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INTRODUCTION |
Contraction of smooth muscle is regulated primarily by the
cytosolic Ca2+ concentration
([Ca2+]i)1
and phosphorylation of the 20-kDa myosin light chain (MLC20) by
Ca2+-calmodulin-dependent myosin light chain
kinase (MLCK) (1-4). However, the relationship between
[Ca2+]i and myosin phosphorylation is not
fixed and under certain conditions may shift. Increased phosphorylation
at submaximal Ca2+ levels, referred to as increased
Ca2+ sensitivity, frequently occurs following agonist
stimulation (4). With respect to the balance of kinase and phosphatase activities, this effect could reflect either increased activity of the
Ca2+·calmodulin·MLCK complex or, inhibition of myosin
phosphatase (MP). The latter was found to be the case (5, 6) and it was
suggested that the inhibition of MP involved a G-protein-linked pathway
(4). In addition, several studies showed that the small GTPase Rho,
specifically RhoA (7), was important in smooth muscle function and the
increased Ca2+ sensitivity following agonist stimulation
(for reviews, see Refs. 4 and 8).
Recently several target proteins of Rho have been identified, including
protein kinase N (9, 10), Rho-associated kinase (Rho-kinase) (11-13),
rhophilin (10), rhotekin (14), citron (15), p140mDia (16), and MP
target subunit 1, termed MYPT1 (17). Phosphorylation of MYPT1 by
Rho-kinase inhibited MP and this was suggested as a mechanism to
increase the Ca2+ sensitivity in smooth muscle (17). In
addition, it was found that bovine brain Rho-kinase could directly
phosphorylate myosin in vitro (18) and thus the change in
Ca2+ sensitivity could reflect the two phosphorylation
mechanisms, i.e. MYPT1 and myosin. Indeed, introduction of
the constitutively active recombinant fragment of bovine Rho-kinase
into Triton X-100-permeabilized smooth muscle provoked a
Ca2+-independent contraction via MLC20 phosphorylation
(19). Also, a specific inhibitor of Rho-kinase, Y-27632, was reported
to inhibit the agonist-induced Ca2+-sensitization of smooth
muscle contraction (20). Rho-kinase has been reported to be involved in
other cellular functions including the formation of stress fibers and
focal adhesions (21, 22) and cardiac hypertrophic responses (23).
Rho-kinase has been classified into two isoforms: ROK
and ROK
(12, 21), also referred to as ROCK-II and ROCK-I (24), respectively.
The isoforms are relatively large proteins (150-160 kDa) consisting of
an amino-terminal kinase domain, a central coiled-coil domain that
includes the Rho-binding domain, and a carboxyl-terminal putative
pleckstrin homology domain separated by the insertion of a
cysteine-rich zinc finger domain (11-13, 21, 24). Purification
protocols have been described for the isolation of Rho-kinases from rat
brain (11), bovine brain (12), and human platelets (13). However, it is
surprising that despite the numerous reports supporting a role for RhoA
in smooth muscle there are no data available on the smooth muscle
Rho-kinase. In the present study, we have purified the smooth muscle
Rho-kinase from chicken gizzard. It is suggested that the gizzard
Rho-kinase is the ROK
isoform. The biochemical properties of the
smooth muscle Rho-kinase are outlined and its possible role(s) in
smooth muscle function are discussed.
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EXPERIMENTAL PROCEDURES |
Materials--
Many of the chemicals and vendors were as listed
previously (25), others are as follow: [
-35S]GTP
S,
NEN Life Science Products Inc.;
L-1-tosylamido-2-phenylethyl clhoromethyl ketone-treated
trypsin, staurosporine, wortmannin, and W-7, Sigma; H-7, H-9, and H-89,
Seikagaku Kogyo, Tokyo, Japan; A3 and
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole, Biomol; chelerythrine chloride, Cascade Biochemical Ltd.; arachidonic acid,
Sigma, Cascade Biochemical Ltd., Nu-Chek-Prep, and Cayman Chemical;
sphingosine and sphingosine 1-phosphate, Biomol; psychosine, Doosan
Serdary Research Lab.; C2-ceramide and
C6-ceramide, Cayman Chemical; all other lipids were
obtained from Sigma; polyethylene glycol 6000 (average
Mr 7500), lysyl endopeptidase
(Achromobacter Protease 1), and microcystin-LR, from Wako
Pure Chemical, Osaka, Japan;
(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) was a generous gift from Yoshitomi Pharmaceutical Industries Ltd. (Iruma, Japan).
Preparation of Proteins--
Full-length MYPT1 (Met1
to Ile1004) and a COOH-terminal fragment
(Ser667 to Ile1004) were expressed as GST
fusion proteins and purified (25). These are referred to as
rG-MYPT11-1004 and rG-MYPT1667-1004,
respectively. Preparation of the NH2-terminal fragment of
MYPT1 (Met1 to Asp674), referred to as
rMYPT11-674, was described previously (26). Other protein
preparations from chicken gizzard were: smooth muscle myosin (27),
MLC20 (28), MLCK (27), caldesmon (29), tropomyosin (30), and 32P-labeled MLC20 (26). GST-RhoA (full-length human RhoA
cDNA was cloned into pGEX2T vector) and GST-RhoAA37 (a
mutation of Thr to Ala resulting in a dominant negative mutant in the
effector-interacting domain; structurally equivalent to H-RasA35) were expressed in Escherichia coli
BL21 strain and purified on glutathione-Sepharose 4B according to the
manufacturer's instructions. MP was prepared from chicken gizzard (31)
and purified further to remove contaminant protein kinase(s) using a
polyclonal antibody against chicken MYPT1 (17) linked to cyanogen
bromide-activated Sepharose 4B. A 1-ml column of antibody-Sepharose 4B
was prepared (about 0.5 mg of antibody/ml of resin) and equilibrated
with TBS. MP was applied and the column was washed with 5 ml of TBS
then 10 ml of TBS plus 1 M MgCl2 (this step
removed the endogenous kinase). Phosphatase was eluted with 10 ml of
TBS, 4.5 M MgCl2, and 2 mg/ml bovine serum
albumin, dialyzed versus 30 mM Tris-HCl (pH
7.5), 30 mM KCl, 1 mM dithiothreitol, and 5%
sucrose and stored at
20 °C.
Subcellular Fractionation of Chicken Gizzard--
10-20 g of
frozen chicken gizzard was homogenized with 5 volumes (w/v) of buffer A
(20 mM Tris-HCl, pH 7.5, 4 mM EDTA, 1 mM EGTA, 0.1 mM dithiothreitol, 1 mM benzamidine, 0.2 µM
(p-amidinophenyl)methanesulfonyl fluoride and 0.25 M sucrose) using a Waring blender and centrifuged at
1,000 × g for 10 min. The pellet was used as the crude
nuclear fraction. The supernatant was centrifuged at 100,000 × g for 1 h to separate cytosol (supernatant) and
membrane fraction (pellet). The crude membrane and nuclear fractions
were solublized in buffer A plus 1% Triton X-100 and centrifuged at
100,000 × g for 30 min. The supernatants were used as
the membrane and nuclear fractions. These fractions were analyzed by
Western blotting using a bovine Rho-kinase antibody. The content of
Rho-kinase in each fraction was estimated by densitometry
(Densitograph, Atto, Japan) using purified Rho-kinase from chicken
gizzard as a standard. The sample loads for the fractions and standard
were varied to obtain a linear response with the enhanced
chemiluminescence (Amersham Pharmacia Biotech) detection procedure.
Purification of Rho-kinase from Chicken Gizzard--
All
procedures were carried out at 4 °C. 200 g of frozen chicken
gizzard were homogenized with 5 volumes (w/v) of buffer A using a
Waring blender and centrifuged at 15,000 × g for 15 min. The pellet was rehomogenized with the same volume of buffer A and
centrifuged. The supernatants were combined and polyethylene glycol
6000 added to 6% (v/v). After 30 min, the precipitate was collected by
centrifugation at 15,000 × g for 15 min and dissolved in 200 ml of buffer B (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1 mM
dithiothreitol, 1 mM benzamidine, 0.2 µM
(p-amidinophenyl)methanesulfonyl fluoride, and 5% glycerol)
containing 0.1 M NaCl using a Dounce homogenizer. The
suspension was clarified by centrifugation at 100,000 × g for 30 min and the supernatant was applied to a
Q-Sepharose Fast Flow column (2.6 × 14 cm) equilibrated with
buffer B plus 0.1 M NaCl. The column was washed extensively
with buffer B plus 0.1 M NaCl and eluted with a 400-ml
linear gradient of NaCl (0.1-0.6 M) in buffer B. The
fractions containing Rho-kinase activity were pooled and dialyzed
against 5 liters of buffer C (10 mM PIPES-NaOH, pH 6.0, 1 mM EDTA, 0.1 mM dithiothreitol, 1 mM benzamidine, 0.2 µM
(p-amidinophenyl)methanesulfonyl fluoride, and 5% glycerol) for 2 h. After brief centrifugation (10,000 × g
for 5 min) the sample was loaded onto a phosphocellulose column
(1.6 × 10 cm) equilibrated in buffer C. The column was washed
with 5 column volumes of buffer C plus 0.15 M NaCl and
Rho-kinase was eluted (at approximately 0.45 M NaCl) by
application of a 180-ml linear gradient of NaCl (0.15-0.7
M) in buffer C. Fractions were pooled and dialyzed
overnight against buffer D (10 mM PIPES-NaOH, pH 6.0, 5 mM MgCl2, 0.1 mM dithiothreitol, 1 mM benzamidine, 0.2 µM
(p-amidinophenyl)methanesulfonyl fluoride, and 5%
glycerol). The dialyzed sample was applied to a 1-ml
glutathione-Sepharose 4B column immobilized with 5 mg of the
"active" form of RhoA, i.e. GTP
S·GST-RhoA (12). The
column was washed with 10 volumes of buffer D containing 0.5 M NaCl. Rho-kinase was eluted in two steps: initially by
elution at higher pH (30 mM Tris-HCl, pH 8.3, 0.1 mM dithiothreitol) and subsequently by elution with
glutathione (30 mM Tris-HCl, pH 7.5, 10 mM
glutathione, and 0.1 mM dithiothreitol).
Protein Kinase Assay--
Unless otherwise indicated, Rho-kinase
activity was assayed in a reaction mixture (final volume of 50 µl)
containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl,
0.1 mM dithiothreitol, 5 mM MgCl2, 1 mM EDTA, 1 µM microcystin-LR, enzyme, and
substrate as indicated. MLCK activity was assayed in a reaction mixture
(final volume of 50 µl) containing 20 mM Tris-HCl, pH
7.5, 100 mM KCl, 0.1 mM dithiothreitol, 1 mM MgCl2, 0.1 mM CaCl2,
1 µM microcystin-LR, 2 µg/ml calmodulin, 0.1 µg/ml
MLCK, and MLC20 as indicated. Reactions were initiated by addition of
[
-32P]ATP to a final concentration of 100 µM. After incubation at 30 °C for 5 min (enzyme
concentration was adjusted to ensure linear kinetics at this time)
40-µl aliquots were removed and added to phosphocellulose paper. The
paper was washed immediately for 10 min with 75 mM
phosphoric acid (4 times), dried, and 32P incorporation
determined by Cerenkov counting. Alternatively, reactions were
terminated by addition of Laemmli sample buffer and boiled for 3 min.
Samples were subjected to SDS-PAGE (7.5-20%), followed by
autoradiography or 32P determination in excised gel slices.
MP Assay--
Phosphatase assays were carried out at 30 °C
using 32P-labeled MLC20 (final concentration 5 µM) as substrate. Assay conditions were: 30 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.2 mg/ml
bovine serum albumin, and 0.3 mM CoCl2
(26).
Phosphorylation of MP and Myosin--
Phosphorylation of MP
holoenzyme by Rho-kinase was carried out at 30 °C for different
times in 20 mM Tris-HCl, pH 7.5, 100 mM KCl,
0.1 mM dithiothreitol, 5 mM MgCl2,
1 mM EDTA, 30 µg/ml MP, 2 µg/ml Rho-kinase, 2 µM GTP
S·GST-RhoA, and 100 µM
[
-32P]ATP in the presence or absence of 1 µM microcystin-LR in a final volume of 50 µl. The
reactions were initiated by addition of [
-32P]ATP and
terminated by addition of Laemmli sample buffer and immediately boiled
for 3 min. These samples were subjected to SDS-PAGE (7.5-20%),
followed by autoradiography or 32P determination in excised
gel slices, positions corresponding to MYPT1, PP1c
, or M20. To
determine the effects of phosphorylation on MP activity,
phosphorylation of MP was carried out under the similar conditions
without microcystin-LR using ATP
S instead of ATP. At each time
point, an aliquot was removed, diluted 100-fold with a buffer
containing 30 mM Tris-HCl, pH 7.5, 0.2 mg/ml bovine serum
albumin, and 0.1 mM EDTA and assayed for phosphatase assay. Phosphorylation of the recombinant fragments of MYPT1 (30 µg/ml rG-MYPT1667-1004, 30 µg/ml rMYPT11-674, and
20 µg/ml GST) was carried out in the presence or absence of 2 µM GTP
S·GST-RhoA and in the absence of
microcystin-LR under the above conditions.
To determine the stoichiometry of phosphorylation of myosin, myosin (1 mg/ml) was incubated with 250 µM
[
-32P]ATP in 20 mM Tris-HCl, pH 7.5, 300 mM KCl, 0.1 mM dithiothreitol, 1 mM
MgCl2, 2 µg/ml Rho-kinase, and 2 µM
GTP
S·GST-RhoA, final volume 50 µl. After incubation for
different times, reactions were terminated by addition of
trichloroacetic acid and sodium pyrophosphate to 5 and 1%,
respectively. The precipitated protein was collected and washed with
the trichloroacetic acid/sodium pyrophosphate solution, using plastic
funnels (Sepacol columns, Seikagaku Kogyo, Tokyo, Japan) fitted with a
fiberglass disc and cotton plug (27). 32P incorporation was
estimated by Cerenkov counting. Assays of actin-activated
Mg2+-ATPase activity of myosin were as described previously
(32).
Peptide Sequence of Smooth Muscle Rho-kinase--
Purified
Rhokinase was subjected to 6% SDS-PAGE and transferred to a
ProblotTM membrane (Applied Biosystems). The band
corresponding to Rhokinase was digested by lysyl endopeptidase. The
resulting peptides were separated by high performance liquid
chromatography and subjected to a gas-phase sequencer (Shimadzu PSQ-1,
Tokyo, Japan) as described previously (33).
Other Procedures--
Lipids were routinely prepared as
chloroform, ethanol, or dimethyl sulfoxide stocks, and vesicles were
prepared as described earlier (25). For dimethyl sulfoxide stocks,
lipids were directly diluted into the same buffer. SDS-PAGE was carried
out with the discontinuous buffer system of Laemmli (34). Western
blotting analyses were carried out as described previously (25, 35). The polyclonal antibody to the NH2-terminal region of MYPT1
(17) and to the coiled-coil region of bovine Rho-kinase (36) were prepared as described earlier. The anti-Rho-kinase antibody recognized both ROK
and ROK
isoforms. Protein concentrations were determined with either the BCA (Pierce) or Bradford (Bio-Rad) procedures, using
bovine serum albumin as a standard.
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RESULTS |
Purification of Rho-kinase from Chicken Gizzard--
The
distribution of Rho-kinase in various fractions from gizzard was
investigated using the bovine Rho-kinase antibody. Most of the kinase
was present in the cytosolic fraction (75.3 ± 3.3%; mean ± S.E., n = 3) compared with the membrane (20.4 ± 2.9%) and nuclear fractions (4.3 ± 1.0%).
Gizzard cytosol was used as source for purification of Rho-kinase. The
purification procedure was monitored by Western blots and also by assay
with GTP
S·GST-RhoA and rG-MYPT1667-1004 as substrate.
An efficient (high yield) method was developed for purification of
Rho-kinase as outlined in Table I. The
yield of Rho-kinase from 200 g of chicken gizzard was
approximately 33 µg and this was purified over 20,000-fold to
apparent homogeneity.
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Table I
Purification of Rho-kinase
Smooth muscle Rho-kinase was purified from 200 g of chicken
gizzard. The kinase activity was assayed using 0.1 mg/ml
rG-MYPT1667-1004 as substrate as described under
"Experimental Procedures." Values present means ± S.E.
(n = 4).
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The initial step was precipitation with polyethylene glycol 6000 and
this recovered about 80% of the cytosolic Rho-kinase. The elution
profiles of the subsequent chromatography procedures are shown in Fig.
1, A and B. These
are reproducible and the kinase activity was closely correlated with
the presence of immunoreactive bands on Western blots. The final step
was affinity chromatography on GTP
S·GST-RhoA glutathione-Sepharose
(Fig. 1C). Two elution steps were used. A step to pH 8.3 eluted a single band of apparent molecular mass 160 kDa (p160). This
was identified as Rho-kinase by Western blots (Fig. 1D). The
purity of Rho-kinase was greater than 90%, as judged by SDS-PAGE, and
this fraction was taken as the final product. A second elution step
with 10 mM glutathione was applied. This removed components
of 160 kDa (p160), a doublet at about 130 kDa (termed p130 in Fig.
1D) and GST-RhoA (Fig. 1D). By Western blots
(Fig. 1D), p160 was identified as Rho-kinase and p130 was
identified as the MYPT1 doublet (apparent masses 133 and 130 kDa (Ref.
31)). The fraction eluted by 10 mM glutathione also
contained the PP1 catalytic subunit (PP1c
) and the small noncatalytic subunit of MP, M20 (data not shown).

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Fig. 1.
Purification of smooth muscle
Rho-kinase. A, Q-Sepharose Fast Flow chromatography.
B, phosphocellulose chromatography. C,
GTP S·GST-RhoA affinity chromatography on glutathione-Sepharose 4B.
Aliquots of fractions from each were assayed in the presence of 2 µM GTP S·GST-RhoA ( ) or GDP·GST-RhoA ( ) using
0.1 mg/ml rG-MYPT1667-1004 as substrate under standard
assay conditions as described under "Experimental Procedures."
D, eluate by pH 8.3 (a) or 10 mM
glutathione (b) from GTP S·GST-RhoA affinity column.
Lanes 1 and 3, Coomassie Blue stains; lanes
2 and 4, Western blots using anti-Rho-kinase antibody;
lane 5, Western blot using anti-MYPT1 antibody.
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The co-purification of MP and Rho-kinase from the Rho affinity column
suggested that both interacted with the active form of RhoA,
i.e. GTP
S·GST-RhoA. To confirm this, a precipitation assay using GTP
S·GST-RhoA-coupled to glutathione-Sepharose 4B and
an overlay assay using [35S]GTP
S·GST-RhoA were
carried out. These assays showed that Rho-kinase and MYPT1 interacted
specifically with GTP
S·GST-RhoA, but interactions were not
detected with either GDP·GST-RhoA nor
GTP
S·GST-RhoAA37 (data not shown). These results are
in agreement with our previous report (12).
To identify the isoform of the purified smooth muscle Rho-kinase, the
p160 was subjected to proteolysis and sequence analysis of derived
peptides. The sequences of 3 peptides were determined and compared with
matching sequences from rat ROK
(Refs. 11 and 21; also referred to
as ROCK-II, see Ref. 24) and rat ROK
(also referred to as ROCK-I).
As shown in Fig. 2, the p160 more closely
matched ROK
(28 of 30 residues identical) than ROK
(21 of 30 residues identical). Thus the Rho-kinase isolated from gizzard was the
ROK
isoform.

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Fig. 2.
Alignment of the smooth muscle
Rho-kinase-derived peptide sequences with ROK
and ROK . Three peptides sequences
derived from smooth muscle Rho-kinase revealed a highly significant
homology with rat ROK . Residues are shown white on black when at
least two out of three are identical.
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Enzymatic Properties of Smooth Muscle Rho-kinase--
Smooth
muscle Rho-kinase phosphorylates a number of proteins, as do several
protein kinases. As shown in Table II the
smooth muscle Rho-kinase phosphorylated MYPT1, myosin, MLC20, myelin basic protein, protamine, histone IIIs, and histone VIIs. Among these
MYPT1, myosin and MLC20 appeared to be better substrates. MLCK,
caldesmon, and tropomyosin were not phosphorylated by smooth muscle
Rho-kinase under the same conditions. The activity of smooth muscle
Rho-kinase toward each substrate was stimulated by GTP
S·GST-RhoA 1.5-2-fold (Table II). A similar result (1.94 ± 0.34-fold
activation, n = 4) was obtained using
baculovirus-expressed RhoA, which was genanylgenanylated at the COOH
terminus. Km, Vmax, and kcat values of smooth muscle Rho-kinase for
MLC20 and rG-MYPT1667-1004 are shown in Table
III. These were estimated at 100 µM ATP. For Rho-kinase and MLC20 the
Km and Vmax values obtained at 200 µM ATP were similar (Table III). In general,
GTP
S·GST-RhoA causes a slight decrease in Km
(reduced by about half) and an increase in Vmax
and kcat (1.4-1.6-fold). The
Km value of smooth muscle Rho-kinase for ATP was
30.8 ± 6.2 µM (mean ± S.E., n = 4). This value is considerably higher than that reported for the
recombinant human p160ROCK, i.e. 0.1 µM
(20).
To examine the effects of pH on Rho-kinase activity, assays were
carried out over a pH range of 4 to 9.3. As shown in Fig. 3A, the basal activity of
Rho-kinase (in the absence of GTP
S·GST-RhoA) was maximum at pH
6.0. An interesting point is that at pH 6.0 the activity of Rho-kinase
was independent of GTP
S·GST-RhoA, whereas at pH 7.5 the dependence
on RhoA was observed (inset of Fig. 3A). The
activity of Rho-kinase at pH 6.0, either in the presence or absence of
GTP
S·GST-RhoA, was similar to that shown in the presence of
GTP
S·GST-RhoA at pH 7.5. The effects of ionic strength on
Rho-kinase activity with myosin, MLC20, and the MP holoenzyme as
substrates are shown in Fig. 3B. With intact myosin (and 1 mM MgCl2) activity was increased with
increasing ionic strength and reached a maximum at 0.3 M
KCl. A sharp decline in activity occurred after 0.5 M KCl.
With MLC20 or MP as substrate, increasing ionic strength caused a
gradual decrease in Rho-kinase activity.

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Fig. 3.
Effects of pH and KCl on Rho-kinase
activity. A, effects of pH. Rho-kinase was assayed in
the absence of GTP S·GST-RhoA using 0.1 mg/ml
rG-MYPT1667-1004 as substrate under standard assay
conditions as described under "Experimental Procedures." Buffers
used were 50 mM Tris-HCl, pH 6.5-9.1 ( ); 100 mM GTA-HCl/NaOH (100 mM 3,3-dimethylglutaric
acid, 100 mM Tris base, 100 mM
2-amino-2-methyl-1,3-propanediol), pH 4-9.3 ( ); 50 mM
PIPES-NaOH, pH 5.2-7.4 ( ). Inset, Rho-kinase was assayed
in 100 mM GTA-HCl/NaOH at pH 7.5 or 6.0 in the presence
(dark) or absence (open) of 2 µM
GTP S·GST-RhoA. The kinase activity at pH 7.5 in the absence of
GTP S·GST-RhoA was expressed as 100%. Error bars
indicate means ± S.E. (n = 3). B,
effects of KCl. Rho-kinase was assayed using 1 mg/ml myosin ( ), 0.1 mg/ml MLC20 ( ), or 0.1 mg/ml MP ( ) as substrate. Other conditions
are: 30 mM Tris-HCl, pH 7.5, 1 mM
MgCl2, 0.1 mM dithiothreitol, 0.1 mM [ -32P]ATP and enzyme. The kinase
activity at 20 mM KCl was expressed as 100%. Error
bars indicate means ± S.E. (n = 4).
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Next the effect of various protein kinase inhibitors on gizzard
Rho-kinase was investigated. Ki values for smooth muscle Rho-kinase were estimated by Dixon plots and are summarized in
Table IV. Staurosporine (a wide-range
inhibitor of protein kinases (37)) was the most potent inhibitor and
its Ki value was about 0.02 µM.
Y-27632, an inhibitor of p160ROCK (20) and H-89 (an inhibitor of
protein kinase A (38)), also strongly inhibited smooth muscle
Rho-kinase. Other inhibitors were less effective and these included:
H-7 and A3 (38); W-7 (a calmodulin antagonist (39)); and
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole (an
inhibitor of casein kinase II (40)). H-9 (38) even at 100 µM showed only slight inhibition. The smooth muscle
Rho-kinase was not inhibited (data not shown) by millimolar
concentrations of chelerythrine (an inhibitor of protein kinase C (41))
and wortmannin (an inhibitor of phosphatidylinositol 3-kinase and MLCK
(42)).
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Table IV
Effects of various protein kinase inhibitors on Rho-kinase activity
The kinase activity was assayed using 0.1 mg/ml
rG-MYPT1667-1004 as substrate as described under
"Experimental Procedures." Values are mean ± S.E.
(n = 4).
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Effects of Phosphorylation of MP and Myosin--
The time course
and stoichiometry of phosphorylation for MYPT1 of MP by smooth muscle
Rho-kinase is shown in Fig.
4A. MYPT1 was phosphorylated
up to 1.8 and 1.3 mol of Pi/mol of MYPT1 in the presence
and absence of 1 µM microcystin-LR, respectively. M20 was
not an effective substrate and PP1c
was not phosphorylated under the
same conditions (Fig. 4A). Thiophosphorylation of MYPT1 of
MP by Rho-kinase decreased the phosphatase activity toward 32P-MLC20 (Fig. 4B). Using the recombinant
fragments of chicken MYPT1 (M133 isoform (31)) as substrates, it was
shown that the smooth muscle Rho-kinase phosphorylated the
COOH-terminal part of MYPT1 (residues 667-1004) and the
NH2-terminal fragment was not phosphorylated (Fig.
4C). These data indicate that there are at least 2 phosphorylation sites for Rho-kinase in the COOH-terminal part
(Ser667 to Ile1004) of MYPT1.

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Fig. 4.
Effect of phosphorylation of myosin
phosphatase. A, myosin phosphatase holoenzyme was
phosphorylated in the presence of GTP S·GST-RhoA by Rho-kinase with
( , , ) or without ( ) 1 µM microcystin-LR, and
32P incorporation in the excised gel slices corresponding
to the position of MYPT1 ( , ), M20 ( ), and PP1c ( ) were
determined as described under "Experimental Procedures."
B, myosin phosphatase holoenzyme were preincubated with
ATP S ( ) for the indicated time without microcystin-LR and then
phosphatase assays were carried out as described under "Experimental
Procedures." Myosin phosphatase activity, prior to preincubation with
ATP S (0 min), was expressed as 100%. C, phosphorylation
of recombinant fragments of MYPT1. GST (lanes 1, 2, 7, and
8), rMYPT11-674 (lanes 3, 4, 9, and
10), and rG-MYPT1667-1004 (lanes 5, 6, 11, and 12) were phosphorylated by Rho-kinase for 30 min in the presence (lanes even number) or absence of
GTP S·GST-RhoA (lanes uneven number) as described under
"Experimental Procedures." The phosphorylated samples were then
subjected to 7.5-20% SDS-PAGE and visualized by Coomassie Blue
staining (lanes 1-6) and autoradiography (lanes
7-12).
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The time course and stoichiometry of myosin phosphorylation by
Rho-kinase are shown in Fig.
5A. In the presence of
GTP
S·GST-RhoA, myosin was maximally phosphorylated by Rho-kinase
to 1.8 mol of Pi/mol of myosin at 0.3 M KCl. A
semilogarithmic plot of the time course indicated that the
phosphorylation of myosin by Rho-kinase followed two first-order
processes. Autoradiograms showed that only MLC20 was phosphorylated
(data not shown), and phosphoamino acid analysis showed that the major
site of phosphorylation was phosphoserine (data not shown).
Two-dimensional phosphopeptide mapping analysis indicated only one
major phosphopeptide and the phosphopeptide map was identical to that
observed on phosphorylation of myosin (or MLC20) with MLCK (data not
shown). These results suggested that smooth muscle Rho-kinase
phosphorylates MLC20 at Ser19, i.e. the major
site for MLCK (1-3). The effect of phosphorylation of myosin by
Rho-kinase on actin-activated ATPase activity was determined and
compared with that by MLCK. As shown in Fig. 5B, phosphorylation of myosin by Rho-kinase increased the actin-activated ATPase activity and, as expected, the extent of activation by both
kinases was similar. Some kinetic data for MLCK and MLC20 are included
in Table III for comparison with Rho-kinase. The important points are
that Vmax and kcat values
for MLCK are comparable to values obtained with Rho-kinase.

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Fig. 5.
Effect of phosphorylation of myosin.
A, time course of phosphorylation of myosin. Myosin was
phosphorylated in the presence of GTP S·GST-RhoA as described under
"Experimental Procedures." Inset shows a semilogarithmic
plot of the time course data. Pmax is the
maximum extent of phosphorylation and Pt is the
phosphorylation level at time t. Error bars
indicate mean ± S.E. (n = 4). B,
effects of phosphorylation of myosin on actin-activated ATPase
activity. Myosin (1 mg/ml) was phosphorylated by Rho-kinase ( ) or
MLCK ( ). The levels of phosphorylation by Rho-kinase and MLCK were
1.8 and 1.9 mol of Pi/mol of myosin, respectively. The
actin-activated ATPase activity was measured as described previously
(32).
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Regulation of Smooth Muscle Rho-kinase--
Incubation of the
purified enzyme with [
-32P]ATP under the standard
assay conditions resulted in autophosphorylation of Rho-kinase. About
1.6 and 1.2 mol of phosphate/mol of Rho-kinase were incorporated in the
presence and absence of GTP
S·GST-RhoA, respectively (data not
shown). The activity of Rho-kinase and the activation by
GTP
S·GST-RhoA were not affected by autophosphorylation (data not shown).
Digestion of Rho-kinase with trypsin (1:500, w/w, trypsin:Rho-kinase)
resulted in a marked increase of activity. As shown in Fig.
6, the dependence of Rho-kinase activity
on GTP
S·GST-RhoA initially was lost (after approximately 7 min
digestion) and continued proteolysis caused an increase in the
GTP
S·GST-RhoA-independent activity. The increase in constitutive
activity, i.e. in the absence of GTP
S·GST-RhoA, reached
a maximum that was 5-6-fold higher than the native enzyme. Further
proteolysis decreased the Rho-kinase activity (Fig. 6). SDS-PAGE
analyses of the tryptic digests showed that the 160-kDa band was
degraded initially into a 125-kDa fragment, and further cleavage
generated 3 major products of 112, 88, and 77 kDa (inset of
Fig. 6). Maximum activation of Rho-kinase activity was closely
correlated with the appearance of the 112- and 88-kDa fragments,
indicating that these fragments contained the catalytic domain.

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Fig. 6.
Proteolysis of Rho-kinase by trypsin.
Rho-kinase (30 µg/ml) was digested at 25 °C with trypsin (1:500
weight ratio, trypsin:Rho-kinase) in 30 mM Tris-HCl, pH
7.5, 1 mM dithiothreitol. Proteolysis was stopped by
addition of diisopropyl fluorophosphate to 1 mM. Kinase
activities were determined in the presence ( ) or absence ( ) of 2 µM GTP S·GST-RhoA using 0.1 mg/ml
rG-MYPT1667-1004 as substrates as described under
"Experimental Procedures." The kinase activity, prior to digestion
(0 min) in the absence of GTP S·GST-RhoA was expressed as 100%.
Inset shows corresponding SDS-PAGE gels. Lane 1,
native Rho-kinase; lanes 2-11, aliquots taken at 1, 4, 7, 12, 20, 30, 45, 60, 75, and 90 min, respectively.
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The pleckstrin homology domain has been reported to be involved in
lipid binding (43, 44). This prompted an investigation of the effects
of lipids on the activity of Rho-kinase (Figs. 7A and 6B).
Arachidonic acid caused a marked stimulation of Rho-kinase activity and
some acidic phospholipids induced a smaller activation (Fig.
7A) with MLC20 as substrate. The concentration dependence for arachidonic acid and the acidic phospholipids are shown in Fig.
7B. An activation of about 6-fold was achieved by 30-50
µM arachidonic acid and over the same concentration range
phosphatidylinositol induced about a 2.5-fold activation. The level of
Rho-kinase activity achieved by arachidonic acid was about the same as
that induced by tryptic hydrolysis. The activation of Rho-kinase was
observed with arachidonic acids obtained from several commercial
sources. Activation of Rho-kinase by arachidonic acid was observed also using the MYPT1 substrate, rG-MYPT1667-1004, as shown in
Table III. kcat was increased about 12-fold,
compared with basal activity, by 50 µM arachidonic acid.
Phosphatidylserine, phosphatidic acid, lysophosphatidylserine,
lysophosphatidic acid, and phosphatidylinositol 4,5-bisphosphate also
stimulated Rho-kinase activity (2-3-fold) but at higher lipid
concentrations (Fig. 7B). Sphingosine and sphingosine
1-phosphate slightly stimulated Rho-kinase activity (Fig.
7A). Other lipids did not affect the activity of Rho-kinase
(Fig. 7A). The stimulation of Rho-kinase activity by arachidonic acid, or acidic phospholipids, was independent of GTP
S·GST-RhoA and the maximum activation by arachidonic acid was
not affected by the presence of GTP
S·GST-RhoA. Thus activation of
Rho-kinase by lipids offers an alternative potential regulatory mechanism that is independent of the small G proteins.

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Fig. 7.
Activation of Rho-kinase activity by various
lipids. Rho-kinase activities were determined using 0.1 mg/ml
MLC20 as substrate in the absence of GTP S·GST-RhoA as described
under "Experimental Procedures" unless otherwise indicated. The
activity of Rho-kinase in the absence of lipids was expressed as 100%.
A, effect of various lipids: C, buffer control;
RhoA, 2 µM GTP S·GST-RhoA; PC,
300 µM phosphatidylcholine; PE, 300 µM phosphatidylethanolamine; PS, 300 µM phosphatidylserine; PA, 300 µM phosphatidic acid; PI, 50 µM
phosphatidylinositol; LPS, 300 µM
lysophosphatidylserine; LPA, 300 µM
lysophosphatidic acid; PIP2, 300 µM
phosphatidylinositol-4, 5-bisphosphate; DG, 300 µM diacylglycerol; Psy, 50 µM
psychosine; C2-Cer, 300 µM
C2-ceramide; C6-Cer, 300 µM C6-ceramide; SM, 300 µM sphingomyelin; SphI-P, 50 µM
sphingosine 1-phosphate; Sph, 50 µM
sphingosine; IP3, 100 µM inositol
1,4,5-trisphosphate; AA, 50 µM arachidonic
acid. Error bars indicate means ± S.E.
(n = 4). B, dose-response activation of
Rho-kinase by various lipids. Arachidonic acid ( ),
phosphatidylinositol ( ), phosphatidylserine ( ), phosphatidic acid
( ), and phosphatidylinositol 4,5-bisphosphate ( ). The data
plotted represent mean ± S.E. (n = 4).
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DISCUSSION |
The purification and partial characterization of Rho-kinase from
chicken gizzard is described above and to our knowledge is the first
report of the isolation of Rho-kinase from smooth muscle. The isolation
procedure is relatively simple and provides Rho-kinase close to
homogeneity and in reasonable yields. Most of the Rho-kinase was
present in the cytosolic fraction and previous preparations of
Rho-kinase have used both the cytosol (e.g. from platelets (13)) and the membrane fraction (e.g. from brain (12)). The enzyme isolated from gizzard was the ROK
isoform. The 2 isoforms of
Rho-kinase have some differences in tissue distribution. ROK
(ROCK-I) was detected in many tissues, although at higher levels in
liver, lung, and testis. In contrast, ROK
(ROCK-II) was enriched in
brain and skeletal muscle (11-13, 21, 24).
The properties of the gizzard Rho-kinase are similar to those reported
earlier. The mass (as judged from SDS-PAGE) is about 160 kDa and it
binds to RhoA in the GTP, or GTP
S, bound form. Binding of the active
form of RhoA results in activation of Rho-kinase activity and this
reflects a decrease in Km and an increase in
Vmax (or kcat). However,
this is a relatively modest degree of activation, 1.5-2-fold, compared
with regulatory influences with other protein kinases. The reported
effects of GTP·RhoA are variable, ranging from zero activation (11),
2-fold activation (13) to about 15-fold activation using a fragment of
MYPT1 (12). Several factors may influence basal activity (in the
absence of GTP·RhoA) and activation by GTP·RhoA. These include: the
substrate used (12), preactivation during Rho-affinity chromatography (11), and other modifications incurred during isolation, and autophosphorylation. A precedent to support the latter is the autophosphorylation of p21-activated serine/threonine kinase that reduces binding of Cdc42 or Rac and activates kinase activity (45).
Autophosphorylation of Rho-kinase has been reported (see Refs. 11-13,
and above data) but from our results this did not affect kinase
activity or the binding of GTP
S bound RhoA. It cannot be eliminated
that autophosphorylation of the gizzard Rho-kinase occurred before its
isolation. It might also be argued that translocation of Rho-kinase
from the cytosol to the membrane-bound GTP·RhoA could result in a
more effective regulation by RhoA. The assembly of a multiprotein
complex (46, 47) at the membrane may result in altered protein
conformations or the contribution to the regulatory mechanism of
membrane components.
The above discussion raises the possibility that there could be other
regulatory mechanisms for Rho-kinase, either independent of or
complementary to RhoA. Limited digestion of Rho-kinase by trypsin
causes 5-6-fold activation of RhoA-independent kinase activity. It was
suggested (21) that the pleckstrin homology/cysteine-rich zinc finger
domain contained an autoinhibitory sequence(s) and thus activation via
tryptic hydrolysis could be explained by loss of this inhibitory
sequence and the generation of a constitutively active
NH2-terminal kinase fragment. The activity of this fragment is considerably higher than that achieved by GTP
S·GST-RhoA and raises the question of whether another modulator/regulator might be
involved in realizing full activation of Rho-kinase. This, together
with the possibility that membrane components could be involved,
prompted our investigation of the effects of various lipids on
Rho-kinase activity. An interesting finding was that Rho-kinase was
activated 5-6-fold by arachidonic acid and to a lesser extent by
certain acidic phospholipids. The activation was independent of RhoA.
The activation of Rho-kinase by arachidonic acid is intriguing since
the first mechanism proposed for increased Ca2+ sensitivity
in smooth muscle involved arachidonic acid. It was shown in
permeabilized muscle fibers that arachidonic acid increased force and
MLC20 phosphorylation at constant Ca2+ concentration (48).
The MP rate was also decreased and it was suggested that this reflected
dissociation of the MP holoenzyme by arachidonic acid (48).
Subsequently it was shown that GTP
S induced the release of
arachidonic acid and that the time course and concentration of
arachidonic acid release were consistent with a physiological role of
arachidonic acid (49). Also several agonists that affect
Ca2+ sensitivity in smooth muscle release arachidonic acid
at constant Ca2+ levels (49). The basal concentration of
arachidonic acid in
-toxin-permeabilized fibers was 20-30
µM and increased to 60-90 µM on
stimulation with GTP
S (49). This is within the range of arachidonic
acid concentration required to activate the gizzard Rho-kinase
(i.e. 30-50 µM). More recently it has been
suggested that arachidonic acid might activate an atypical protein
kinase C isoform or related kinase (50). Based on this discussion it is
possible that arachidonic acid acts as an additional (alternative) mechanism to activate Rho-kinase. However, activation of Rho is established as a critical step in the Ca2+-sensitization
process in smooth muscle and therefore any role for arachidonic acid
would probably be downstream of Rho activation.
Potential substrates for the bovine ROK
isoform include: MYPT1 (17),
MLC20 (18),
-adducin (36), and ERM family (ezin, radixin, and moesin
(51)). With respect to the Ca2+ sensitization of smooth
muscle the focus is on MYPT1 and MLC20 and the gizzard Rho-kinase
showed high activity toward both. With the
rG-MYPT1667-1004 fragment the Km for
gizzard Rho-kinase was considerably lower than the estimated
concentration of MYPT1 in smooth muscle, about 1 µM (8)
and thus MYPT1 is a reasonable in vivo target for
Rho-kinase. As shown above, thiophosphorylation of MYPT1 by Rho-kinase
inhibited the MP holoenzyme, confirming earlier observations (17). The
sites of phosphorylation on MYPT1 have not been established and this is
under investigation. Thus consistent with earlier suggestions (12, 17)
it is proposed that a major target for Rho-kinase is the MYPT1 subunit.
In a previous report it was found that MYPT1 was phosphorylated by an
endogenous kinase in the MP holoenzyme preparation (52).
Phosphorylation at Thr654/Thr695 of the
M130/M133 isoforms, respectively, inhibited phosphatase activity. The
endogenous kinase was inhibited by chelerythrine (IC50 5 µM) but not by H-7. In contrast, the smooth muscle
Rho-kinase was inhibited by H-7 (Ki 0.45 µM) but not by millimolar concentration of chelerythrine.
Clearly, Rho-kinase and the endogenous kinase are not identical. It is
apparent, therefore that more than one kinase can phosphorylate MYPT1
and cause inhibition of MP.
The second substrate for Rho-kinase that potentially is important in
the contractile process of smooth muscle is myosin. It is shown above
that Rho-kinase phosphorylates MLC20 and intact myosin and that the
time course of phosphorylation of myosin followed two rates, implying
sequential phosphorylation of the two heads of myosin. This is similar
to the situation with myosin and MLCK (1, 53). The increase in the
myosin phosphorylation rate on increasing ionic strength may reflect a
change in myosin conformation, namely the 10 S to 6 S transition that
occurs between 0.2 and 0.3 M KCl (54). Previously it was
reported that brain Rho-kinase phosphorylated myosin (18) and our data
confirm this observation with smooth muscle Rho-kinase. The light chain
phosphorylation site for Rho-kinase and MLCK is Ser19 and
thus the effect of phosphorylation by the two kinases on actin-activated ATPase activity is the same. The critical question is
whether myosin is phosphorylated by Rho-kinase under physiological conditions? An earlier report (19) showed that a constitutively-active fragment of Rho-kinase could induce contraction of Triton X-100-skinned portal vein via the phosphorylation of myosin, but to date this has not
been demonstrated using the native, or full-length enzyme. Using
Rho-kinase inhibitors the important role of Rho-kinase in smooth muscle
function recently was shown (20) but these effects could result from
phosphorylation of MYPT1 or myosin, or both. Our data with the native
enzyme from smooth muscle suggests that direct phosphorylation of
myosin by Rho-kinase is feasible. The kcat
values for MLCK and Rho-kinase also are comparable. Presumably, if this
occurs in situ it would be under conditions where MLCK activity is reduced, possibly at low Ca2+ concentrations. A
signal to activate Rho-kinase obviously is required. The cascade
involving translocation of RhoA to the membrane (47) and subsequent
recruitment of Rho-kinase has the problem that myosin or MYPT1 may not
be accessible to a membrane-bound kinase. A cytosolic active form of
Rho-kinase may be more effective and it is possible that arachidonic
acid activates the cytosolic pool of Rho-kinase.
In summary, a Rho-kinase of chicken gizzard smooth muscle has
been isolated and shown to be a ROK
isoform. It exhibits a broad
substrate specificity, but two good substrates (based on phosphorylation rates) are the MP subunit, MYPT1 and myosin.
Thiophosphorylation of MYPT1 causes inhibition of the MP holoenzyme and
phosphorylation of the myosin light chains on Ser19
increases the actin-activated ATPase of myosin.
kcat values for MLCK and Rho-kinase with myosin
as substrate are comparable. An interesting finding was that
arachidonic acid activated Rho-kinase independent of RhoA and it is
suggested that this activation is a potential physiological mechanism.