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(Received for publication, May 15, 1996, and in revised form, June 19, 1996)
From the Division of Signal Transduction, Nara Institute of Science
and Technology, 8916-5 Takayama, Ikoma 630-01, the ABSTRACT The small GTPase Rho is implicated in
physiological functions associated with actin-myosin filaments such as
cytokinesis, cell motility, and smooth muscle contraction. We have
recently identified and molecularly cloned Rho-associated
serine/threonine kinase (Rho-kinase), which is activated by GTP·Rho
(Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito,
M., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996)
EMBO J. 15, 2208-2216). Here we found that Rho-kinase
stoichiometrically phosphorylated myosin light chain (MLC). Peptide
mapping and phosphoamino acid analyses revealed that the primary
phosphorylation site of MLC by Rho-kinase was Ser-19, which is the site
phosphorylated by MLC kinase. Rho-kinase phosphorylated recombinant
MLC, whereas it failed to phosphorylate recombinant MLC, which
contained Ala substituted for both Thr-18 and Ser-19. We also found
that the phosphorylation of MLC by Rho-kinase resulted in the
facilitation of the actin activation of myosin ATPase. Thus, it is
likely that once Rho is activated, then it can interact with Rho-kinase
and activate it. The activated Rho-kinase subsequently phosphorylates
MLC. This may partly account for the mechanism by which Rho regulates
cytokinesis, cell motility, or smooth muscle contraction.
INTRODUCTION Rho is a small GTPase, which exhibits both GDP/GTP binding and
GTPase activities (for reviews, see Refs. 1 and 2). Rho has GDP-bound
inactive and GTP-bound active forms, which are interconvertible by
GDP/GTP exchange and GTPase reactions (1, 2). Rho is implicated in the
cytoskeletal responses to extracellular signals including
lysophosphatidic acid and certain growth factors, which form stress
fibers and cause focal adhesion (3, 4). Rho is also implicated in other
physiological functions associated with cytoskeletal rearrangements
such as cell morphology (5), cell aggregation (6), cell motility (7),
and cytokinesis (8, 9). Recent studies indicate that Rho is also
involved in the regulation of phosphatidylinositol 3-kinase (10, 11, 12),
phosphatidylinositol 4-phosphate-5-kinase (13), and c-fos
expression (14). Upon stimulation with certain extracellular signals,
GDP·Rho may be converted to GTP·Rho, and then it can bind to
specific targets and cause its effects. We have recently purified three
putative targets for Rho (p128, p138, and p164) from the bovine brain
(15, 16). p128 was identified as serine/threonine kinase, protein
kinase N (15). p138 was identified as the
MBS1 of myosin phosphatase (16). p164 was
identified as a novel serine/threonine kinase, named Rho-kinase (17),
which is also known as ROK (18).
MLC phosphorylation plays pivotal roles in smooth muscle contraction
(for reviews, see Refs. 19, 20, 21) and in the actin-myosin interaction for
stress fiber and contractile ring formation in non-muscle cells (for a
review, see Ref. 22). This also has an effect on cytokinesis and cell
motility (22). MLC kinase primarily phosphorylates MLC at Ser-19
(19, 20, 21, 23). Any protein kinases so far obtained, besides specific
kinases such as MLC kinase, do not phosphorylate this site (24). When
smooth muscles are stimulated by agonists such as vasoconstrictors,
Ca2+ is mobilized into the cytoplasm. Ca2+
activates the calmodulin-dependent MLC kinase. The MLC
phosphorylation induces myosin-actin interaction and thereby activates
myosin ATPase (19, 20, 21), which then induces smooth muscle contraction
(19, 20, 21). However, the cytosolic Ca2+ level is not always
proportional to the contraction level, and an additional mechanism that
can regulate the Ca2+ sensitivity of the smooth muscle
contraction has been proposed (25). Since GTP To extend these observations, we have examined if Rho-kinase
phosphorylates MLC. We found that Rho-kinase stoichiometrically
phosphorylated MLC at the site that is phosphorylated by MLC kinase,
which causes the activation of myosin ATPase.
EXPERIMENTAL PROCEDURES MLC (30), Myosin and MLC kinase
(23) were purified from the frozen chicken gizzard. F-actin was
purified from the rabbit skeletal muscle (31). Rho-kinase was purified
from the bovine brain (17). [ The cDNA encoding the catalytic
fragment of Rho-kinase (6-553 amino acids) was inserted into the
BamHI site of pAcYM1-GST to produce GST-Rho-kinase.
GST-Rho-kinase was purified from Sf9 cells by use of a baculovirus
system (32) by means of a glutathione-Sepharose column (17).
GST-Rho-kinase was constitutively active.2
The cDNA fragment encoding MLC was amplified by polymerase chain
reaction from rat brain Quick clone cDNA (Clontech) with the
primers 5 The kinase reaction for Rho-kinase
was carried out in 50 µl of the reaction mixture (50 mM
Tris/HCl at pH 7.5, 2 mM EDTA, 1 mM DTT, 7 mM MgCl2, 0.15% CHAPS, 250 µM
[ Myosin ATPase assay was carried out as
described (33) with minor modifications. Briefly, 0.1 mg/ml myosin was
phosphorylated by GST-Rho-kinase (4.5 µg of protein) in 0.45 ml of
the reaction mixture (50 mM Tris/HCl at pH 7.5, 1 mM DTT, 5 mM MgCl2, 1 mM EGTA, 85 mM KCl, and 500 µM
ATP (80-200 MBq/mmol)) for 20 min at 30 °C. Myosin was
phosphorylated by MLC kinase (4.5 µg of protein) under similar
conditions except in the presence of 0.1 mM
CaCl2 and 10 µg/ml calmodulin. The myosin ATPase reaction
was carried out in 0.1 ml of ATPase buffer (0.05 mg/ml phosphorylated
myosin, 50 mM Tris/HCl at pH 7.5, 0.5 mM DTT,
10 mM MgCl2, 0.5 mM EGTA, 85 mM KCl, and 1 mM ATP (80-200 MBq/mmol)) at the
indicated concentrations of F-actin for 30 min at 30 °C. An aliquot
(80 µl) of the reaction mixture was added into the stop solution
(1.3% charcoal, 0.12 M NaH2PO4,
and 0.33 M perchloric acid) and filtrated. Inorganic
phosphate that was liberated from [ SDS-PAGE was performed as described
previously (34). A phosphopeptide mapping analysis of MLC was carried
out as described (35). A phosphoamino acid analysis of MLC was carried
out as described (36).
RESULTS We examined whether Rho-kinase phosphorylates isolated MLC in a
cell-free system and found that Rho-kinase phosphorylated MLC (Fig.
1A). GTP
About 1 mol of phosphate could be maximally incorporated into 1 mol of
isolated MLC or MLC of intact myosin by Rho-kinase in the presence of
GTP The apparent affinity of isolated MLC for Rho-kinase was estimated by
measuring the phosphorylation of various concentrations of MLC (Fig.
1B). The apparent Km values for MLC in
the presence and absence of GTP MLC is phosphorylated primarily at Ser-19 and secondarily at Thr-18 by
MLC kinase (23), and the phosphorylation of Ser-19 is essential to
facilitate actin activation of myosin ATPase (39, 40). MLC is
phosphorylated at Ser-1, Ser-2, and Thr-9 by protein kinase C, and this
phosphorylation by protein kinase C inhibits the actin activation of
myosin ATPase (41, 42, 43). To determine the primary phosphorylation site
of MLC by Rho-kinase, we performed peptide mapping of the
phosphorylated MLC by either Rho-kinase, MLC kinase, or protein kinase
C in vitro. The pattern of two-dimensional peptide mapping
of MLC phosphorylated by Rho-kinase was identical to that produced by
MLC kinase and different from that produced by protein kinase C (Fig.
2A). A phosphoamino acid analysis revealed
that phosphorylation occurred mainly on the serine residue and
partially on the threonine residue of the MLC that was phosphorylated
by Rho-kinase and that the phosphorylation occurred only on the serine
residue of the MLC that was phosphorylated by the MLC kinase. It may be
noted that the MLC kinase preferentially phosphorylates MLC at Ser-19
in these conditions. Essentially identical results were obtained when
GST-Rho-kinase was used instead of Rho-kinase.
We fused GST proteins with wild type MLC and with MLC containing a
substitution for the alanine residues for Thr-18 and Ser-19, and then
we examined if Rho-kinase and MLC kinase could phosphorylate these
recombinant proteins. Rho-kinase, GST-Rho-kinase, and MLC kinase
phosphorylated GST-MLC but did not phosphorylate GST or
GST-MLCA18A19 (Fig. 2B). Protein kinase C
phosphorylated both GST-MLC and GST-MLCA18A19 (data not
shown). These results indicate that Rho-kinase phosphorylates MLC
mainly at Ser-19, which is the same site phosphorylated by MLC
kinase.
To examine whether Rho-kinase functions equivalently to MLC kinase in a
cell-free system, we performed the actin-activated MgATPase assay.
Purified intact myosin was phosphorylated to 1 mol/mol by
GST-Rho-kinase, and then the actin-activated MgATPase activity was
measured. The MgATPase activity of the phosphorylated myosin increased
in a F-actin-dependent manner to the extent similar to that
increased by MLC kinase (Fig. 3). The apparent
Ka values for actin and the molecular activity of
the phosphorylated myosin were 0.56 ± 0.05 µM and
0.18 ± 0.02 s
We showed here that Rho-kinase phosphorylated both isolated MLC and MLC
of intact myosin in a GTP·Rho-dependent manner. The
primary phosphorylation site of MLC by Rho-kinase was at Ser-19, which
is the same site phosphorylated by MLC kinase. The phosphorylation of
MLC of intact myosin increased the MgATPase activity of myosin. These
results indicate that Rho-kinase phosphorylates the MLC of intact
myosin and activates its MgATPase activity in a
GTP·Rho-dependent manner. The Km value
of Rho-kinase for MLC was lower than that of MLC kinase, but the
molecular activity of Rho-kinase was lower than that of MLC kinase.
This indicates that Rho-kinase efficiently phosphorylates MLC at lower
concentrations.
When smooth muscles are stimulated by an agonist such as
vasoconstrictors, Ca2+ is mobilized into the cytoplasm.
Ca2+ activates the calmodulin-dependent MLC
kinase. Because smooth muscles contain large amounts of myosin (about
10% of the total protein, about 50 µM) and MLC kinase
(about 0.1% of the total protein) (19), MLC kinase is believed to
phosphorylate MLC in smooth muscles. However, non-muscle tissue such as
liver contains much smaller amounts of myosin and MLC kinase (data not
shown). Rho-kinase is ubiquitously expressed in various tissues (17).
We have speculated that Rho-kinase phosphorylates the MLC of intact
myosin and activates MgATPase in a GTP·Rho-dependent
manner when non-muscle cells are stimulated by an agonist such as
lysophosphatidic acid and certain growth factors and that the
phosphorylated myosin interacts with actin leading to stress fiber and
contractile ring formation (Fig. 4). In fact, we have
recently found that overexpression of dominant activated RhoA
(RhoAV14) in NIH3T3 cells results in an increase in MLC
phosphorylation as well as stress fiber formation (16). This may be
partly explained by the fact that GTP·RhoA interacts with Rho-kinase
and MBS of myosin phosphatase and activates Rho-kinase; the activated
Rho-kinase subsequently phosphorylates the MBS, thereby inactivating
myosin phosphatase (Fig. 4) (16). We have assumed that phosphorylation
of MLC by Rho-kinase also contributes to the increased level of MLC
phosphorylation in these cells. Further studies are necessary to
understand the roles of Rho-kinase in controlling MLC
phosphorylation.
FOOTNOTES Acknowledgments We are grateful to M. Nishimura for
secretarial assistance. We also thank Dr. J. T. Stull (University of
Texas Southwestern Medical Center) for providing us the anti-myosin
antibody, Drs. K. Okawa and A. Iwamatsu (Kirin Brewery Co. Ltd.) for
peptide sequencing, and Dr. M. Inagaki (Aichi Cancer Center Research
Institute) for helpful discussions.
REFERENCES
Volume 271, Number 34,
Issue of August 23, 1996
pp. 20246-20249
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
,,
First
Department of Internal Medicine, Mie University School of Medicine, Tsu
514, the § Second Department of Anatomy, Kyoto University
Faculty of Medicine, Kyoto 606, and the ¶ Department of Virology
II, National Institute of Health, Tokyo 162, Japan
S, a non-hydrolyzable
GTP analog, lowers the Ca2+ concentrations necessary for
the contraction of permeabilized smooth muscles, a GTP-binding protein
was presumed to regulate the Ca2+ sensitivity (26, 27). Rho
has been shown to be involved in the GTP-enhanced Ca2+
sensitivity of the smooth muscle contraction (28). Recent evidence
suggests that GTPS increases MLC phosphorylation at submaximal
Ca2+ concentrations presumably by inhibiting myosin
phosphatase through Rho (29). We have recently shown that GTP·Rho
activates Rho-kinase, and then Rho-kinase phosphorylates MBS and
thereby inactivates myosin phosphatase (16). This may increase MLC
phosphorylation and induce the consequent contraction of the smooth
muscles.
-32P]ATP was purchased
from Amersham Corp. All materials used in the nucleic acid study were
purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). Other materials
and chemicals were obtained from commercial sources.
-AATAGGATCCGATTTAACCGCCACCATGTCG-3 and
5-ATAAGGATCCTCAGTCATCTTTGTCTTTCGCTC-3. Substitution of alanine
residues for threonine 18 and serine 19 was performed by polymerase
chain reaction. The cDNA fragments were cloned into the
BamHI site of pGEX-2T. GST-MLC and GST-MLCA18A19
were purified as described (17).
-32P]ATP (1-20 GBq/mmol), purified enzyme, and
indicated amounts of MLC or myosin) with or without 1 µM
GTPS·GST-RhoA. The kinase reaction for MLC kinase was carried out
in 50 µl of the reaction mixture (50 mM Tris/HCl at pH
7.5, 1 mM MgCl2, 85 mM KCl, 250 µM [-32P]ATP (0.5-5 GBq/mmol), purified
enzyme, and indicated amounts of MLC or myosin) with or without 0.1 mM CaCl2 and 10 µg/ml calmodulin. 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 by an image analyzer (Fuji).
-32P]ATP was
assessed by a scintillation counter.
S·GST-RhoA enhanced the
phosphorylation of MLC by Rho-kinase, but GDP·GST-RhoA or
GTPS·GST-RhoAA37 did not (Fig. 1A).
RhoA37 is structurally equivalent to RasA35,
which contains an amino acid substitution in the effector domain that
causes it to fail to interact with its target (37, 38).
GTPS·GST-Rac1 had no effect. The recombinant Rho-kinase
(GST-Rho-kinase), which is constitutively active, phosphorylated MLC.
Under similar conditions, MLC kinase phosphorylated MLC in a
Ca2+-calmodulin-dependent manner (Fig.
1A). We also found that Rho-kinase phosphorylated the MLC of
intact myosin in a GTPS·GST-RhoA-dependent manner
(Fig. 1A).
Fig. 1.
Phosphorylation of MLC by Rho-kinase.
A, isolated MLC (0.5 µg of protein) was phosphorylated by
Rho-kinase (20 ng of protein) in the presence of either GST (lane
1), GDP·GST-RhoA (lane 2), GTPS·GST-RhoA
(lane 3), GTPS·GST-RhoAA37 (lane
4), GDP·GST-Rac1 (lane 5), or GTPS·GST-Rac1
(lane 6), by GST-Rho-kinase (5 ng of protein) (lane
7), or by MLC kinase (10 ng of protein) in the absence (lane
8) or presence (lane 9) of Ca2+ and
calmodulin. Intact myosin (5 µg of protein) was phosphorylated by
Rho-kinase in the absence (lane 10) or presence (lane
11) of GTPS·GST-RhoA. The phosphorylated MLC was resolved by
a SDS-PAGE and visualized by an image analyzer. The results are
representative of three independent experiments. B, various
doses of MLC were phosphorylated by Rho-kinase (25 ng of protein) in
the absence (
) or presence (
) of GTPS·GST-RhoA, by
GST-Rho-kinase (7.5 ng of protein) (
), or by MLC kinase (7.5 ng of
protein) in the absence (
) or presence (
) of Ca2+ and
calmodulin. The values shown are means ± S.E. of
triplicates.
S·GST-RhoA and by GST-Rho-kinase (data not shown). It is noted
that a limited number of kinases such as MLC kinase and protein kinase
C are known to phosphorylate intact myosin stoichiometrically (24).
S·GST-RhoA were 2.6 ± 0.4 and
12.6 ± 1.6 µM, and the molecular activities were
0.26 ± 0.03 and 0.15 ± 0.02 s
1, respectively.
Thus, it is likely that GTPS·GST-RhoA increases the affinity of
Rho-kinase for MLC and produces the maximum velocity of the
phosphorylation reaction. The apparent Km value and
molecular activity of GST-Rho-kinase were 0.91 ± 0.07 µM and 0.67 ± 0.09 s1, respectively.
The apparent Km value and molecular activity of MLC
kinase for MLC were 52.1 ± 7.1 µM and 2.0 ± 0.36 s1, respectively, under the conditions. The
Km value of Rho-kinase for MLC was lower than that
of MLC kinase, indicating that Rho-kinase phosphorylates myosin at
lower concentrations, but the molecular activity of Rho-kinase was
lower than that of MLC kinase. The lower molecular activity of
Rho-kinase than that of GST-Rho-kinase may be explained by the fact
that Rho-kinase lost its activity to some extent during the
purification.
Fig. 2.
Identification of the phosphorylation site of
MLC by Rho-kinase. A, a phosphopeptide mapping analysis of
MLC. MLC (0.5 µg of protein) was phosphorylated by Rho-kinase, MLC
kinase, or protein kinase C. Phosphorylated MLC was digested with
trypsin, and each sample was loaded onto a silica gel plate.
Phosphopeptides were separated by electrophoresis (horizontal
dimension) and chromatography (vertical dimension) and then were
visualized by an image analyzer. Asterisks denote origins.
B, phosphorylation of recombinant MLC. MLC, GST, GST-MLC, or
GST-MLCA18A19 (2 µM each) was phosphorylated
by Rho-kinase (20 ng of protein), GST-Rho-kinase (10 ng of protein), or
MLC kinase (10 ng of protein) as indicated. Lanes 1-4, by
Rho-kinase; lanes 5-8, by GST-Rho-kinase; lanes
9-12, by MLC kinase. Lanes 1, 5, and
9, MLC; lanes 2, 6, and 10,
GST; lanes 3, 7, and 11, GST-MLC;
lanes 4, 8, and 12,
GST-MLCA18A19. The results are representative of three
independent experiments.
1, respectively. These values were
roughly the same as those for the myosin phosphorylated by MLC kinase.
We used GST-Rho-kinase instead of native Rho-kinase in this experiment
because high concentrations of myosin were necessary to detect the
myosin ATPase activity and the stoichiometrical phosphorylation of
myosin by native Rho-kinase was difficult under the conditions.
Fig. 3.
Effect of phosphorylation of myosin by
Rho-kinase on the MgATPase activity that was activated by actin.
Myosin was incubated with GST-Rho-kinase (), with MLC kinase (
),
or without kinase (). After incubation, the ATPase activity was
measured at the various concentrations of F-actin. The values shown are
means ± S.E. of triplicates.
Fig. 4.
Model for the regulation of MLC
phosphorylation by Rho, Rho-kinase, and myosin phosphatase.
Cat, catalytic subunit of myosin phosphatase.
To whom correspondence should be addressed. Tel.:
81-7437-2-5440; Fax: 81-7437-2-5449; E-mail:
kaibuchi{at}bs.aist-nara.ac.jp.
1
The abbreviations used are: MBS, myosin-binding
subunit; MLC, myosin light chain; GTPS, guanosine
5-(3-O-thio)-triphosphate; GST, glutathione
S-transferase; DTT, dithiothreitol; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE,
polyacrylamide gel electrophoresis.
2
A detailed analysis concerning recombinant
Rho-kinase will be described elsewhere.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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J. S. Dhaliwal, D. B. Casey, A. J. Greco, A. M. Badejo Jr., T. B. Gallen, S. N. Murthy, B. D. Nossaman, A. L. Hyman, and P. J. Kadowitz Rho kinase and Ca2+ entry mediate increased pulmonary and systemic vascular resistance in L-NAME-treated rats Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1306 - L1313. [Abstract] [Full Text] [PDF]
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ABSTRACT