Inhibition of sustained smooth muscle contraction by PKA and
PKG preferentially mediated by phosphorylation of RhoA
Karnam S.
Murthy,
Huiping
Zhou,
John R.
Grider, and
Gabriel M.
Makhlouf
Departments of Physiology and Medicine, Medical College of
Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
The role of RhoA in myosin
light-chain (MLC)20 dephosphorylation and smooth muscle
relaxation by PKA and PKG was examined in freshly dispersed and
cultured smooth muscle cells expressing wild-type RhoA, constitutively
active RhoV14, and phosphorylation site-deficient
RhoA188. Activators of PKA
(5,6-dichloro-1-
-ribofuranosyl benzimidazole 3',5'-cyclic
monophosphothionate, Sp-isomer; cBIMPS) or PKG
[8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate
(8-pCPT-cGMP), sodium nitroprusside (SNP)] or both PKA and PKG (VIP)
induced phosphorylation of constitutively active RhoV14 and
agonist (ACh)- or GTP
S-stimulated wild-type RhoA but not RhoA188. Phosphorylation was accompanied by translocation
of membrane-bound wild-type RhoA and RhoV14 to the cytosol
and complete inhibition of ACh-stimulated Rho kinase and phospholipase
D activities, RhoA/Rho kinase association, MLC20
phosphorylation, and sustained muscle contraction. Each of these events
was blocked depending on the agent used, by the PKG inhibitor KT5823 or
the PKA inhibitor myristoylated PKI. Inhibitors were used at a
concentration (1 µM) previously shown by direct measurement of kinase
activity to selectively inhibit the corresponding kinase. In muscle
cells overexpressing the active phosphorylation site-deficient mutant
RhoA188, MLC20 phosphorylation was partly
inhibited by SNP, VIP, cBIMPS, and 8-pCPT-cGMP, suggesting the
existence of an independent inhibitory mechanism downstream of RhoA.
Results demonstrate that dephosphorylation of MLC20 and
smooth muscle relaxation are preferentially mediated by PKG- and
PKA-dependent phosphorylation and inactivation of RhoA.
myosin light chain; myosin light chain phosphatase; regulatory myosin light chain; relaxation
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INTRODUCTION |
CONTRACTION OF VASCULAR
AND visceral smooth muscle is mediated by phosphorylation of
Ser19 on the regulatory myosin light chain
(MLC)20 (14, 32) and consists of a transient
initial phase followed by a prolonged, sustained phase. The initial
phase is mediated by inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent Ca2+
release and Ca2+/calmodulin-dependent activation of MLC
kinase (14, 23, 32). The sustained phase is RhoA
dependent and reflects inhibition of MLC phosphatase (6,
32). On activation, RhoA is translocated to the membrane in
which it initiates two converging pathways involving activation of Rho
kinase and phospholipase D (PLD) (7, 22, 27). Rho kinase
inhibits MLC phosphatase via phosphorylation of the 130-kDa regulatory
myosin phosphatase targeting subunit-1 (MYPT1) (6, 8, 32,
35). Hydrolysis of phosphatidylcholine by PLD yields
phosphatidic acid, which is dephosphorylated to diacylglycerol, leading
to activation of PKC; PKC phosphorylates an endogenous 17-kDa
inhibitory protein (CPI-17) which binds directly to and
strongly inhibits MLC phosphatase (3, 20, 27).
Relaxation of the initial or sustained phase of contraction is mediated
by dephosphorylation of Ser19 on MLC20 by the
catalytic subunit of MLC phosphatase (21, 37). Relaxant
agonists inhibit smooth muscle contraction by activating cAMP- and/or
cGMP-dependent protein kinase (PKA and PKG) (1, 15, 21, 29,
34). Low levels of cAMP activate PKA exclusively, whereas higher
levels can cross-activate PKG-I (12, 13, 24). When both
cyclic nucleotides are generated concurrently, for example, in response
to VIP, cAMP activates PKA as well as PKG (24). PKA and
PKG inhibit the initial contraction by acting on two critical targets
involved in Ca2+ mobilization. Both protein kinases inhibit
PLC-
1-dependent Ins(1,4,5)P3 formation by
phosphorylating RGS-4 and accelerating the inactivation of GTP-bound
G
q (28). Only one kinase, PKG-I, inhibits
Ins(1,4,5)P3-induced Ca2+ release
by direct phosphorylation of the sarcoplasmic
Ins(1,4,5)P3 receptor/Ca2+ channel
(16, 21).
Mechanisms invoked for relaxation of sustained contraction involve
stimulation of MLC phosphatase activity via inhibition of RhoA and/or
RhoA-dependent targets, such as MYPT1 and CPI-17 by PKG and/or PKA.
Indirect evidence (30) suggests that PKG induces
relaxation by phosphorylating RhoA: contraction of permeabilized vascular myocytes by exogenous geranylgeranylated RhoA was inhibited by
exogenous PKG-I and 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP), whereas contraction by a RhoA mutant (RhoA188)
that is not susceptible to phosphorylation by PKG was not affected, suggesting the absence of a mechanism of relaxation downstream of RhoA.
Inhibition of histamine-stimulated contraction and MLC20 phosphorylation by PKG is accompanied by a decrease in CPI-17 phosphorylation that parallels the increase in MLC phosphatase activity, consistent with inhibition of RhoA activity upstream of the
PLD/PKC/CPI-17 pathway (4). However, there is also
evidence that PKG stimulates MLC phosphatase activity independently of RhoA (32, 37). The NH2-terminal zipper
sequence of PKG-I
binds selectively to and induces phosphorylation
of MYPT1. In vitro studies (32) suggest that this
phosphorylation has no effect on MLC phosphatase activity; however,
blockade of PKG-I
binding to MYPT1 prevented MLC20
dephosphorylation by 8-Br-cGMP. Phosphorylation of Thr850
in the COOH terminus of MYPT1 by PKA has been demonstrated in vitro
only and appears to be associated with an increase in MLC phosphatase
activity that could lead to MLC20 dephosphorylation and relaxation (10, 11).
In the present study, we examined the ability of both PKA and PKG to
phosphorylate RhoA in freshly dispersed and cultured smooth muscle
cells and determined the effect of this phosphorylation on
agonist-stimulated RhoA, Rho kinase, and PLD activities and on RhoA
translocation to the cytosol and dissociation from membrane-bound Rho
kinase. Parallel studies were done in cultured smooth muscle cells
expressing GTPase-resistant, constitutively active RhoV14
to distinguish the effects of RhoA inactivation and translocation and
in cells expressing phosphorylation site-deficient RhoA188
to identify the effects of PKG and PKA on targets downstream of RhoA.
The results showed that both kinases phosphorylate wild-type RhoA,
stimulate its translocation back to the cytosol, and inhibit its
activity and the activity of membrane-bound Rho kinase and PLD at the
same time as they inhibited sustained MLC20 phosphorylation and muscle contraction. In smooth muscle cells expressing
RhoA188, MLC20 phosphorylation was
significantly inhibited by PKG and PKA in the absence of RhoA
phosphorylation, suggesting the existence of a subsidiary mechanism of
relaxation downstream of RhoA.
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MATERIALS AND METHODS |
Preparation of dispersed gastric smooth muscle cells.
Smooth muscle cells were isolated from the circular muscle layer of
rabbit stomach by sequential enzymatic digestion, filtration, and
centrifugation as described previously (23-26). The
partly digested strips were washed, and muscle cells were allowed to disperse spontaneously for 30 min. Cells were harvested by filtration through 500 µm Nitex and centrifuged twice at 350 g for 10 min. For some experiments, the cells were cultured in DMEM containing 10% fetal bovine serum until they attained confluence and were then
passaged once for use in various studies (27).
Expression of dominant-negative RhoN19,
constitutively active RhoV14, and phosphorylation
site-deficient RhoA188 in cultured smooth muscle cells.
RhoA mutant cDNA was subcloned into the multiple cloning site
(EcoRI) of the eukaryotic expression vector (pEXV),
and a myc tag was incorporated into the NH2
terminus. Recombinant plasmid cDNAs (2 µg each) were transiently
transfected into cultured smooth muscle cells using Lipofectamine Plus
reagent. The cells were cotransfected with 1 µg of pGreen Lantern-1
DNA to monitor expression. Control cells were cotransfected with 2 µg
of pEXV and 1 µg of pGreen Lantern-1 DNA. Transfection efficiency was
monitored by the expression of green fluorescent protein using FITC
filters. Expression was demonstrable in over 80% of the cells.
Phosphorylation of RhoA.
Phosphorylation of RhoA was measured from the amount of 32P
incorporated into the enzyme after immunoprecipitation with specific RhoA antibody. Smooth muscle cell suspensions (10 ml; 3 × 106 cells/ml) were incubated with
[32P]orthophosphate for 4 h at 31°C.
One-milliliter samples were then incubated with various agents for 1 min in the presence or absence of specific PKA or PKG inhibitors. The
reaction was terminated with an equal volume of lysis buffer (final
concentrations in mM: 150 NaCl, 10 MgCl2, 1 PMSF, 10 EDTA,
10 Na2P2O7, 50 NaF, 0.2 Na3VO4, plus 1% Triton X-100, 0.5% SDS,
0.75% deoxycholate, 10 µg/ml leupeptin, and 100 µg/ml aprotinin).
The cell lysates were separated by centrifugation at 13,000 g for 15 min at 4°C, and they were incubated with
polyclonal RhoA antibody for 2 h at 4°C and with 40 µl of
protein A-Sepharose for another 1 h. The immunoprecipitates were
washed, extracted with Laemmli buffer, and separated by electrophoresis on 10% SDS-PAGE. After transfer to polyvinylidine difluoride
membranes, 32P-labeled RhoA was visualized by
autoradiography, and the amount of radioactivity in the band
was measured by liquid scintillation and expressed as counts/min
(cpm)/mg protein.
Assay for activated RhoA.
Activated RhoA was measured in freshly dispersed or cultured muscle
cells incubated for 3 h in low-phosphate medium containing 10 mCi
32PO4 as described previously
(27). Aliquots (2 × 106 cells) were
treated with ACh in the presence or absence of PKA and PKG activators,
and lysates were prepared. RhoA was immunoprecipitated with RhoA
antibody in freshly dispersed cells with myc antibody in
cultured cells expressing wild-type or RhoA188. The
immunoprecipitates were washed three times with lysis buffer and boiled
for 20 min at 68°C in buffer containing 5 mM EDTA, 2 mM
dithiothreitol, 0.2% SDS, 0.5 mM GTP, and 0.5 GDP. GTP and GDP were
separated on polyethylene-cellulose plates developed with 1 M
KH2PO4 (pH 3.4) and measured by autoradiography.
Activated GTP-bound RhoA was also assayed by a different technique
using Rhotekin (Rho binding domain). The GST-tagged fusion protein
corresponding to residues 7-89 of Rhotekin was used to measure selectively active GTP-bound RhoA. Muscle cell lysates (100 µg of protein) were incubated with glutathione-agarose slurry of
Rhotekin at 4°C for 45 min. The beads were washed three times with
the washing buffer containing (in mM) 50 Tris · HCl (pH 7.2), 150 NaCl, 10 MgCl2, and 0.1 PMSF, plus 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 1% Triton X-100. GTP-bound RhoA was solubilized in Laemmli sample buffer and analyzed by 15% SDS-PAGE followed by Western blot analysis and chemiluminescence.
Assay for PLD activity.
PLD activity was determined by the formation of the PLD-specific
product, phosphatidylethanol (PEt), as described previously (27). Smooth muscle cells (2 × 106
cells/ml) were incubated with [3H]myristic acid (2 µCi/ml) for 3 h and then with 150 mM ethanol for 15 min at
31°C in HEPES medium. The cells were centrifuged and resuspended in
fresh medium. After stimulation with ACh for 10 min, the reaction was
terminated with chloroform/methanol/HCl (100:200:2; vol/vol/vol), and
the organic phase was extracted and analyzed for [3H]PEt
by thin-layer chromatography. [3H]PEt was identified
using unlabeled standards and visualized under ultraviolet light at 357 nm. Spots corresponding to [3H]PEt were scraped and
counted by liquid scintillation.
Assay for Rho kinase activity.
Rho kinase activity was determined by immunokinase assay in cell
extracts as described previously (27). Rho kinase was
immunoprecipitated with Rho kinase antibody, and the immunoprecipitates
were washed with phosphorylation buffer and incubated for 5 min on ice
with 5 µg of myelin basic protein. Kinase assays were initiated by the addition of 10 µCi of [
-32P]ATP (3,000 Ci/mmol)
and 20 µM ATP, followed by incubation for 10 min at 37°C.
[32P]myelin basic protein was absorbed onto
phosphocellulose discs, and free radioactivity was removed by repeated
washings with 75 mM phosphoric acid. The amount of radioactivity on the
discs was measured by liquid scintillation.
Immunoblot analysis of RhoA, Rho kinase, and phosphorylated
MLC20.
RhoA was measured in membrane and cytosolic fractions by Western blot
analysis after treatment with ACh in the presence or absence of PKA and
PKG activators. RhoA-Rho kinase association was determined by
immunoblot in RhoA immunoprecipitates using Rho kinase antibody.
Phosphorylated MLC20 was determined by immunoblot analysis
using a phospho-Ser19-specific antibody. The proteins were
resolved by SDS-PAGE and electrophoretically transferred onto
polyvinylidene difluoride membranes. Membranes were incubated for
12 h with appropriate antibody and then incubated for 1 h
with a horseradish peroxidase-conjugated secondary antibody. The bands
were identified by enhanced chemiluminescence.
Measurement of relaxation in dispersed smooth muscle cells.
Inhibition of ACh-induced contraction (i.e., relaxation) by sodium
nitroprusside (SNP) or VIP was expressed as the decrease in maximal
cell contraction induced by 0.1 µM ACh as described previously
(24-26). A 0.5-ml aliquot of cell suspension was
added to 0.2 ml HEPES medium containing ACh alone and with SNP or VIP, and the reaction was terminated with 1% acrolein. Mean cell length of
50 muscle cells treated with various agents was measured by scanning
micrometry and compared with the length of untreated muscle cells (mean
control cell length: 116 ± 3 µm).
Materials.
[32P]orthophosphate was obtained from Amersham Pharmacia
Biotech (Piscataway, NJ); collagenase and soybean trypsin inhibitor was
from Worthington Biochemical (Freehold, NJ); Western blotting and
chromatography material was from Bio-Rad Laboratories (Hercules, CA);
RhoA antibody (Sc-119), MLC20 phospho-antibody (Sc-12896), and Rho kinase antibody (Sc-5561) were from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were obtained from Sigma (St.
Louis, MO). RhoA cDNA was a gift from Dr. Lee Slice, University of
California (Los Angeles, CA).
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RESULTS |
Phosphorylation of RhoA by PKG and PKA.
In the basal state, RhoA is mainly present in the cytosol in inactive
form bound to GDP and Rho guanine nucleotide dissociation inhibitor
(GDI) and is translocated to the plasma membrane on activation
by agonists (7, 17). Treatment of freshly dispersed smooth
muscle cells with ACh (0.1 µM), SNP (1 µM), or VIP (1 µM) alone
did not induce RhoA phosphorylation (Fig.
1). After treatment with ACh, however,
both SNP and VIP induced RhoA phosphorylation, suggesting that the
substrate is activated, membrane-bound RhoA (Fig. 1). RhoA
phosphorylation induced by SNP was concentration dependent and was
abolished by the PKG inhibitor KT5823 but was not affected by PKA
inhibitor myristoylated PKI (Figs. 2 and
3). RhoA phosphorylation induced by VIP,
which activates both PKA and PKG in gastric smooth muscle cells
(24), was partly inhibited by KT5823 (55 ± 6%
inhibition) and myristoylated PKI (43 ± 4% inhibition) and
abolished by a combination of both kinase inhibitors (93 ± 5%
inhibition) (Fig. 3).

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Fig. 1.
Phosphorylation of activated RhoA by activators of PKG
and PKA in smooth muscle cells. Freshly dispersed gastric smooth muscle
cells labeled with 32P were incubated with sodium
nitroprusside (SNP; 1 µM) or VIP (1 µM) (A), and with
8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate
(8-pCPT-cGMP; 10 µM) or 5,6-dichloro-1- -ribofuranosyl
benzimidazole 3',5'-cyclic monophosphothionate, Sp-isomer (cBIMPS; 10 µM) (B). The agents were added alone and after stimulation
with ACh (0.1 µM) for 5 min. RhoA immunoprecipitates were separated
on SDS-PAGE and identified by autoradiography. Radioactivity in the
bands was expressed as counts/min (cpm). Immunoblots were performed to
determine comigration of radioactive bands with RhoA (not shown).
Values are means ± SE of 3 experiments.
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Fig. 2.
Concentration-dependent phosphorylation of RhoA by
SNP in smooth muscle cells. Freshly dispersed gastric smooth muscle
cells labeled with 32P were stimulated with ACh (0.1 µM)
and were then treated with various concentrations of SNP.
Phosphorylation of RhoA was identified in immunoprecipitates by
autoradiography as described in MATERIALS AND METHODS (see
Fig. 1 legend). Immunoblots were performed to determine comigration of
radioactive bands with RhoA (not shown). Values are means ± SE of
4 experiments.
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Fig. 3.
Inhibition of RhoA phosphorylation by PKA and PKG
inhibitors. Freshly dispersed gastric smooth muscle cells were treated
with ACh (0.1 µM) and then with SNP (1 µM) or VIP (1 µM)
(A) and with 8-pCPT-cGMP (10 µM) or cBIMPS (10 µM)
(B) in the presence or absence of the PKG inhibitor KT5823
(1 µM) or the PKA inhibitor myristoylated PKI (1 µM).
Phosphorylation of RhoA was identified in immunoprecipitates by
autoradiography as described in MATERIALS AND METHODS.
Immunoblots were performed to determine comigration of radioactive
bands with RhoA (not shown). Values are means ± SE of 4 experiments.
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Nonhydrolyzable membrane-permeable analogs of cGMP
[8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate;
8-pCPT-cGMP] and cAMP (5,6-dichloro- 1-
-ribofuranosyl
benzimidazole 3',5'-cyclic monophosphothionate, Sp-isomer; cBIMPS) that
selectively activate PKG and PKA, respectively, also induced
phosphorylation of RhoA (Fig. 1). Phosphorylation by 8-pCPT-cGMP was
inhibited by KT5823 only, whereas phosphorylation by cBIMPS was
inhibited by myristoylated PKI only (Fig. 3). As previously shown by
direct measurements of PKA and PKG activities in gastric smooth muscle
cells, KT5823 and PKI, used at concentrations of 1 µM and less,
selectively inhibited PKG and PKA activity, respectively (24,
25).
Phosphorylation of RhoA in cultured muscle cells overexpressing
RhoA mutants.
As shown in freshly dispersed smooth muscle cells, SNP and VIP induced
RhoA phosphorylation in cultured smooth muscle cells overexpressing
wild-type RhoA but only after stimulation with ACh (Fig.
4A). However, both SNP and VIP
induced phosphorylation of RhoA in the absence of ACh in permeabilized
cultured smooth muscle cells overexpressing myc-tagged
wild-type RhoA after treatment with GTP
S or in cells overexpressing
myc-tagged constitutively active RhoV14 (Fig.
4). SNP and VIP did not induce RhoA phosphorylation in cells
overexpressing dominate-negative RhoN19 (data not shown).
Additional studies were done in cultured muscle cells overexpressing
wild-type RhoA or RhoV14. Phosphorylation was induced by
8-pCPT-cGMP or cBIMPS, and immunoprecipitation was performed separately
with myc antibody and RhoA antibody. The results with RhoA
antibody showed slightly greater phosphorylation indicative of
endogenous RhoA (Fig. 4B). The results implied that PKA and PKG preferentially phosphorylated active, membrane-bound RhoA.
Inactive, cytosolic RhoA, bound to GDI at its COOH terminus, is
probably protected from phosphorylation by PKA or PKG
(17).

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Fig. 4.
Phosphorylation of RhoA in cultured cells overexpressing wild-type
RhoA or RhoV14. A: cultured smooth muscle cells
overexpressing wild-type RhoA were treated with SNP (1 µM) or VIP (1 µM) alone or after stimulation with ACh (0.1 µM) or GTP S (1 µM). For activation with GTP S, the cells were permeabilized with
35 µg/ml of saponin for 5 min. Cells overexpressing constitutively
active RhoV14 were treated only with SNP or VIP.
Phosphorylation of RhoA was identified in myc
immunoprecipitates by autoradiography from cells labeled with
32P as described in MATERIALS AND METHODS.
Immunoblots were performed to determine comigration of radioactive
bands with RhoA (not shown). No phosphorylation was observed in cells
expressing dominate-negative RhoN19 (data not shown).
Values are means ± SE of 4 experiments. B: cultured
smooth muscle cells overexpressing wild-type RhoA (top) or
RhoV14 (bottom) were treated with ACh and with
either PKA or PKG activators. Phosphorylation of RhoA was identified
separately in immunoprecipitates obtained with either myc
antibody or RhoA antibody. Values are means ± SE of 4 experiments.
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Inhibition of RhoA activity by PKA and PKG.
Acetylcholine-stimulated RhoA activity was inhibited by SNP (82 ± 4%) and 8-pCPT-cGMP (92 ± 5%) (Figs. 5 and
6). Inhibition by either agent was
completely blocked by KT5823 but was not affected by myristoylated PKI.
ACh-stimulated RhoA activity was inhibited also by cBIMPS (73 ± 6%), and the inhibition was completely blocked by myristoylated
PKI but was not affected by KT5823 (Fig.
6). VIP inhibited ACh-stimulated RhoA
activity by 94 ± 6%; the inhibition was partly blocked by
myristoylated PKI and KT5823 and was completely blocked by a
combination of both kinase inhibitors, consistent with the ability of
VIP to activate both PKA and PKG (Fig. 5).

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Fig. 5.
Inhibition of ACh-stimulated RhoA activation by SNP and
VIP. Freshly dispersed gastric smooth muscle cells labeled with
32P were stimulated with ACh (0.1 µM) and then treated
with SNP (1 µM) (A) or VIP (1 µM) (B) in the
presence or absence of KT5823 (1 µM) or myristoylated PKI (1 µM).
Activity was determined in RhoA immunoprecipitates from the ratio of
[32P]GTP/GDP as described in MATERIALS AND
METHODS. Results are expressed as %GTP incorporation. Values are
means ± SE of 4 experiments.
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Fig. 6.
Inhibition of ACh-stimulated RhoA activation by selective
activators of PKA and PKG. Freshly dispersed gastric smooth muscle
cells labeled with 32P were stimulated with ACh (0.1 µM)
and were then treated with 8-pCPT-cGMP (10 µM) (A) or
cBIMPS (10 µM) (B) in the presence or absence of KT5823 (1 µM) or myristoylated PKI (1 µM). Activity was determined in RhoA
immunoprecipitates from the ratio of [32P]GTP/GDP as
described in MATERIALS AND METHODS. Results are expressed
as %GTP incorporation. Values are means ± SE of 4 experiments.
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Absence of PKA/PKG-dependent phosphorylation and inhibition of RhoA
activity in phosphorylation site-deficient RhoA188.
Previous studies (17, 18, 30, 31) have shown that RhoA can
be phosphorylated in vitro by PKA and PKG at Ser188 close
to its COOH terminus and that mutation of this residue to Ala prevents
phosphorylation by either kinase. In cells overexpressing myc-tagged wild-type RhoA, both SNP and VIP inhibited
ACh-stimulated activation of RhoA. Neither SNP nor VIP had any effect
on RhoA activation in cells overexpressing RhoA188 (Fig.
7A). Similar results were
obtained when RhoA activation was measured directly using the
GST-tagged fusion protein Rhotekin (Fig. 7B). The effect of
SNP and VIP on RhoA phosphorylation was examined in cells
overexpressing myc-tagged RhoA188. No
phosphorylation was observed when immunoprecipitation was performed
using myc antibody (Fig. 7C). Significant
phosphorylation of endogenous wild-type RhoA was observed when
immunoprecipitation was performed using RhoA antibody (Fig.
7C). The extent of phosphorylation was small when compared
with phosphorylation obtained when wild-type RhoA was overexpressed.
Results shown in Fig. 7 confirm that PKA and PKG phosphorylate RhoA at
Ser188 in vivo and that phosphorylation at this site is
essential for PKA- and PKG-dependent inactivation of RhoA.

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Fig. 7.
Effect of SNP and VIP on RhoA phosphorylation and
activity in cells expressing phosphorylation site-deficient
RhoA188. A: cultured smooth muscle cells
overexpressing myc-tagged wild-type RhoA or
RhoA188 were treated with SNP (1 µM) or VIP (1 µM)
after stimulation with ACh (0.1 µM). RhoA activation was determined
in RhoA immunoprecipitates from the ratio of [32P]GTP/GDP
as described in MATERIALS AND METHODS. B:
cultured smooth muscle cells overexpressing myc-tagged
wild-type RhoA or RhoA188 were treated with SNP (1 µM) or
VIP (1 µM) after stimulation with ACh (0.1 µM). RhoA activation was
determined using the GST-tagged fusion protein Rhotekin, as described
in MATERIALS AND METHODS. C: cultured smooth
muscle cells overexpressing myc-tagged RhoA188
were treated with SNP (1 µM) or VIP (1 µM) after stimulation with
ACh (0.1 µM). RhoA phosphorylation (expressed as cpm) was measured
separately in immunoprecipitates obtained with myc or RhoA
antibody. Phosphorylation detected in RhoA antibody immunoprecipitates
reflected phosphorylation of endogenous RhoA. Values are means ± SE of 3 experiments.
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Effects of PKA- and PKG-dependent phosphorylation on translocation
of RhoA.
Activation of RhoA requires translocation of inactive RhoA to the
membrane in which p115RhoGEF mediates the exchange of GTP for GDP
(7-9). We examined whether phosphorylation by PKG and PKA accelerated translocation of membrane-bound RhoA back to the cytosol. Treatment of ACh-stimulated cells with SNP or VIP decreased the amount of membrane-bound RhoA, suggesting that RhoA phosphorylation accelerated the translocation of RhoA back to the cytosol (Fig. 8). To examine further whether inhibition
of RhoA activity could be distinguished from the effect of
accelerated translocation of RhoA back to the cytosol, the
effects of SNP and VIP on translocation were measured in cells
overexpressing GTPase-resistant RhoV14, which is
predominantly membrane bound. Treatment of these cells with SNP and VIP
decreased the amount of membrane-bound RhoA, suggesting that
translocation was independent of inactivation (exchange of GDP for GTP)
(Fig. 8). Phosphorylation probably decreases the binding of wild-type
RhoA or RhoV14 to the membrane by increasing RhoA binding
to GDI. The latter appears to extract RhoA from the membrane by
competing with membrane lipids for binding to the geranylgeranylated
COOH terminus of RhoA (2, 5, 12). Consistent with this
notion, SNP and VIP had no effect on the amount of membrane-bound RhoA
in cells overexpressing phosphorylation site-deficient
RhoA188 (Fig. 8).

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Fig. 8.
Effect of PKG- and PKA-dependent phosphorylation on
translocation of RhoA. Freshly dispersed smooth muscle cells expressing
wild-type RhoA (A) and cultured smooth muscle cells
expressing RhoV14 (B) or RhoA188
(C) were stimulated with ACh (0.1 M) and were then treated
with SNP (1 µM) or VIP (1 µM). The amount of RhoA in the cytosolic
(C) and particulate (M) fractions was determined by Western blot. A
decrease in ACh-induced RhoA binding to membrane was observed with SNP
and VIP in freshly dispersed cells and in cultured cells expressing
constitutively active RhoV14 but not in cells expressing
phosphorylation site-deficient RhoA188. Values are
means ± SE of 3 experiments.
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Inhibition of ACh-stimulated RhoA/Rho kinase association by PKA and
PKG.
Translocation of RhoA back to the cytosol should prevent its
association with and activation of two membrane-bound downstream effectors, Rho kinase and PLD. Treatment of dispersed smooth muscle cells with SNP or VIP decreased ACh-stimulated association of RhoA with
Rho kinase (Fig. 9). In cultured smooth
muscle cells overexpressing RhoV14, Rho kinase was
predominantly associated with membrane-bound RhoA. Treatment of these
cells with SNP or VIP decreased RhoA/Rho kinase association
concurrently with translocation of RhoA back to the cytosol. Inhibition
of RhoA/Rho kinase association by SNP and VIP was absent in cultured
smooth muscle cells overexpressing phosphorylation site-deficient
RhoA188 (Fig. 9).

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Fig. 9.
Inhibition of RhoA/Rho kinase association by SNP and VIP.
Cultured gastric smooth muscle cells overexpressing wild-type RhoA,
RhoV14, or RhoA188 were stimulated with ACh
(0.1 µM) and were then treated with SNP (1 µM) or VIP (1 µM).
Association of RhoA with Rho kinase was analyzed by immunoblotting Rho
kinase in myc immunoprecipitates. Values are means ± SE of 3 experiments.
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Inhibition of ACh-stimulated Rho kinase and PLD activities by PKA
and PKG.
We (27) have previously shown that ACh and other
contractile agonists stimulated Rho kinase and PLD activities and that both activities were strongly inhibited in cultured smooth muscle cells
overexpressing dominate-negative RhoN19. Here we show that
ACh-stimulated Rho kinase and PLD activities in freshly dispersed
smooth muscle cells were inhibited by SNP (86 ± 4 and 81 ± 7% inhibition, respectively), and the inhibition was completely
reversed by KT5823 but not by myristoylated PKI (Fig.
10). ACh-stimulated Rho kinase and PLD
activities were inhibited also by VIP (78 ± 6 and 74 ± 4%,
respectively). Inhibition was partly reversed by KT5823
(42 ± 3 and 50 ± 6%) and myristoylated PKI (33 ± 5 and 42 ± 3%) and completely reversed by a combination of both
kinase inhibitors (Fig. 10).

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Fig. 10.
Inhibition of ACh-stimulated phospholipase D (PLD) and
Rho kinase activities by PKG and PKA activators. Freshly dispersed
gastric muscle cells were stimulated with ACh (0.1 µM) and then
treated with SNP (1 µM) or VIP (1 µM) in the presence or absence of
KT5823 (1 µM) or myristoylated PKI (1 µM). PLD activity was
measured as described in MATERIALS AND METHODS and
expressed as [3H]phosphatidylethanol
([3H]PEt cpm/mg protein). Rho kinase activity was
measured in Rho kinase immunoprecipitates as described in
MATERIALS AND METHODS and expressed as cpm/mg protein.
Values are means ± SE of 4 experiments.
|
|
Both SNP and VIP inhibited Rho kinase activity in smooth muscle cells
overexpressing constitutively active RhoV14 but had no
effect in cells overexpressing phosphorylation site-deficient RhoA188 (Fig. 11). The
pattern of inhibition paralleled the translocation of RhoA to the
cytosol and its dissociation from its targets in the membrane.

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Fig. 11.
ACh-stimulated Rho kinase activity in cells
expressing RhoA188, RhoV14, and
RhoN19. Cultured muscle cells overexpressing
RhoA188, RhoV14, or RhoN19 were
stimulated with ACh (0.1 µM) and were then treated with SNP (1 µM)
or VIP (1 µM). Rho kinase activity was measured as described in the
MATERIALS AND METHODS and expressed as cpm/mg protein.
Values are means ± SE of 4 experiments.
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Inhibition of sustained MLC20 phosphorylation and
muscle contraction by PKA and PKG.
Others (32, 35, 36) and we (27) have
previously shown that sustained MLC20 phosphorylation and
smooth muscle contraction induced by contractile agonists including ACh
were significantly inhibited by the Rho kinase inhibitor Y-27632 and/or
by overexpression of dominate-negative RhoN19. In the
present study, Rho-dependent sustained MLC20
phosphorylation and muscle contraction were measured 5 min after the
addition of ACh, that is, after termination of the initial
Rho-independent, Ca2+-dependent phase of contraction. Both
SNP and VIP inhibited ACh-induced sustained muscle cell contraction in
freshly dispersed smooth muscle cells (Fig.
12). The inhibition of muscle
contraction (i.e., relaxation) by SNP was selectively blocked by
KT5823, whereas inhibition of contraction induced by VIP was partly
blocked by KT5823 and myristoylated PKI and was completely blocked by a
combination of both kinase inhibitors (Fig. 12). ACh-induced sustained
contraction was also inhibited by 8-pCPT-cGMP and cBIMPS. Inhibition by
8-pCPT-cGMP and cBIMPS was selectively blocked by KT5823 and
myristoylated PKI, respectively (Fig.
13).

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Fig. 12.
Inhibition of ACh-induced sustained muscle contraction
by SNP and VIP. Freshly dispersed muscle cells were treated with ACh
(0.1 µM) for 5 min and were then treated with SNP (1 µM) or VIP (1 µM) in the presence or absence of KT5823 or myristoylated PKI. Muscle
contraction was measured by scanning micrometry as described in
MATERIALS AND METHODS and expressed as %decrease in
control cell length (mean control cell length: 112 ± 3 µm;
decrease in muscle cell length at 5 min: 29 ± 2 µm or 25 ± 3%). Values are means ± SE of 4-5 experiments.
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Fig. 13.
Inhibition of ACh-induced sustained muscle contraction
by selective activators of PKG and PKA. Freshly dispersed muscle cells
were treated with ACh (0.1 µM) for 5 min and then treated with
8-pCPT-cGMP (10 µM) or cBIMPS (10 µM) in the presence or absence of
KT5823 or myristoylated PKI. Muscle contraction expressed as %decrease
in control cell length: 106 ± 4 µm; decrease in muscle cell
length at 5 min: 24 ± 3 µm or 21 ± 2%. Values are
means ± SE of 4-5 experiments.
|
|
To determine whether PKG and/or PKA could inhibit sustained
MLC20 phosphorylation and muscle contraction by acting on
effectors downstream of RhoA, MLC20 phosphorylation was
measured in smooth muscle cells overexpressing wild-type RhoA and
phosphorylation site-deficient RhoA188. As shown in Figs.
7, 10, and 11, ACh-stimulated RhoA and Rho kinase activities were
similar in smooth muscle cells overexpressing wild-type RhoA and
RhoA188, suggesting that RhoA-dependent downstream
mechanisms were not affected by the mutation. VIP and SNP, as well as
cBIMPS and 8-pCPT-cGMP, virtually abolished ACh-stimulated
MLC20 phosphorylation in cells overexpressing wild-type
RhoA but only partly inhibited MLC20 phosphorylation in
cells overexpressing RhoA188 (Fig.
14). Complete inhibition of
MLC20 phosphorylation in cells overexpressing wild-type
RhoA was accompanied by complete inhibition of RhoA and Rho kinase
activities (Figs. 7 and 10), precluding the involvement of mechanisms
downstream of RhoA. However, partial inhibition by PKG and PKA in
muscle cells overexpressing RhoA188 suggested the existence
of a subsidiary inhibitory mechanism downstream of RhoA normally masked
by upstream inactivation of RhoA.

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Fig. 14.
Inhibition of ACh-induced myosin light-chain
(MLC)20 phosphorylation by SNP and VIP. Cultured smooth
muscle cells overexpressing wild-type RhoA or RhoA188 were
stimulated with ACh (0.1 µM) and then treated with SNP (1 µM) or
VIP (1 µM) (A) and with 8-pCPT-cGMP (10 µM) or cBIMPS
(10 µM) (B). MLC20 phosphorylation was
determined using phosphospecific antibody. ACh-stimulated sustained
MLC20 was partly inhibited in muscle cells expressing
RhoA188. Values are means ± SE of 3 experiments.
|
|
 |
DISCUSSION |
The crucial role of RhoA in mediating sustained
MLC20 phosphorylation and muscle contraction has focused
attention on the ability of PKA and/or PKG to induce relaxation by
inactivating RhoA and/or RhoA-dependent downstream effectors (4,
30). Two RhoA-dependent convergent pathways that lead to
inhibition of MLC phosphatase and enhanced phosphorylation of
MLC20 have been characterized. The two major effectors
within these pathways are the regulatory subunit of MLC phosphatase
MYPT1 and the PKC-dependent inhibitor of MLC phosphatase CPI-17
(3, 6, 32, 35). Here we show that, whereas RhoA is the
main target of PKG and PKA, there exists a subsidiary target downstream
of RhoA normally masked by inactivation of RhoA.
PKG and PKA induced relaxation by phosphorylating activated,
membrane-bound RhoA and accelerating its inactivation (i.e., exchange
of GDP for GTP) and its dissociation from membrane-bound substrates
(i.e., translocation back to the cytosol). In cultured smooth muscle
cells overexpressing GTPase-resistant RhoV14,
phosphorylated RhoA was translocated to the cytosol without GDP, GTP
exchange implying that inactivation and translocation of RhoA were
distinct processes. In cells overexpressing phosphorylation site-deficient RhoA188, ACh-stimulated Rho kinase activity
and MLC20 phosphorylation were similar to those in cells
overexpressing wild-type RhoA. PKA and PKG abolished Rho kinase
activity and MLC20 phosphorylation in cells overexpressing
wild-type RhoA, but they had little or no effect on Rho kinase activity
in cells overexpressing RhoA188, yet they partly inhibited
MLC20 phosphorylation. The partial inhibition of
MLC20 phosphorylation suggested the existence of an
independent inhibitory mechanism downstream of RhoA normally masked in
cells expressing wild-type RhoA by PKA- and PKG-dependent phosphorylation and inactivation of RhoA (29, 33).
These results differ from those of Sauzeau et al. (30),
who used a different experimental approach in which endogenous RhoA was
not activated by agonists but was added exogenously. In these experiments, both exogenous RhoA and RhoA188 induced
contraction in permeabilized vascular muscle strips, but only
contraction induced by RhoA was inhibited by 8-Br-cGMP, suggesting the
absence of mechanisms distal to RhoA. In contrast, Surks et al.
(33) detected a PKG-dependent mechanism that could potentially function downstream of RhoA. They showed that blocking the
interaction of the NH2-terminal leucine zipper sequence of PKG-I
with MYPT1 partly reversed the dephosphorylation of
MLC20 by 8-Br-cGMP, but they did not determine whether
phosphorylation of RhoA contributed to dephosphorylation of
MLC20.
The ability of RhoA phosphorylation to abolish MLC20
phosphorylation precludes the necessity but does not eliminate the
possibility for inactivation of targets downstream of RhoA by PKG or
PKA. A likely candidate is the Rho kinase target, MYPT1, which binds selectively to PKG-I
but can be phosphorylated by PKA at
Thr853 (10, 33). Phosphorylation by both
kinases is assumed to enhance MLC phosphatase activity by uncoupling
the catalytic subunit from membrane-bound MYPT1. The possibility that
CPI-17, which is also located downstream of RhoA and is known to
regulate MLC phosphatase activity, may, in turn, be regulated by PKG
and/or PKA has not been explored.
It is not clear how phosphorylation of RhoA at Ser188
accelerates its inactivation and translocation from the membrane. In
the resting state, inactive GDP-bound RhoA is mainly present in the cytosol bound to GDI for which it has high affinity (7,
17). GDI binds to RhoA via its geranylgeranylated COOH terminus,
close to the site of phosphorylation (Ser188) by PKA and
PKG (17). Consequently, little or no phosphorylation by
PKA or PKG was observed in the present study in the basal state. Inactivation of membrane-bound RhoA involves GTP hydrolysis and translocation of RhoA back to the cytosol via a noncatalytic
association with GDI; the latter appears to compete with membrane
phospholipids for binding to inactivated RhoA (5, 17).
Though a distinct process, translocation of RhoA back to the cytosol
was tantamount to inactivation, since it diverted RhoA from its
membrane-bound substrates, chiefly, Rho kinase and PLD. It is possible
that other components involved in the process of inactivation or
translocation are susceptible to regulatory phosphorylation by PKG and
PKA. Phosphorylation of GDI, a known substrate for PKG, is reported to
stabilize the inactive Rho-GDI complex in the cytosol (2).
In summary, this study shows that both PKG and PKA act preferentially
on RhoA to inhibit sustained MLC20 phosphorylation and muscle contraction. Both kinases phosphorylate RhoA, causing its prompt
inactivation and translocation back to the cytosol away from its
membrane-bound substrates, Rho kinase and PLD. The two pathways, Rho
kinase/MYPT1 and PLD/PKC/CPI-17, that converge to inhibit MLC
phosphatase are blocked, resulting in dephosphorylation of
MLC20 and muscle relaxation. The direct inactivation of
RhoA masks a possible action of PKG and PKA on targets downstream of RhoA.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-28300.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
K. S. Murthy, P.O. Box 908711, Medical College of Virginia,
Virginia Commonwealth University, Richmond, VA 23298 (E-mail:
skarnam{at}hsc.vcu.edu).
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.
10.1152/ajpgi.00465.2002
Received 28 October 2002; accepted in final form 17 January 2003.
 |
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