expressed
alone, several radioactive spots were observed, whereas spots
corresponding to spots a or b were not detected
(Fig. 7B). When HA-
-adducin was coexpressed with CAT, the
spot corresponding to spot a was detected, and the spot corresponding to spot b was weakly detected (Fig. 7,
C and D). Similar results were obtained when
HA-
-adducin was coexpressed with the dominant active RhoA (data not
shown). These findings suggest that the site of adducin corresponding
to spot a was phosphorylated by Rho-kinase both in
vitro and in vivo.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Complex Formation between MBS and Adducin--
We purified
MBS-interacting proteins by GST-MBS-ANK affinity column chromatography
and identified them as adducin. Adducin is a membrane skeletal protein
that associates with F-actin and spectrin-F-actin complexes and
promotes the association of spectrin with F-actin (33). Adducin is
localized at cell-cell contact sites in some epithelial cells (35).
Adducin is thought to participate in the assembly of the spectrin-actin
network. We showed that -adducin interacts with the ankyrin-repeat
domain of MBS in vitro and that some population of MBS
interacts with
-adducin in vivo (Fig. 2). We also showed
that the localization of MBS is similar to that of
-adducin at
cell-cell contact sites in confluent MDCK epithelial cells (Fig. 3) and
that myosin phosphatase dephosphorylates the
-adducin phosphorylated
by Rho-kinase (Fig. 6). We confirmed that the interaction of
-adducin with MBS is not modulated by the activated RhoA and that
MBS is co-immunoprecipitated with
-adducin from the bovine brain
cytosol where the activated RhoA is absent (data not shown). Thus, it
is likely that MBS constitutively binds to adducin and that myosin
phosphatase efficiently regulates the state of phosphorylation of
adducin.
Dual Regulation of the Phosphorylation State of Adducin-- Rho regulates MLC phosphorylation via two pathways through its targets, Rho-kinase and MBS, as follows (21, 24). Activated Rho interacts with Rho-kinase and the MBS of myosin phosphatase and activates Rho-kinase. The activated Rho-kinase subsequently phosphorylates MBS, thereby inactivating myosin phosphatase (21). Rho-kinase by itself phosphorylates MLC at the same site that is phosphorylated by MLC kinase and activates myosin ATPase (24). Both pathways appear to be important for an increase of the phosphorylation of MLC (29).
Here we found that Rho-kinase phosphorylatesPhosphorylation of Adducin by Rho-kinase--
Adducin is a
substrate for PKC and PKA (35, 41, 42). The phosphorylation of adducin
by PKA reduces the activity of adducin to associate with F-actin and
spectrin-F-actin complexes and to promote the binding of spectrin to
F-actin. As mentioned earlier, phosphorylation by PKC has little effect
on this activity (40). -Adducin is primarily phosphorylated at
Ser-408, Ser-436, Ser-481, and Ser-726 by PKA and at Ser-726 by PKC
(40).
Roles of Rho in the Regulation of Adducin
Activity--
Accumulating evidence indicates that Rho participates in
signaling pathways that regulate actin cytoskeletons such as stress fibers and in cell substratum adhesions such as focal adhesions in
fibroblasts (3). Rho is also involved in the regulation of cell
morphology (4) and cell aggregation (5). It has recently been shown
that activated Rho is required for maintaining cadherin-mediated
cell-cell adhesion (6). Rho appears to be involved in the assembly of
adhesion molecules such as cadherin and peripheral proteins including
cortical actin filaments, ERM (ezrin, radixin, and moesin), and
vinculin at cell-cell contact sites (6, 52). Cortical actin filaments
consist of a number of proteins including spectrin-F-actin-adducin
complexes. Adducin is thought to promote the formation of this complex
(33). We showed herein that Rho-kinase phosphorylates -adducin, and
this phosphorylation is dually regulated by the Rho targets Rho-kinase and MBS and that the phosphorylation of
-adducin by Rho-kinase enhances its binding activity to F-actin. Taken together, these findings suggest that Rho-kinase and MBS can regulate the F-actin binding activity of adducin through its phosphorylation downstream of
Rho, thereby resulting in the assembly of the spectrin-F-actin network.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Masato Nakafuku for providing cDNA of rat Notch1, Dr. Masaaki Ito for providing chicken gizzard myosin phosphatase, and Dr. Masaki Inagaki for valuable discussions. We are also grateful to Akemi Takemura for secretarial assistance and Nagatoki Kinoshita for technical assistance.
![]() |
FOOTNOTES |
---|
* This investigation was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, and Culture of Japan (1997) and by grants from the Mitsubishi Foundation and Kirin Brewery Co. Ltd.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. Tel.:
81-743-72-5440; Fax: 81-743-72-5449; E-mail:
kaibuchi{at}bs.aist-nara.ac.jp.
1
The abbreviations used are: Rho-kinase,
Rho-associated kinase; MBS, myosin-binding subunit; MLC, myosin light
chain; PKC, protein kinase C; PKA, protein kinase A; pAb, polyclonal
antibody; GST, glutathione S-transferase; A-PMSF,
(p-amidinophenyl)-methanesulfonyl fluoride; GTPS,
guanosine 5'-(3-O-thio)-triphosphate; ATP
S, adenosine
5'-O-(3-thiophosphate); PAGE, polyacrylamide gel
electrophoresis; Ab, antibody; DTT, dithiothreitol; PBS,
phosphate-buffered saline; HA, hemagglutinin; MDCK, Madin-Darby canine
kidney; aa, amino acid(s); DMEM, Dulbecco's modified Eagle's
medium.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|