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J. Biol. Chem., Vol. 277, Issue 40, 37382-37393, October 4, 2002
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From the Departments of Medicine and
Received for publication, May 8, 2002, and in revised form, June 25, 2002
RhoA, in its active GTP-bound form, stimulates
transcription through activation of the serum-response factor (SRF). We
found that cGMP inhibited serum-induced Rho·GTP loading and
transcriptional activation of SRF-dependent reporter genes
in smooth muscle and glial cells in a cGMP-dependent
protein kinase (G-kinase)-dependent fashion. Serum
stimulation of the SRF target gene vinculin was also blocked by
cGMP/G-kinase. G-kinase activation inhibited SRF-dependent transcription induced by upstream RhoA activators including
G Rho proteins are small GTPases of the Ras family that cycle
between an active, GTP-bound form and an inactive, GDP-bound form and
regulate diverse cellular functions such as cytoskeletal organization, smooth muscle contraction, muscle and neuronal differentiation, cell
cycle progression, and gene expression (1-5). RhoA is activated downstream of multiple membrane receptors, for example the thrombin and
lysophosphatidic acid receptors, which couple to the heterotrimeric G-proteins G As a regulator of gene expression, RhoA modulates the activity of the
ubiquitous serum-response factor (SRF), which binds to the sequence
CC(A/T)6GG in the core of the serum-response element (SRE)
(3, 7). Induction of immediate early genes, e.g.
c-fos, by serum and growth factors is mediated by SRF in
cooperation with a ternary complex factor (TCF) that is controlled by
the Ras-Raf-MEK-Erk pathway (8). SRF target genes also include vinculin
and many muscle-specific genes, but promoters of these genes lack
TCF-binding sites, and SRF appears to cooperate with other cell
type-specific transcription factors (4, 5, 8, 9). Activated RhoA
increases transcription of smooth muscle-specific promoters and
SRF-dependent reporter genes, whereas RhoA inactivation by
C3 exoenzyme abolishes their transcription. RhoA activation has little
effect on the c-fos promoter, but RhoA inactivation inhibits
c-fos induction by various growth factors, indicating that
at least basal RhoA activity is required for c-fos induction (8, 9). Although the mechanism(s) by which RhoA regulates SRF activity
are incompletely defined, recent studies (8-10) suggest that depletion
of the cellular G-actin pool by RhoA-induced actin polymerization is
necessary and sufficient for stimulation of SRF-dependent transcription. Others (11) have
suggested that SRF activation by RhoA requires the cooperation of SRF
with NF-6B and C/EBP The nitric oxide (NO)/cGMP/cGMP-dependent protein kinase I
(G-kinase I) signal transduction pathway plays an important role in
vascular biology, regulating smooth muscle tone as well as SMC
proliferation and differentiation (15, 16); G-kinase I also regulates
endothelial cell permeability and motility and platelet aggregation
(16, 17). Homozygous deletion of the G-kinase I gene in mice abolishes
NO/cGMP-dependent relaxation of smooth muscle, resulting in
severe vascular and intestinal dysfunction with death at an early age
(18). G-kinase I affects smooth muscle tone by decreasing the release
of calcium from intracellular stores and by reducing calcium
sensitivity of the contractile apparatus (16); the latter effect is due
to a direct inhibition of RhoA-induced calcium sensitization of SMC
contraction by G-kinase I (19). Additional reports have confirmed
inhibition of RhoA functions by the NO/cGMP/G-kinase signaling pathway.
Insulin-induced relaxation of vascular SMCs is mediated via
NO/cGMP/G-kinase inhibition of RhoA (20), and SMC contraction induced
by constitutively active mutants of G G-kinase regulates gene expression, both at the transcriptional and
post-transcriptional level, and has been shown to increase the
expression of some genes, for example c-fos,
junB, and mitogen-activated protein kinase phosphatase 1, as
well as decrease expression of others, for example, thrombospondin,
gonadotropin-releasing hormone, and soluble guanylate cyclase (15, 23,
24). The precise mechanism of G-kinase action is not known; however, in
fibroblasts and neuronal/glial cells, transactivation of the
c-fos promoter by G-kinase I requires an intact cAMP
response element (CRE), nuclear translocation of the kinase, and
phosphorylation of the CRE-binding protein (25-27). To our knowledge,
the effect of G-kinase on RhoA-mediated gene expression has not yet
been examined.
We now show that G-kinase inhibits SRE/SRF-dependent
transcription induced by serum or by constitutively active forms of
G Materials and DNA Constructs--
The expression vector
pGEX-2T-TRBD, which encodes glutathione S-transferase (GST)
coupled to the Rho binding domain (RBD) of rhotekin, was from M. A. Schwartz, with GST-RBD affinity-purified from bacteria as described
(28). Materials for measurement of GTP/GDP bound to Rho were from
sources described previously (29, 30). A RhoA-specific antibody
(SC-179), the monoclonal anti-Myc antibody (clone 9E10, SC-40), and the
anti-Erk1/2 antibody (SC-94) were from Santa Cruz Biotechnology; the
phospho-specific anti-active Erk antibody (V6671) was from Promega. The
anti-C-terminal G-kinase I antibody (KAP-PK005) was from StressGen; the
anti-FLAG epitope antibody (F7425) and purified myosin light chain were
from Sigma, and the hybridoma line producing the anti-EE epitope
antibody was from G. Walter (31). The ROK inhibitor Y27632 was from
Calbiochem.
Human G-kinase I
The reporter constructs pSRE-Luc and pSRF-Luc were from Stratagene;
they contain five copies of the full c-fos serum-response element (5'-TAGGATGTCCATATTAGGACATC-3') or the isolated SRF-binding site (5'-GTCCATATTAGGAC-3') upstream of the minimal thymidine kinase
promoter and luciferase coding sequence, respectively.
The human vinculin promoter (38) was amplified using the primers
5'-GAGCTCGAGTACAAAGCGTGAGGGAACTG-3' and
5'-GGCAAGCTTCGTATGAAACACTGGCTTCG-3' with genomic DNA isolated
from 1321-N1 astrocytoma cells. The amplification product encoded
nucleotide Cell Culture and Transfections--
Bovine aortic smooth muscle
cells (BoASMCs) were from Cell Applications, Inc. (San Diego, CA), and
were used between passages 4 and 7; they were routinely cultured in DME
medium supplemented with 10% fetal bovine serum (FBS), penicillin,
streptomycin, and fungizone at 37 °C in a 95% air, 5%
CO2 atmosphere. Subconfluent cells in 12-well cluster
dishes were transfected with 0.6 µg of DNA and 4 µl of
PolyfectTM (Promega) in DME supplemented with 2% FBS;
24 h later they were transferred to DME without serum for 16 h, treated with 250 µM 8-chlorophenylthio-cGMP (CPT-cGMP)
for 8 h, and stimulated with 2% serum during the last 4 h.
Primary neonatal rat cardiomyocytes were provided by W. Dillmann (39);
they were plated on gelatin-coated 12-well dishes and treated as
described for BoASMCs, except that 10% FBS was used for stimulation.
Rat CS54 pulmonary arterial smooth muscle cells (provided by A. Rothman
(40)) and C6 glioma cells (provided by M. Ellisman) were transfected as
described previously (27, 41) using FuGENETM (Roche
Molecular Biochemicals) and LipofectAMINETM Plus
(Invitrogen), respectively. After transfection, cells were recovered
for 2 h in DME containing 10% FBS before they were transferred to
DME without serum for 24 h; 250 µM CPT-cGMP was
added for the last 8 h, and cells were stimulated with 10% serum
for the last 4 h as indicated. To measure the effect of G-kinase I
on the activity of endogenous RhoA, C6 cells were transfected with the
G-kinase I Reporter Gene Assays--
In BoASMCs, firefly luciferase
activity was normalized to Renilla luciferase expressed from
the co-transfected control vector pTK-RL (Promega); firefly and
Renilla luciferase activities were measured using the
Dual-luciferase Reporter Assay SystemTM from Promega. In
CS54 and C6 cells, firefly luciferase activity was normalized to
G-kinase Assay--
G-kinase activity was measured using the
synthetic peptide Kemptide and the specific cAMP-dependent
protein kinase (protein kinase A) inhibitor PKI as described previously
(26).
Western Immunoblots--
Western blots were generated and
developed using horseradish peroxidase-coupled secondary antibodies and
enhanced chemiluminescence as described previously (26).
Assays for RhoA Activity--
Cells were incubated in DME medium
without serum for 48 h, treated with 250 µM CPT-cGMP
for 2 h, and serum-stimulated for 3 min. The rhotekin pull-down
assay for Rho·GTP was performed essentially as described (28); cells
were harvested by lysis in situ, and 30 µg of GST-RBD (or
the same amount of GST) coupled to glutathione-Sepharose beads were
added to the samples. After a 1-h incubation at 4 °C with gentle
shaking, beads were washed, and bound Rho was eluted in SDS sample
buffer; samples were analyzed by SDS-PAGE and electroblotting with the
amount of RhoA estimated by Western blotting using an RhoA-specific antibody.
To measure absolute amounts of GTP and GTP + GDP bound to Rho, we
modified a procedure we developed previously to measure GTP and GTP + GDP bound to Ras and Rap1 (29, 42); this method will be described in
detail elsewhere.2 Cells were
lysed in a buffer containing 50 mM Tris-HCl, pH 7.4, 1%
Nonidet P-40, 1% CHAPS, 200 mM NaCl, 1 mM
MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.2 mM phenylmethylsulfonyl fluoride. After centrifugation at
10,000 × g for 2 min, one portion of the supernatant
was added to tubes containing 10 mM MgSO4 (to
measure GTP bound to Rho and inhibit exchange activity), and another
portion was added to tubes containing 2 µM GTP and 10 mM EDTA (to stimulate nucleotide exchange and convert all
of the Rho to the GTP-bound state, and thus measure total nucleotides
bound to Rho). To both portions was added 30 µg of GST-RBD bound to
glutathione beads, and samples were incubated for 1 h at 4 °C
with gentle shaking. After washing the beads four times in 50 mM Tris-HCl, pH 7.4, 2% Nonidet P-40, 500 mM
NaCl, 10 mM MgSO4, and two times in 20 mM TrisPO4, pH 7.4, 5 mM
MgSO4, Rho-bound GTP was released by heating the beads for
3 min at 100 °C in 5 mM TrisPO4, pH 7.4, 2 mM EDTA, 2 mM dithiothreitol. GTP eluted from
both samples was measured in a coupled enzymatic assay by conversion to
ATP in the presence of ADP and nucleoside diphosphate kinase (29). The
resulting ATP was measured by the firefly luciferase method in a
photon-counting luminometer (MGM instruments). In control experiments
to be described elsewhere,2 we showed that the nucleotide
exchange on Rho is complete and that the GTP is quantitatively eluted
from Rho bound to RBD under the conditions used. The assay is linear
with respect to cell lysate input, and the percent Rho activation,
calculated as [GTP]/[total nucleotides] bound to Rho, is
constant over a wide range of lysate input.
To measure the amount of GTP and GDP bound to EE epitope-tagged RhoA,
cells were extracted in situ as described above, and after
centrifugation, the supernatants were split in half and added to tubes
containing protein G-agarose beads coated with either an anti-EE
antibody or control mouse IgG. After 1 h of gentle shaking at
4 °C, the beads were washed, and GTP and GDP were released from the
immunoprecipitated Rho as described above. In one aliquot of the
sample, GTP was measured as described above, and in another aliquot the
sum of GDP plus GTP was measured by first converting GDP to GTP using
pyruvate kinase and phosphoenolpyruvate as described previously (30).
Control experiments demonstrated that the amount of GTP and the sum of
GTP plus GDP eluted from the immunoprecipitated RhoA was the same in
the absence or presence of soluble GST-RBD and yielded an activation
state of RhoA similar to that measured for endogenous Rho under various
conditions.2
Phalloidin Staining--
Cells were plated on glass coverslips
coated with fibronectin (10 µg/ml), serum-starved for 36 h, and
treated with 250 µM CPT-cGMP for 2 h prior to
stimulation with 10% FBS for 30 min. Cells were fixed for 10 min in
freshly prepared 1% para-formaldehyde (in
phosphate-buffered saline (PBS)), permeabilized for 10 min in PBS
containing 0.1% Triton X-100, and blocked for 1 h in PBS containing 3% bovine serum albumin prior to staining with Texas Red-labeled phalloidin (1:50, Molecular Probes). Cells were visualized using a Zeiss fluorescence microscope.
Northern Blots--
Cytoplasmic RNA was isolated and resolved by
denaturing agarose gel electrophoresis, and Northern blots were
hybridized with a radioactively labeled vinculin cDNA and a 28 S
rRNA probe as described previously (43).
ROK Assay--
C6 cells were transfected in 6-well dishes with
expression vectors encoding G-kinase I Statistical Analysis--
Unless stated otherwise, the data
presented in bar graphs represent the mean ± S.D. of at
least three independent experiments performed in duplicate. Other data
showing blots or photographs of a typical experiment represent data
that were reproduced at least three times in independent experiments.
Statistical analyses were performed using the Student's t
test. A two-tailed value of p < 0.05 was considered to
indicate statistical significance.
Effects of cGMP on Serum-induced RhoA Activation, Cytoskeletal
Changes, and SRE-dependent Transcription in
BoASMCs--
Several studies have suggested inhibition of RhoA
function by the NO/cGMP/G-kinase signal transduction pathway (19-22,
44). This inhibition could be through the following: (i) an
"upstream" mechanism, i.e. a step preceding RhoA
activation; (ii) direct RhoA phosphorylation by G-kinase; (iii) a
"downstream" mechanism, i.e. a step subsequent to RhoA
activation; or (iv) a combination of these mechanisms. Changes in the
distribution of RhoA between membrane and cytosol have been used as an
indication for Rho activation with increased membrane association
occurring when RhoA is activated, e.g. by the addition of
GTP
We therefore decided to examine the effect of cGMP on serum-induced
RhoA activation in early passage BoASMCs. The specific activity of
G-kinase in near-confluent cultures of these BoASMCs was 0.36 ± 0.20 nmol/min/mg protein. We used two methods to assess Rho activation.
In the first assay, developed by Ren et al. (28), Rho·GTP
is isolated from cell lysates using the rhotekin Rho binding domain
(RBD), and the amount of RhoA bound to the RBD is determined by
semi-quantitative Western blotting (28). Serum stimulation of BoASMCs
increased the amount of Rho·GTP, and pretreating cells with the
membrane-permeable cGMP analog CPT-cGMP almost completely prevented
this increase (Fig.
1A, upper
panel). Control blots of whole cell lysates (10% of the assay
input) demonstrated similar amounts of total RhoA and G-kinase I
present under all conditions (Fig. 1A, lower two
panels).
The second method allows quantitation of Rho activation by measuring
the amount of GTP on RBD-bound Rho in an enzymatic assay.2
As before, BoASMCs were stimulated with serum in the absence and
presence of CPT-cGMP, and one part of the sample was subjected to the
RBD pull-down to isolate Rho·GTP directly, whereas in another part of
the sample, Rho was first completely loaded with GTP prior to the RBD
pull-down to yield the total amount of Rho-bound nucleotides. Determination of GTP bound to Rho in both samples allowed us to calculate the percent Rho activation as [GTP]/[total
nucleotides] bound to Rho. By using this method, we found that 4-5%
of Rho was in the GTP-bound state in serum-starved BoASMCs, and serum stimulation increased the amount of Rho·GTP about 2.5-fold (Fig. 1B, open bars). Treating cells with CPT-cGMP
marginally decreased basal Rho activity but inhibited serum-stimulated
Rho activation by more than 50% (Fig. 1B, filled
bars; in this and all subsequent figures, open and
filled bars represent cells cultured in the absence and
presence of cGMP, respectively).
Consistent with these findings and with the findings of others in rat
aortic SMCs (19), we observed that CPT-cGMP treatment inhibited
serum-induced stress fiber formation in BoASMCs, a process that is well
known to depend on RhoA activation (Fig. 1C). Of note,
CPT-cGMP treatment also appeared to decrease the amount of stress
fibers in serum-starved cells, although we did not find a statistically
significant decrease in basal RhoCGTP under these conditions,
suggesting that cGMP/G-kinase may inhibit stress fibers by additional mechanisms.
Next, we examined the effect of CPT-cGMP on serum-induced transcription
of an SRE-dependent reporter gene. Serum stimulation of
BoASMCs increased SRE-dependent transcription about 7-fold (Fig. 1D, open bars). Treating cells with
CPT-cGMP had little effect on basal transcription of the
SRE-dependent reporter but inhibited serum-stimulated
reporter gene activity by >50% (Fig. 1D, filled
bars). At levels that did not impair cell viability, C3 exozyme,
which ADP-ribosylates and inactivates RhoA (6), completely inhibited
serum-induced transcription (Fig. 1D). Thus, as described
for other cell types (8, 46), serum induction of the pSRE-Luc appeared
to be dependent on RhoA function in BoASMCs.
Changes in Erk1/2 activity could influence SRE-dependent
transcription through regulation of TCF, which cooperates with SRF (7,
8), and the cGMP/G-kinase signaling pathway has been reported either to
inhibit, stimulate, or not affect the Raf/MEK/Erk1/2 pathway in
different cell types (43, 47-52). We therefore examined the effect of
CPT-cGMP on serum-induced Erk1/2 activation in BoASMCs, but we found no
significant effect of cGMP on Erk1/2 phosphorylation in the absence or
presence of serum stimulation (Fig. 1E shows 5- and 10-min
time points at which serum stimulation was maximal; similarly, there
was no effect of cGMP at longer incubation times). Thus, inhibition of
serum-induced transcription of pSRE-Luc by cGMP in BoASMCs is
associated with inhibition of serum-stimulated RhoA activity but not
with modulation of Erk1/2 activity, suggesting that cGMP regulates SRF
rather than TCF activity.
Effects of cGMP on SRE/SRF-dependent Transcription in
CS54 Pulmonary Artery SMCs--
To examine the effect of cGMP on
SRF-dependent transcription further, we used CS54 rat
pulmonary artery SMCs which, unlike BoASMCs, maintain a differentiated
phenotype and endogenous G-kinase I expression over multiple passages
(40). The specific activity of G-kinase in near-confluent cultures of
CS54 cells was 0.31 ± 0.52 nmol/min/mg protein. The transfection
efficiency of CS54 cells was higher than that of BoASMCs and allowed us
to compare the effect of cGMP on two different reporters as follows:
pSRE-Luc under control of the full fos SRE, and pSRF-Luc
containing an isolated SRF-binding site without the adjacent
TCF-binding site. Serum stimulation of CS54 cells increased luciferase
expression from pSRE-Luc 17-fold and from pSRF-Luc 4.5-fold (Fig.
2, A and B, show
results obtained with pSRE-Luc and pSRF-Luc, respectively). The effects
of serum were completely inhibited in cells co-transfected with an
expression vector encoding C3 exoenzyme, indicating Rho dependence
(data not shown). Although treating cells with CPT-cGMP had no effect
on basal luciferase activity, it significantly inhibited serum-induced
luciferase expression from both reporters (Fig. 2, A and
B, filled bars). As in BoASMCs, treating CS54
cells with CPT-cGMP largely prevented the serum-induced increase in
Rho·GTP levels and formation of phalloidin-stained stress fibers, and CPT-cGMP had no effect on serum-induced Erk1/2 phosphorylation (data
not shown). These results indicate that cGMP inhibits
SRF-dependent transcription as well as decreasing
serum-induced RhoA activation and stress fiber formation in smooth
muscle cells.
Effect of G-kinase on Vinculin mRNA Expression and Vinculin
Promoter Activity--
Vinculin is an SRF target gene
transcriptionally induced by serum in a RhoA-dependent
fashion (8, 10). In contrast to the fos promoter, where SRF
and TCF bind to adjacent sites and cooperate with each other, the
vinculin promoter lacks TCF binding, and serum induction of the
promoter is independent of MEK/Erk/TCF signaling (8, 38).
We examined the effect of serum and cGMP on vinculin mRNA
expression in CS54 cells and BoASMCs. Whereas cGMP did not detectably change basal vinculin mRNA levels in serum-starved cells, serum induction of vinculin mRNA was inhibited by cGMP in both cell types
(Fig. 3A shows CS54 cells, but
similar results were obtained with BoASMCs; equal loading of RNA was
demonstrated by reprobing the blot with an rRNA probe shown in the
lower panel).
We also examined the effect of cGMP/G-kinase on a luciferase reporter
construct under control of the human vinculin promoter (pVP-Luc). We
found that serum stimulation increased luciferase expression from
pVP-Luc 2-3-fold in CS54 cells and BoASMCs (Fig. 3B shows
CS54 cells), similar to the 3-fold serum induction of the promoter
reported in NIH3T3 cells (38). When CS54 or BoASMCs were pre-treated
with CPT-cGMP, serum induction of pVP-Luc was significantly inhibited.
Taken together, these results indicate that G-kinase activation
inhibits serum induction of the SRF target gene vinculin.
Effects of cGMP on Serum-induced RhoA Activation and
SRE/SRF-dependent Transcription in C6 Glial Cells--
C6
glioma cells contain very little endogenous G-kinase activity and,
therefore, provide the opportunity to determine whether the effects of
cGMP are dependent upon the presence of G-kinase, or could be
attributable to other cGMP effectors or cross-activation of
cAMP-dependent protein kinase (protein kinase A). To test
the effect of cGMP on Rho activity, we stably transfected C6 cells with
a G-kinase I expression vector and isolated a clone (C6-GKI) that
expressed G-kinase at a physiological level; the specific G-kinase
activity of C6-GKI cells was 0.38 ± 0.06 nmol/min/mg protein
compared with 0.02 ± 0.01 nmol/min/mg protein in parental C6
cells. Fig. 4A shows that CPT-cGMP
largely prevented the serum-induced increase in Rho·GTP without
lowering basal Rho·GTP levels in C6-GKI cells. When we compared the
effect of cGMP in parental and G-kinase-expressing C6 cells, we found
that cGMP had no significant effect on serum-induced Rho·GTP levels
in the G-kinase-deficient parental cells, whereas it lowered
serum-induced Rho·GTP levels by about 40% in C6-GKI cells (Fig.
4B, open bars, cells stimulated with serum;
filled bars, cells stimulated with serum in the presence of
CPT-cGMP).
Next, we compared the effect of cGMP on SRE-dependent
transcription in transiently transfected parental C6 cells
co-transfected with either empty vector or G-kinase vector. The
expression and specific activity of G-kinase I in transiently
transfected C6 cells was slightly higher than that found in the stably
transfected C6-GKI cells (41) (Fig. 4C, compare lanes
2 and 4, C6-GKI cells and C6 cells transiently
transfected with G-kinase; lanes 1 and 3 show
untransfected C6 cells and cells transfected with empty vector). In
G-kinase-deficient C6 cells co-transfected with empty vector, cGMP had
no significant effect on either basal or serum-stimulated luciferase
activity, whereas in G-kinase-expressing cells, cGMP inhibited
serum-stimulated luciferase expression by >50% (Fig. 4D).
Thus, inhibition of SRE-dependent transcription by cGMP, like inhibition of serum-stimulated Rho activation by cGMP, was strictly G-kinase-dependent and not due to cross-activation
of protein kinase A or other effects of cGMP in C6 cells.
Control experiments showed that cGMP treatment had no significant
effect on serum-induced increases in Erk-1 activity in
G-kinase-expressing C6 cells (data not shown). Basal and serum-induced
Erk1/2 phosphorylation and luciferase expression from pSRE-Luc were
inhibited by the MEK inhibitor U0126 to the same degree; cGMP treatment
further inhibited luciferase expression in serum-stimulated cells,
suggesting that the G-kinase-mediated inhibition was independent of the
Raf/MEK/Erk/TCF pathway (data not shown). Serum stimulation increased
luciferase expression from the SRF-specific reporter pSRF-Luc by
3.6 ± 1.1-fold in the absence and 2.3 ± 0.8-fold in the
presence of CPT-cGMP in G-kinase-expressing C6 cells, confirming the
inhibition of SRF function by cGMP/G-kinase.
Effect of G-kinase on SRF-dependent Transcription and
Rho Activation Induced by Constitutively Active G
Transfecting a constitutively active G
Transfection of a constitutively active p115RhoGEF construct
(
The high transfection efficiency of C6 cells allowed us to determine
the effect of constitutively active G
We also examined the effect of cGMP on G Transcriptional Effects of Constitutively Active RhoA Mutants Are
Inhibited by G-kinase--
To determine whether G-kinase may inhibit
transcriptional effects downstream of RhoA, we used two
GTPase-deficient, constitutively active RhoA mutants. Transfection of
RhoA(63L) into G-kinase-expressing C6 cells increased pSRE-Luc
transcription about 9-fold (Fig.
6A), with similar results having
been reported in other cell types (7, 46, 56). Treating the cells with
CPT-cGMP significantly inhibited RhoA(63L)-induced reporter gene
activity (Fig. 6A), although it did not affect the
expression level of the epitope-tagged RhoA(63L) (Fig. 6B,
compare lanes 3 and 4, cells transfected with EE-RhoA(63L) cultured in the absence and presence of cGMP,
respectively, to lanes 1 and 2, cells transfected
with empty vector). Moreover, cGMP treatment did not affect the high
activation state of RhoA(63L) (Fig. 6C). In
G-kinase-deficient C6 cells, CPT-cGMP treatment did not affect
RhoA(63L)-induced reporter gene activity; however, activation of
protein kinase A by CPT-cAMP inhibited RhoA(63L)-induced luciferase
expression by 55 ± 4% (n = 3). Results similar
to those described in Fig. 6A were obtained in CS54 cells,
and CPT-cGMP also inhibited the effect of RhoA(V14) on pSRE-Luc
expression (data not shown). These data suggest that cGMP/G-kinase
inhibit steps downstream of RhoA.
G-kinase, like protein kinase A, phosphorylates RhoA on
Ser188 in vitro, and it has been suggested that
protein kinase A phosphorylation of Ser188 may uncouple
RhoA from interaction with downstream effectors such as ROK (57). To
test the hypothesis that RhoA Ser188 phosphorylation by
G-kinase may mediate inhibition of RhoA-dependent transcription, we examined the effect of cGMP/G-kinase on the transcriptional effects of RhoA(63L/188A), a constitutively active RhoA
mutant that cannot be phosphorylated by G-kinase. We confirmed that
purified G-kinase phosphorylated bacterially expressed
RhoA(63L) but not RhoA(63L/188A) in vitro (data not shown).
As demonstrated in Fig. 6A, RhoA(63L/188A) induced
SRE-dependent transcription to the same degree as
RhoA(63L), and this effect was inhibited by cGMP to a similar extent as
with RhoA(63L). The expression level and activation state of
RhoA(63L/188A) were similar to that of RhoA(63L) and were not affected
by cGMP (Fig. 6, B and C). We conclude that
inhibition of RhoA-dependent transcription by cGMP/G-kinase
does not require phosphorylation of RhoA on Ser188.
Expression of a Constitutively Active RhoA Mutant Prevents the
Effects of G-kinase on Glial Cell Morphology--
In neuronal and
astroglial cells, cytoskeletal changes induced by RhoA activation cause
rounding of the cell body and retraction of cell processes, whereas
inactivation of RhoA by C3 exoenzyme leads to extension of neurite-like
processes (6, 58-60). To examine the effect of cGMP on glial cell
morphology, we transiently transfected parental C6 cells with either
empty vector or G-kinase I vector and co-transfected an expression
vector for
Next, we examined the effect of cGMP/G-kinase on RhoA(63L)-induced
changes in glial cell morphology. As described for other cell types of
glial/neuronal origin (58-60), transfection of constitutively active
RhoA(63L) induced cell rounding in G-kinase-expressing C6 cells,
producing pancake-shaped cells without extensions (Fig. 7, upper
panels, <5% of the cells showed neurite-like extensions). Treating these cells with CPT-cGMP failed to induce extension of cell
protrusions in contrast to the morphological changes observed in cells
expressing G-kinase alone (Fig. 7, lower panels, <5% of
the cGMP-treated RhoA(63L)- and G-kinase-expressing cells demonstrated neurite-like extensions). Of note, the amounts of G-kinase I and RhoA(63L) vector DNA transfected in these experiments were the same as
those used in the experiments where G-kinase activation inhibited the
transcriptional effects of RhoA(63L) (Fig. 6A). Thus, the
effects of RhoA(63L) on glial cell morphology are dominant over the
effects of G-kinase, whereas the downstream effectors of RhoA(63L)
mediating SRE-dependent transcription are sensitive to
cGMP/G-kinase inhibition.
Transcriptional Effects of RhoA Effector Kinases Are Cell
Type-specific and Inhibited by G-kinase--
The RhoA effectors
mediating SRF-dependent transcriptional activation have not
been clearly defined, although it is likely that RhoA activates SRF
through a combination of effectors and that some of the Rho effectors
involved in transcription also induce cytoskeletal changes (6, 10, 12,
13, 56, 61-63). Constitutively active forms of ROK, PKN, and PRK-2
have been shown to stimulate transcription from the fos SRE
in different cell types (56, 61, 62). We examined the effect of
constitutively active ROK, PKN, and PRK-2 on pSRE-Luc in SMCs and glial
cells and studied the effect of G-kinase. In G-kinase-expressing C6 cells, constitutively active ROK induced reporter gene activity about
8-fold, and this effect was inhibited >50% by cGMP (Fig. 8A). PKN and PRK-2 had no effect
on pSRE-Luc in C6 cells. All Rho effector kinases were expressed at
similar levels in the presence and absence of cGMP (Fig. 8B,
compare lanes 2 and 3, cells transfected with the
indicated kinase and cultured in the absence and presence of cGMP,
respectively, to lane 1, cells transfected with empty vector). In BoASMCs, PKN induced reporter gene activity about 4-fold
with PRK-2 having a small but statistically significant effect and ROK
showing a minimal effect (Fig. 8C, open bars). Both PKN- and PRK-2-induced SRE-dependent transcription was
significantly inhibited by cGMP (Fig. 8C, filled
bars). In CS54 cells, the transcriptional effect of constitutively
active ROK reached statistical significance (2.3-fold increase in
luciferase expression in the absence of cGMP) and was inhibited in the
presence of cGMP (1.6-fold increase in luciferase expression in the
presence of cGMP, mean of two independent experiments performed in
duplicate). Thus, G-kinase activation appears to interfere with the
function of several downstream effectors of RhoA involved in
transcriptional regulation.
Because ROK seemed to be one of the main RhoA effectors able to
increase SRE-dependent transcription in C6 cells, we
examined the effect of the specific ROK inhibitor Y27632 on
G
The transcriptional effects of cGMP and G-kinase I on the full-length
fos promoter require nuclear translocation of the kinase in
C6 cells (41); therefore, we examined whether G-kinase has to
translocate to the nucleus to inhibit SRF-dependent
transcription. We used a G-kinase I G-kinase Does Not Modulate the Activity of Constitutively Active
ROK--
Because G-kinase inhibited transcriptional effects of ROK in
C6 cells, we examined whether G-kinase affected the enzymatic activity
of the constitutively active ROK catalytic domain, which is independent
of Rho·GTP binding (35). We transfected C6 cells with the expression
vector encoding Myc epitope-tagged constitutively active ROK, and we
determined ROK activity in anti-Myc immunoprecipitates using myosin
light chain as a substrate. The amount of 32PO4
incorporation into myosin light chain was the same in ROK immunoprecipitates from G-kinase-expressing C6 cells cultured in the
presence and absence of cGMP (Fig. 9B, upper
panel, compare lanes 4 and 5, cells cultured
in the absence of cGMP, to lanes 6 and 7, cells
cultured in the presence of cGMP). Similar amounts of ROK were present
in all immunoprecipitates (Fig. 9B, lanes 4-7 in
the lower panel; lanes 2 and 3 show
cells transfected with a kinase-dead mutant ROK, and lane 1 shows mock-transfected cells). These results indicate that G-kinase
does not affect the enzymatic activity of the constitutively active,
Rho-independent catalytic domain of ROK. Thus, the inhibition of
ROK-induced transcription by cGMP/G-kinase appears to involve effectors
downstream of ROK.
The data presented in this study demonstrate that the
cGMP/G-kinase signaling pathway inhibits SRE/SRF-dependent
transcription through inhibition of RhoA signaling. Treating cells with
cGMP inhibited SRE-dependent transcription induced by
serum, by upstream activators of RhoA, and by constitutively active
RhoA mutants in a G-kinase-dependent fashion; it also
inhibited serum induction of the SRF target gene vinculin. G-kinase
inhibited RhoA signaling by both upstream and downstream mechanisms:
(i) G-kinase inhibited RhoA activation by serum and G Investigations from multiple laboratories have provided evidence that
activation of either the NO/cGMP/G-kinase or the cAMP/protein kinase A
pathway can antagonize RhoA functions. In vascular SMCs, G-kinase
inhibits RhoA-induced calcium sensitization of the contractile apparatus, which contributes to the vasodilatory effects of NO/cGMP (16, 19-21). In several cell types including vascular SMCs, G-kinase activation induces cytoskeletal disorganization with the break down of
stress fibers and disassembly of focal adhesion complexes (19, 22, 44,
65, 66). Activation of protein kinase A similarly inhibits
agonist-induced smooth muscle contraction and induces stress
fiber/focal adhesion breakdown in different cells; in addition, cAMP
inhibits Rho-dependent leukocyte adhesion and induces
morphological changes in neuronal and glial cells that are reciprocal
to those induced by RhoA activation (21, 57, 60, 67-69).
Several observations indicate that the cGMP- or cAMP-induced effects on
RhoA-dependent SMC contraction and cytoskeletal
organization require phosphorylation of RhoA by G-kinase or protein
kinase A. G-kinase inhibits RhoA but not RhoA(188A)-induced calcium
sensitization of SMC contraction, and cells transfected with RhoA(188A)
are protected from G-kinase-induced stress fiber disassembly (19, 22).
Similarly, transfection of RhoA(14V,188A) is more effective than
transfection of RhoA(14V) in preventing cAMP-induced cytoskeletal changes in SH-EP cells (57). In vitro, Ser188 in
RhoA is efficiently phosphorylated by both G-kinase and protein kinase
A (19, 22, 70). However, demonstration of G-kinase-induced RhoA
phosphorylation in vivo has not been possible, and
difficulties in detecting in vivo phosphorylation of RhoA by
protein kinase A have been reported (19, 71). One group (70) found that cAMP treatment induced low levels of phosphorylation of only
membrane-bound RhoA in lymphoid cells and demonstrated that cAMP
induced translocation of RhoA from the membrane to the cytosol. Whereas
protein kinase A phosphorylation of RhoA in vitro does not
affect the ability of RhoA to bind or hydrolyze GTP (70), cAMP
treatment of intact leukocytes has been shown to inhibit
chemoattractant-stimulated guanine nucleotide exchange on RhoA (67).
These findings have led to the hypothesis that protein kinase
A/G-kinase phosphorylation of RhoA may lead to membrane dissociation
and interfere with Rho/Rho-GEF association leading to decreased Rho
activity (67, 70). Recently, one group (72) demonstrated that
integrin-induced RhoA activation and membrane translocation was
inhibited by increased intracellular cAMP concentrations.
Activation of G-kinase has also been shown to prevent agonist-induced
RhoA membrane translocation suggesting inhibition of RhoA activation,
but RhoA·GTP levels were not measured in these studies (19, 20, 22,
44). We found that G-kinase activation inhibited serum-induced
RhoA·GTP loading in BoASMCs, CS54 cells, and in G-kinase-expressing
but not in G-kinase-deficient C6 cells. The results with C6 cells
exclude the possibility that cross-activation of protein kinase A by
cGMP might be responsible for the reduction in Rho·GTP levels.
Although serum stimulation of RhoA activity was modest in all three
cell types studied, it was similar to that described in other cell
types (28). Transfection of a constitutively active G Although RhoA Ser188 phosphorylation by G-kinase or protein
kinase A may induce changes in RhoA membrane association and interfere with RhoA activation by GEFs (67, 70), other workers have reported that
phosphorylation of RhoA by protein kinase A in vitro decreased the binding of RhoA to ROK, leading to the notion that RhoA
phosphorylation may interfere with RhoA association with downstream
effectors (57). We found that G-kinase inhibited the transcriptional
effects of RhoA(63L) and RhoA(63L,188A) to a similar extent without
affecting the high activation state of both mutants; these results
indicate that G-kinase inhibited downstream effects of RhoA independent
of Rho phosphorylation. G-kinase inhibition of
RhoA(63L)-dependent transcription contrasted with the
finding that RhoA(63L)-transfected C6 cells were resistant to the
morphological changes induced by cGMP; similarly, RhoA(14V)-transfected
glial cells are resistant to the morphological changes induced by cAMP (60). Although the morphological changes induced by cGMP (or cAMP) in
glial cells are similar to the changes induced by C3 exoenzyme-mediated
Rho inactivation (60, 69) (Fig. 7), cGMP treatment did not
significantly decrease basal Rho·GTP levels in serum-starved C6
cells, suggesting that the cGMP-induced morphological changes occur
independently of Rho·GTP levels. The cytoskeletal effectors
responsible for RhoA(63L)-induced morphology appeared to be less
sensitive to G-kinase inhibition than the RhoA effectors involved in
transcriptional regulation, supporting the notion that RhoA effectors
involved in cytoskeletal organization only partly overlap with
effectors involved in transcriptional regulation (12, 13).
The effectors of RhoA that mediate transcriptional activation of SRF
are incompletely defined. Confirming the observations of others (56,
61, 62), we found that constitutively active ROK, PKN, and PRK-2
activated the SRE-dependent reporter gene to different
degrees in different cell types. All three kinases have been found to
induce cytoskeletal changes (1, 35-37). Notably, the transcriptional
effects of the three constitutively active kinase constructs were
inhibited by cGMP/G-kinase, suggesting that G-kinase inhibited step(s)
distal or parallel to these Rho effectors which is (are) required for
SRF activation. We found that the catalytic activity of constitutively
active ROK was not affected by G-kinase. Sandu et al. (20)
reported that cGMP treatment of SMCs prevented thrombin-induced ROK
activation, presumably by preventing thrombin-induced RhoA activation.
Likewise, cGMP treatment of SMCs inhibited thrombin-induced RhoA
activation and Rho/ROK-dependent DNA
synthesis.3 On the other hand,
stress fiber induction by constitutively active ROK is resistant to
inhibition by cAMP/protein kinase A (57).
A recently developed model for SRF regulation via actin dynamics
suggests that reduction of the soluble G-actin pool by RhoA-induced actin polymerization may be coupled to activation of SRF (10). G-kinase
could inhibit RhoA signaling to SRF by increasing the G/F-actin ratio
through phosphorylation of cytoskeletal components distal to the known
RhoA effector kinases. We found that G-kinase activation reduced
phalloidin-stainable F-actin in serum-starved BoASMCs and CS54 cells,
without significantly decreasing basal Rho·GTP levels. Others (19,
22, 44, 65, 73) have also observed decreased F-actin staining and/or an
increase in the G-actin fraction in NO/cGMP-stimulated cells. One
possible mechanisms whereby G-kinase could inhibit actin polymerization
and increase G-actin levels independently of RhoA·GTP levels is
through phosphorylation of the vasodilator-stimulated phosphoprotein
VASP. VASP is found in focal adhesions and along F-actin fibers, and
VASP phosphorylation down-regulates its F-actin binding and
actin-polymerization-promoting activity (74). Work is underway to
examine the role of VASP phosphorylation in the regulation of
SRF-dependent transcription.
We thank Dr. W. Dillmann for providing
cardiomyocytes; Drs. A. Rothman and M. Ellisman for providing other
cell lines; Drs. J. H. Brown, A. Hall, K. Kaibuchi, Y. Ono,
M. A. Schwartz, J. Settleman, and M. Simon for providing DNA
constructs; Dr. J. Feramisco for assistance with fluorescence
microscopy and photography; and G. Nguyen for expert technical assistance.
*
This work was supported in part by United States Public
Health Service Grants GM55586 (to R. B. P.) and CA89828 (to
G. R. B.), University of California Cancer Research Coordinating
Committee Grant 6-444818-36240 (to R. B. P.), and California
Tobacco-related Disease Research Grants 9KT-0170H and 8DT-0169 (to
T. G. and D. E. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 858-534-8805;
Fax: 858-534-1421; E-mail: rpilz@ucsd.edu.
Published, JBC Papers in Press, July 15, 2002, DOI 10.1074/jbc.M204491200
2
J. C. Chen, S. Zhuang, T. H. Nguyen,
G. R. Boss, and R. B. Pilz, submitted for publication.
3
T. M. Seasholtz, unpublished observations.
The abbreviations used are:
ROK, Rho kinase;
BoASMC, bovine aortic smooth muscle cells;
CPT-cGMP, 8-chlorophenylthio-cGMP;
CRE, cAMP-response element;
DME, Dulbecco's
modified Eagle's medium;
FBS, fetal bovine serum;
GEF, guanine
nucleotide exchange factor;
G-kinase, cGMP-dependent
protein kinase;
LPA, lysophosphatidic acid;
MLC, myosin light chain;
NO, nitric oxide;
SMC, smooth muscle cell;
PBS, phosphate-buffered
saline;
SRE, serum-response element;
SRF, serum-response factor;
TCF, ternary complex factor;
RBD, rhotekin Rho binding domain;
CHAPS, 3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulfonate;
GST, glutathione S-transferase;
Erk, extracellular
signal-regulated protein kinase;
MEK, mitogen-activated protein
kinase/ERK kinase;
GTP
cGMP-dependent Protein Kinase Inhibits
Serum-response Element-dependent Transcription by
Inhibiting Rho Activation and Functions*
, Pharmacology and
the Cancer Center, University of California, San Diego,
La Jolla, California 92093-0652
ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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13 and p115RhoGEF, with
G13-induced Rho·GTP loading inhibited by G-kinase.
G-kinase had no effect on the high activation levels of RhoA(63L) or
the double mutant RhoA(63L,188A) but inhibited transcriptional
activation by these two RhoA mutants to a similar extent, suggesting an
effect downstream of RhoA and independent of RhoA Ser188
phosphorylation. Constitutively active forms of the Rho effectors Rho
kinase (ROK), PKN, and PRK-2 induced SRF-dependent
transcription in a cell type-specific fashion with ROK being the most
efficient; G-kinase inhibited transcription induced by all three
effectors without affecting ROK catalytic activity. G-kinase had no
effect on RhoA(63L)-induced morphological changes in glial cells,
suggesting distinct transcriptional and cytoskeletal effectors of RhoA.
We conclude that G-kinase inhibits SRF-dependent
transcription by interfering with RhoA signaling; G-kinase acts both
upstream of RhoA, inhibiting serum- or G13-induced Rho
activation, and downstream of RhoA, inhibiting steps distal to the Rho
targets ROK, PKN, and PRK-2.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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12 and G13 and activate
RhoA-specific guanine nucleotide exchange factors, e.g.
p115RhoGEF (1, 6). RhoA-induced cytoskeletal changes include formation
of F-actin stress fibers and focal adhesion complexes, and
RhoA-mediated smooth muscle contraction involves calcium sensitization
of the contractile apparatus (1, 2, 6). Downstream effectors of RhoA
include the Rho kinase (ROK)1
family, the protein kinase C-related kinases PRK-1 (also called PKN)
and PRK-2, mDia, and rhotekin (2).
. RhoA signaling to SRF cannot be attributed to
a single known downstream effector, and RhoA effector mutants suggest
distinct but partially overlapping effector pathways for cytoskeletal
reorganization and SRF activation (12, 13). RhoA prenylation may not be
required for induction of SRF-dependent transcription but
is required for the cytoskeletal effects of RhoA, also suggesting
different pathways (14).
12 and
G13 is RhoA-dependent and inhibited by cGMP
analogs (21). Incubation of SMCs or G-kinase-transfected HeLa cells
with NO donors or cGMP analogs prevents membrane translocation of
activated RhoA and induces disassembly of the actin cytoskeleton (19,
22). The effects of NO and cGMP on the actin cytoskeleton are absent in
G-kinase-deficient cells and restored by transfection of cells with
G-kinase I, indicating that they are mediated by G-kinase (19, 22).
G-kinase I phosphorylates RhoA in vitro on
Ser188, and the cytoskeletal effects of G-kinase activation
are inhibited by expression of a mutant RhoA(A188), suggesting that
inhibition of RhoA cytoskeletal functions by G-kinase I is at least
partially due to phosphorylation of serine 188 (19, 22).
12/G13 and p115RhoGEF in smooth muscle
cells and glial cells. G-kinase inhibited the activation of endogenous,
cellular RhoA by serum, had no effect on the high activation level of
mutant RhoA(63L), but inhibited transcriptional activation by RhoA(63L)
and RhoA(63L, 188A). These data suggest that G-kinase acts both
upstream and downstream of RhoA. Of the downstream effectors studied,
constitutively active ROK, PKN, and PRK-2 significantly activated the
SRE-dependent reporter in a cell type-specific manner, and
the transcriptional effects of all three kinases were inhibited by
G-kinase activation.
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cDNA was provided by S. Lohmann and was
expressed from the cytomegalovirus early promoter as described
previously (26). Expression vectors encoding constitutively active
G12 and G13 were from M. Simon (32), and
the vector for C3 exoenzyme was from A. Hall (7). The vector for
p115RhoGEF and the cDNAs encoding wild type RhoA and mutant
RhoA(63L) were from J. H. Brown (33, 34); the RhoA coding sequence was
excised and placed in-frame with the N-terminal epitope tag EEEEYMPME
(EE tag) in the expression vector pcDNA3. To produce double mutant
RhoA(63L/188A), the coding sequence of RhoA(63L) was amplified with
primers encoding the C-terminal Ser188 
Ala substitution
and subcloned downstream of the EE epitope into pcDNA3, after
sequence verification of the amplified product. Myc epitope-tagged
expression vectors encoding constitutively active or kinase-dead mutant
forms of the catalytic domain of ROK were from K. Kaibuchi (see Ref.
35); FLAG epitope-tagged constitutively active PKN was from Y. Ono (see
Ref. 36), and FLAG epitope-tagged constitutively active PRK-2 was from
J. Settleman (see Ref. 37).
1113 to +17 relative to the translation start site with
AAG substituted for ATG at +2
(GenBankTM accession number L04933); it was verified by DNA
sequencing and subcloned into pGL2-basic (Promega) upstream of the
luciferase coding sequence. The vinculin cDNA probe was amplified
using the primers 5'-CAAGTGTGACCGAGTGGACC-3' and
5'-TTGGTATCAATGGCTTCGTC-3' and sequenced (38).
expression vector and selected for growth in G418; a
clone was selected that stably expressed G-kinase I at an activity
level comparable with that found in vascular SMCs.
-galactosidase activity expressed from pRSV-Gal; the reporter
gene activities were measured as described before (27). Histochemical
staining for -galactosidase activity was performed with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside as
described (26).
and either the constitutively
active or inactive (kinase-dead) catalytic domain of ROK (1 µg of
each DNA). After 36 h of culture in serum-free DME, cells were
incubated for 2 h in the presence or absence of 250 µM CPT-cGMP and harvested in 0.3 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1 mM
EGTA, 1 mM dithiothreitol, 10 mM
MgCl2, 50 mM -glycerol phosphate, 1 mM Na3VO4, 100 µM
microcystin-LR, and a protease inhibitor mixture). Cleared
lysates were subjected to immunoprecipitation with 5 µg of 9E10
anti-Myc antibody and protein G-agarose. Washed beads were incubated in
50 µl of kinase buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol), containing 10 µM [
-32PO4]ATP (60 Ci/mmol)
and 80 µg/ml myosin light chain for 30 min at 30 °C. Reaction
products were analyzed by SDS-PAGE/electroblotting/autoradiography, and
the amount of ROK in the immunoprecipitates was determined by Western
blotting with anti-Myc antibody.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
S to cell lysates (6). Activation of G-kinase prevents
agonist-induced RhoA membrane translocation in several cell systems,
suggesting inhibition of RhoA activation by an upstream mechanism (19,
20, 44). However, the only study directly measuring the effect of
G-kinase activation on RhoACGTP levels found no significant effect of
cGMP on thromboxane A2-induced,
G12/13-mediated Rho activation in platelets (45).
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Fig. 1.
Effects of cGMP on serum-induced RhoA
activation, cytoskeletal changes, and SRE-dependent
transcription in BoASMCs. A, BoASMCs were incubated in
serum-free DME for 48 h, treated with 250 µM
CPT-cGMP (cGMP) for 2 h, and serum-stimulated for 3 min as
indicated. The rhotekin pull-down assay was performed as described
under "Experimental Procedures" with the amount of Rho·GTP bound
to rhotekin estimated by Western blotting using a RhoA-specific
antibody (upper panel). The total amount of RhoA present in
cell lysates is shown in the middle panel (10% of input),
and the lower panel shows the same blot probed with a
G-kinase I-specific antibody. B, BoASMCs were
serum-stimulated as described above in the absence (open
bars) or presence (closed bars) of 250 µM
CPT-cGMP. GTP and total nucleotides bound to Rho were measured as
described under "Experimental Procedures" with the percent
Rho activation calculated as [GTP]/[total nucleotides] bound to Rho
(*, p < 0.03 for the comparison between
serum-stimulated Rho activation in the absence versus
presence of cGMP). C, BoASMCs were serum-starved for 36 h and treated with 250 µM CPT-cGMP for 2 h prior to
serum stimulation for 30 min as indicated. F-actin was visualized by
staining with Texas Red-labeled phalloidin as described under
"Experimental Procedures." Magnification was ×40 in all four
panels. D, BoASMCs were transfected with 0.2 µg of
pSRE-Luc and 0.2 µg of pTK-RL as described under "Experimental
Procedures"; some cells were co-transfected with 0.05 µg of an
expression vector encoding C3 exoenzyme, and the total amount of DNA
was adjusted to 0.6 µg. After 16 h of serum starvation, cells
were incubated for 8 h in the absence (open bars) or
presence (filled bars) of 250 µM CPT-cGMP and
stimulated as indicated with 2% serum for the last 4 h prior to
harvesting. Firefly luciferase activity was normalized to
Renilla luciferase activity in each sample, and the relative
luciferase activity measured in untreated cells (1st column)
was assigned the value of 1 (*, p < 0.01).
E, BoASMCs were serum-starved for 24 h and incubated
for 2 h in the absence (lanes 1, 3, and
5) or presence (lanes 2, 4, and
6) of 250 µM CPT-cGMP; some cells were stimulated with 2% serum for 5 (lanes 3 and
4) or 10 min (lanes 5 and 6).
Duplicate Western blots of whole cell lysates were probed with an
antibody specific for activated, Tyr/Thr-phosphorylated Erk-1 and -2 (pErk, upper panel) and with an antibody that
recognizes Erk1/2 irrespective of their phosphorylation status
(Erk, lower panel).
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Fig. 2.
Effects of cGMP on
SRE/SRF-dependent transcription in CS54 pulmonary artery
SMCs. CS54 cells were transfected with 0.1 µg of pSRE-Luc
(A) or 0.2 µg of pSRF-Luc (B) and 0.1 µg of
pRSV- Gal as described under "Experimental Procedures," with the
total amount of DNA adjusted to 0.6 µg. After 16 h of serum
starvation, cells were incubated for 8 h in the absence
(open bars) or presence (filled bars) of 250 µM CPT-cGMP and stimulated as indicated with 10% FBS for
the last 4 h prior to harvesting. Firefly luciferase activities
were normalized to -galactosidase activities in each sample, and the
relative luciferase activity measured in untreated cells (1st
column) was assigned the value of 1 (*, p < 0.03 for the comparison between serum-stimulated luciferase activity in the
absence versus presence of cGMP).
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Fig. 3.
Effect of G-kinase on vinculin mRNA
expression and promoter activity. A, CS54 cells were
cultured in serum-free media for 48 h; some cells were treated
with 250 µM CPT-cGMP for a total of 4 h, and some
cells were stimulated with 10% FBS for the last 2 h prior to
harvesting as indicated. Northern blots were prepared with total
cytoplasmic RNA (20 µg per lane); blots were hybridized to a
radioactively labeled vinculin cDNA probe (upper panel)
and re-probed with an rRNA probe (lower panel) to determine
RNA loading as described under "Experimental Procedures."
B, CS54 cells were transfected with a luciferase reporter
gene under control of the human vinculin promoter (pVP-Luc, 0.2 µg)
and pRSV- Gal (internal control, 0.1 µg); cells were treated with
250 µM CPT-cGMP and serum-stimulated as indicated.
Reporter gene activities were determined as described in Fig. 2 (*,
p < 0.05 for the comparison between the absence and
presence of cGMP in this and all subsequent figures, unless stated
otherwise).
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Fig. 4.
Effects of cGMP on serum-induced RhoA
activation and SRE-dependent transcription in C6 glial
cells. A, stably transfected, G-kinase I -expressing C6
cells were incubated in serum-free DME for 48 h, treated with 250 µM CPT-cGMP for 2 h, and stimulated with 10% FBS
for 3 min as indicated. The amount of Rho·GTP bound to rhotekin was
estimated by pull-down assay and Western blotting with a RhoA-specific
antibody as described in Fig. 1A; the total amount of RhoA
present in cell lysates (10% of input) is shown in the lower
panel. B, G-kinase-deficient wild type C6 cells
(C6-Wt) and G-kinase-expressing stably transfected C6 cells
(C6-GKI) were serum-starved and then serum-stimulated as
described in A; GTP and total nucleotides bound to Rho were
measured as described in Fig. 1B. The Rho activity found in
serum-treated cells cultured in the absence of cGMP (open
bars) was assigned the value of 1 (*, p < 0.05).
C, whole cell lysates of untransfected C6 cells (lane
1), stably transfected C6-GKI cells (lane 2), and C6
cells transiently transfected with empty vector (lane 3) or
G-kinase I vector (lane 4) were analyzed by Western
blotting with a G-kinase (GK) I-specific antibody as
described under "Experimental Procedures" (40 µg of protein/lane
were loaded). D, wild type C6 cells were transfected with
0.1 µg of pSRE-Luc, 0.1 µg of pRSV-Gal, and 0.3 µg of either
empty vector or expression vector encoding G-kinase I as described
under "Experimental Procedures." After 16 h of serum
starvation, cells were incubated for 8 h in the absence
(open bars) or presence (filled bars) of 250 µM CPT-cGMP and stimulated with 10% FBS for the last
4 h prior to harvesting as indicated. Luciferase activities were
normalized to -galactosidase activities as described in Fig. 2 (*,
p < 0.03).
12,
G13, and p115RhoGEF--
Lysophosphatidic acid, the
main RhoA-stimulating component of serum, activates RhoA through the
heterotrimeric G-protein G13, whereas thrombin appears
to activate RhoA through G12 (6). Constitutively active
forms of G12 and G13 have been shown to induce SRF-dependent transcription through activation of
RhoA, which may involve activation of p115RhoGEF as an intermediate step (1, 53, 54). To define more clearly the mechanism(s) by which
G-kinase inhibits serum-induced SRF-dependent
transcription, we decided to examine the effects of G-kinase on
G12, G13, and p115RhoGEF signaling to
SRF.
13(QL) construct
into C6 glioma cells increased pSRE-Luc transcription 12-14-fold (Fig. 5A); similar results have been
reported for NIH3T3 cells (53, 54). cGMP treatment had no significant
effect on G13(QL)-stimulated reporter gene expression in
G-kinase-deficient C6 cells co-transfected with empty vector or a
kinase-dead mutant G-kinase I, but in C6 cells co-transfected with wild
type G-kinase I, cGMP inhibited G13(QL)-stimulated
luciferase expression by >50% (Fig. 5A). Similar results
were obtained when G12(QL) was substituted for
G13(QL) (data not shown). Activation of protein kinase A
in G-kinase-deficient C6 cells, e.g. by addition of 100 µM 8-chlorophenylthio-cAMP, inhibited
G13(QL)-stimulated luciferase expression from pSRE-Luc by 63 ± 2% (n = 3), similar to the effect
observed with cGMP-activated G-kinase. These results are in keeping
with the inhibition of G13(QL)-induced SMC contraction
by cAMP as well as cGMP analogs (21).
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Fig. 5.
Effect of G-kinase on
SRE-dependent transcription and Rho activation induced by
constitutively active G 13 and
p115RhoGEF. A, wild type C6 cells were transfected with 0.1 µg of pSRE-Luc, 0.1 µg of pRSV-Gal, and 0.3 µg of either empty
vector, expression vector encoding kinase-dead (GK(KD)), or
wild type G-kinase I (GK) as indicated. Some cells were
co-transfected with 0.1 µg of expression vector encoding
constitutively active G13(QL) to stimulate Rho activity.
After 16 h of serum starvation, cells were incubated for 8 h
in the absence (open bars) or presence (filled
bars) of 250 µM CPT-cGMP. Luciferase activity was
normalized to -galactosidase activity as described in Fig. 2 (*,
p < 0.01). B, C6 cells were transfected and
treated as described in A but some cells were co-transfected
with 0.1 µg of expression vector encoding constitutively active
N-p115RhoGEF to stimulate Rho activity (*, p < 0.01). C, C6 cells were transfected with expression vectors
encoding EE-tagged wild type RhoA (0.7 µg), G-kinase I (1 µg),
and 0.3 µg of either empty vector, vector encoding constitutively
active G13(QL), or N-p115RhoGEF as indicated. After
16 h of serum starvation, cells were incubated for 8 h in the
absence (open bars) or presence (filled bars) of
250 µM CPT-cGMP. Cell lysates were subjected to
immunoprecipitation with an anti-EE antibody, and GTP and GDP bound to
RhoA in the immunoprecipitates were measured as described under
"Experimental Procedures." Percent Rho activation was calculated as
[GTP]/[GTP + GDP] bound to RhoA (*, p < 0.01).
N-p155-RhoGEF (33)) into C6 cells increased
SRE-dependent transcription about 10-fold, and this effect
was inhibited by cGMP in a G-kinase-dependent fashion (Fig.
5B). For comparison, transfection of full-length, wild type
p115RhoGEF increased reporter gene expression only 2.7 ± 0.4-fold
in the absence and 1.7 ± 0.2-fold in the presence of cGMP in
G-kinase-expressing C6 cells (n = 3).
13 and
N-p115RhoGEF on RhoA activity and examine the effect of
cGMP/G-kinase. We co-transfected C6 cells with G-kinase I and EE
epitope-tagged RhoA together with either empty vector, constitutively
active G13, or N-p115RhoGEF, and we measured the
amount of GTP and GDP bound to RhoA in anti-EE immunoprecipitates. The
results are shown in Fig. 5C; transfection of constitutively
active G13 or N-p115RhoGEF increased RhoA activation
from 4 to about 12%. Treating cells with CPT-cGMP significantly inhibited G13-stimulated Rho activity but did not affect
basal or N-p115RhoGEF-stimulated Rho activity. The effect of cGMP on G13-stimulated Rho activity is in keeping with the
inhibitory effect of cGMP on G13-stimulated
SRE-dependent transcription (Fig. 5A) and the
inhibitory effect of cGMP on G13-stimulated SMC
contraction (21). However, the lack of an effect of cGMP on
N-p115RhoGEF-stimulated Rho activity was surprising. For comparison, transfection of full-length, wild type p115RhoGEF increased the amount
of GTP bound to Rho only 1.8-fold in the absence and 1.4-fold in the
presence of cGMP in G-kinase-expressing C6 cells (n = 2; the difference between the absence and presence of cGMP did not reach statistical significance). These results suggest that
cGMP/G-kinase inhibition of serum- and G13-stimulated
Rho activation may involve functional inhibition of a limiting,
endogenous RhoGEF, which could be overcome by overexpression of
constitutively active N-p115RhoGEF. These results further suggest
that the G-kinase inhibition of N-p115RhoGEF-stimulated
transcription is not due to inhibition of RhoA activation but rather
involves inhibition of downstream effects of RhoA.
12(QL)- and
N-p115RhoGEF-stimulated SRE-dependent transcription in
different types of muscle cells expressing endogenous G-kinase. In
BoASMCs, CS54 pulmonary artery SMCs, and primary neonatal rat
cardiomyocytes, transfection of constitutively active
G12 increased luciferase expression from pSRE-Luc about
7-9-fold, and treating cells with CPT-cGMP inhibited this effect by
~50% (Table I). Similar results were
obtained with G13 (data not shown). Transfection of
constitutively active N-p115RhoGEF increased
SRE-dependent transcription 4-8-fold with about 40%
inhibition in cGMP-treated cells. All three cell types contain
comparable amounts of G-kinase activity (described above for BoASMCs
and CS54 cells, and in Ref. 55 for cardiomyocytes). Of note, the
effects of cGMP on serum- and G12-induced transcription diminished progressively with increasing passage of BoASMCs and were
lost in cells cultured for >11 passages; high passage BoASMCs cease to
express G-kinase I (15). Thus, the effect of cGMP on G12,13- and N-p115RhoGEF-stimulated transcription
occurs in cells at physiological levels of G-kinase I.
Effect of cGMP on SRE-dependent transcription induced by
constitutively active G12 and N-p115RhoGEF in muscle
cells
Gal and were co-transfected with either empty
vector or expression vectors encoding constitutively active
G12 or N-p115-RhoGEF. After 24 h of serum
starvation, cells were cultured for 8 h in the presence (+cG) or
absence (cG) of 250 µM CPT-cGMP as indicated. Firefly
luciferase activity was normalized to either Renilla
luciferase activity (for BoASMCs) or -galactosidase activity, and
the relative luciferase activity measured in untreated cells
transfected with empty vector was assigned a value of 1.
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Fig. 6.
Transcriptional effects of constitutively
active RhoA mutants are inhibited by G-kinase. A, C6 cells
were transfected with 0.1 µg of pSRE-Luc, 0.1 µg of pRSV- Gal,
0.3 µg of G-kinase I vector, and 0.1 µg of either empty vector
(EV) or expression vector encoding constitutively active EE
epitope-tagged RhoA(63L) or RhoA(63L/188A) as indicated. After 16 h of serum starvation, cells were incubated for 8 h in the absence
(open bars) or presence (filled bars) of 250 µM CPT-cGMP, and reporter gene activity was measured as
described in Fig. 2. B, cells were transfected with empty
vector (lanes 1 and 2), EE-RhoA(63L) (lanes
3 and 4), or EE-RhoA(63L/188A) (lanes 5 and
6) and incubated in the absence (lanes 1,
3, and 5) or presence (lanes 2,
4, and 6) of CPT-cGMP as described in A.
Whole cell lysates were analyzed by SDS-PAGE/electroblotting, and blots
were developed with an anti-EE antibody. C, cells were
transfected with EE epitope-tagged wild type RhoA (Rhowt),
RhoA(63L), or RhoA(63L,188A) and treated as described in A,
but cell lysates were subjected to immunoprecipitation with anti-EE
antibody; GTP and GDP bound to the Rho immunoprecipitates were measured
as described under "Experimental Procedures" with percent Rho
activation calculated as [GTP]/[GTP + GDP] bound to Rho.
-galactosidase to identify transfected cells. Cells
transfected with empty vector remained flat, elongated, and polygonal,
in the absence and presence of cGMP (Fig. 7).
In contrast, cells transfected with G-kinase vector and treated with
cGMP became small and dense, with >80% of the cell population
developing neurite-like extensions greater than one cell diameter in
length (Fig. 7). Cells in which RhoA was inhibited by C3 exoenzyme
transfection showed similar morphological changes as those induced by
G-kinase activation, suggesting that some of the same effector
molecules may be involved (Fig. 7, C3 exoenzyme induced neurite-like
extensions in >90% of the cell population).
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Fig. 7.
Expression of a constitutively active RhoA
mutant prevents the effects of G-kinase on glial cell morphology.
Wild type C6 cells were transfected with 0.2 µg of pRSV- Gal and
either empty vector (0.4 µg), G-kinase (GK) I vector
(0.3 µg), G-kinase vector (0.3 µg) plus RhoA(63L) (0.1 µg), or an
expression vector encoding C3 exoenzyme (0.05 µg) as indicated; the
total amount of DNA was adjusted to 0.6 µg. Cells were maintained in
DME with 10% FBS for 24 h and incubated for 1 h in the
absence (upper panels) or presence (lower panels)
of 250 µM CPT-cGMP prior to fixing and staining for
-galactosidase activity to visualize successfully transfected cells.
All cells were photographed at ×40 magnification.
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Fig. 8.
Transcriptional effects of RhoA effector
kinases are cell type-specific and inhibited by G-kinase. A,
C6 cells were transfected with 0.1 µg of pSRE-Luc, 0.1 µg of
pRSV- Gal, and 0.3 µg of G-kinase I as described in Fig.
4D. Cells were co-transfected either with empty vector
(EV) or constitutively active ROK, PKN, or PRK-2, and after
16 h of serum starvation were incubated for 8 h in the
absence (open bars) or presence (filled bars) of
250 µM CPT-cGMP. Luciferase activity was measured as
described in Fig. 2 and was normalized to -galactosidase activity
(*, p < 0.001). B, C6 cells were
transfected and treated as described in A; whole cell
lysates were analyzed by SDS-PAGE/electroblotting, and blots were
developed with an anti-Myc (upper panel) or anti-FLAG
(lower two panels) epitope antibody to examine expression
levels of the epitope-tagged constitutively active ROK, PKN, or PRK-2,
respectively. Lane 1, untreated cells transfected with empty
vector; lanes 2 and 3, cells transfected with
expression vector encoding the indicated epitope-tagged kinase and
incubated for 8 h in the absence or presence of 250 µM CPT-cGMP, respectively. As is apparent from lane
1, both anti-epitope antibodies cross-reacted with other cellular
proteins. C, BoASMCs were transfected with 0.2 µg of
pSRE-Luc and 0.2 µg of pTK-RL as described in Fig. 1D;
cells were co-transfected with 0.1 µg of either empty vector or
expression vectors encoding constitutively active ROK, PKN, or PRK-2 as
indicated. After 16 h of serum starvation, cells were incubated
for 8 h in the absence (open bars) or presence
(filled bars) of 250 µM CPT-cGMP, and firefly
and Renilla luciferase activities were measured as described
in Fig. 1D (*, p < 0.05 for the comparison
between cells cultured in the absence versus presence of
cGMP).
13-and RhoA(63L)-induced reporter gene expression in
these cells (Fig. 9A). By itself, Y27632 inhibited G13- and RhoA(63L)-induced
transcription to a similar extent as did cGMP; the effects of Y27632
and cGMP combined appeared to be additive. These results suggest that
cGMP/G-kinase inhibit downstream effectors of RhoA in addition to ROK
in C6 cells, as suggested by the findings in other cell types (Fig. 8C) (12, 13, 61, 62).
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Fig. 9.
Effect of G-kinase on ROK-inhibited cells and
on the catalytic activity of constitutively active ROK. A,
C6 cells were transfected with pSRE-Luc, pRSV- Gal, and G-kinase I
as described in Fig. 4D and co-transfected with 0.1 µg of
either empty vector, constitutively active G13(QL), or
RhoA(63L) as indicated. Cells were incubated in serum-free medium for a
total of 24 h and treated as indicated with the ROK inhibitor
Y27632 (10 µM) for the last 8 h and with CPT-cGMP
(250 µM) for the last 7 h prior to measuring
luciferase and -galactosidase activities as described in Fig. 2.
G13- and RhoA(63L)-stimulated luciferase activities were
compared in the absence versus presence of cGMP (*,
p < 0.03) or Y27632 (
, p < 0.03);
luciferase activities in Y27632-treated cells were compared in the
absence versus presence of cGMP (#, p < 0.05). B, C6 cells were transfected with expression vectors
encoding G-kinase I and either Myc-tagged constitutively active ROK
(lanes 4-7) or Myc-tagged inactive, kinase-dead ROK
(lanes 2 and 3) as described under
"Experimental Procedures." Mock-transfected cells are shown in
lane 1. After 36 h of culture in serum-free DME, cells
were incubated for 2 h in the absence (lanes 1,
2, 4, and 5) or presence (lanes
3, 6, and 7) of 250 µM
CPT-cGMP. Cleared cell lysates were subjected to immunoprecipitation
with an anti-Myc antibody, and the immunoprecipitates were incubated
with [-32PO4]ATP and myosin light chain
(MLC) as described under "Experimental Procedures";
reaction products were analyzed by
SDS-PAGE/electroblotting/autoradiography to determine the amount of
32PO4 incorporation into MLC (p-MLC,
upper panel). The amount of ROK in the immunoprecipitates
was determined by Western blotting using the anti-Myc antibody
(lower panel, the upper band represents ROK and
the lower band immunoglobulin heavy chain).
variant with two amino acid
substitutions in the nuclear localization signal that prevent nuclear
translocation and fos promoter transactivation
(GKI(K407A/R409A)) without affecting kinase activity (26); we
found that this G-kinase mutant mediated inhibition of RhoA(63L)- and
ROK-induced pSRE-Luc expression by cGMP as efficiently as wild type
G-kinase (data not shown). These results suggest that G-kinase acts on
extranuclear targets to inhibit SRF-dependent transcription.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
13;
and (ii) G-kinase inhibited transcription induced by constitutively
active RhoA mutants and by the constitutively active Rho effector
kinases ROK, PKN, and PRK-2. These effects were observed both in cells
of smooth muscle and neuronal origin. The regulation of
RhoA-dependent transcription by the NO/cGMP/G-kinase
pathway may play an important role during smooth muscle differentiation
and cardiac hypertrophy, because recent studies have demonstrated that
RhoA signaling to SRF is required for expression of smooth
muscle-specific genes during muscle differentiation and is involved in
transcriptional responses associated with cardiac hypertrophy (4, 5, 9,
61, 64). On the other hand, RhoA activation has little effect on the
intact fos promoter, consistent with the finding that
cGMP/G-kinase activates the fos promoter and increases
c-fos mRNA expression in serum-starved cells through
RhoA/SRE-independent mechanisms (8, 23, 27).
13
increased RhoA·GTP loading more dramatically, and the effect was
inhibited by G-kinase. Transfection of the constitutively active
N-p115RhoGEF increased RhoA·GTP loading to a similar extent as
that observed with G13, but in contrast to
G13, the effect of this GEF construct was resistant to
G-kinase inhibition. These results suggest that G-kinase may inhibit
the function of an endogenous GEF responsible for RhoA activation by
serum and G13, e.g. by interrupting the
interaction of the GEF with its upstream regulator or with RhoA. An
overexpressed, constitutively active N-p115RhoGEF could be resistant
to G-kinase inhibition because the GEF activity is independent of
G-protein activation, the altered GEF is no longer sensitive to
G-kinase, or high concentrations of active GEF may force an interaction
with RhoA irrespective of G-kinase phosphorylation. Compared with
N-p115RhoGEF, transfection of full-length, wild type p115RhoGEF
increased RhoA·GTP loading significantly less, and its effect
appeared to be inhibited by cGMP/G-kinase. However, activation of RhoA
by wild type p115 was marginal, and the effect of cGMP did not reach
statistical significance. More work is required to identify the exact
mechanism(s) by which G-kinase inhibits Rho activation.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
S, guanosine
5'-3-O-(thio)triphosphate.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Takai, Y.,
Sasaki, T.,
and Matozaki, T.
(2001)
Physiol. Rev.
81,
153-208 2.
Bishop, A. L.,
and Hall, A.
(2000)
Biochem. J.
348,
241-255[CrossRef][Medline]
[Order article via Infotrieve]
3.
Bar-Sagi, D.,
and Hall, A.
(2000)
Cell
103,
227-238[CrossRef][Medline]
[Order article via Infotrieve]
4.
Wei, L.,
Zhou, W.,
Croissant, J. D.,
Johansen, F. E.,
Prywes, R.,
Balasubramanyam, A.,
and Schwartz, R. J.
(1998)
J. Biol. Chem.
273,
30287-30294 5.
Sobue, K.,
Hayashi, K.,
and Nishida, W.
(1999)
Mol. Cell. Biochem.
190,
105-118[CrossRef][Medline]
[Order article via Infotrieve]
6.
Seasholtz, T. M.,
Majumdar, M.,
and Brown, J. H.
(1999)
Mol. Pharmacol.
55,
949-956 7.
Hill, C. S.,
Wynne, J.,
and Treisman, R.
(1995)
Cell
81,
1159-1170[CrossRef][Medline]
[Order article via Infotrieve]
8.
Gineitis, D.,
and Treisman, R.
(2001)
J. Biol. Chem.
276,
24531-24539 9.
Mack, C. P.,
Somlyo, A. V.,
Hautmann, M.,
and Somlyo, A. P.
(2001)
J. Biol. Chem.
276,
341-347 10.
Sotiropoulos, A.,
Gineitis, D.,
Copeland, J.,
and Treisman, R.
(1999)
Cell
98,
159-169[CrossRef][Medline]
[Order article via Infotrieve]
11.
Montaner, S.,
Perona, R.,
Saniger, L.,
and Lacal, J. C.
(1999)
J. Biol. Chem.
274,
8506-8515 12.
Sahai, E.,
Alberts, A. S.,
and Treisman, R.
(1998)
EMBO J.
17,
1350-1361[CrossRef][Medline]
[Order article via Infotrieve]
13.
Zohar, M.,
Teramoto, H.,
Katz, B.-Z.,
Yamada, K. M.,
and Gutkind, J. S.
(1998)
Oncogene
17,
991-998[CrossRef][Medline]
[Order article via Infotrieve]
14.
Allal, C.,
Favre, G.,
Couderc, B.,
Salicio, S.,
Sixou, S.,
Hamilton, A. D.,
Sebti, S. M.,
Lajoie-Mazenc, I.,
and Pradines, A.
(2000)
J. Biol. Chem.
275,
31001-31008 15.
Lincoln, T. M.,
Dey, N.,
and Sellak, H.
(2001)
J. Appl. Physiol.
91,
1421-1430 16.
Ruth, P.
(1999)
Pharmacol. Ther.
82,
355-372[CrossRef][Medline]
[Order article via Infotrieve]
17.
Vaandrager, A. B.,
and De Jonge, H. R.
(1996)
Mol. Cell. Biochem.
157,
23-30[Medline]
[Order article via Infotrieve]
18.
Pfeifer, A.,
Klatt, P.,
Massberg, S., Ny, L.,
Sausbier, M.,
Hirnei , C.,
Wang, G.-X.,
Korth, M.,
Aszòdi, A.,
Andersson, K.-E.,
Krombach, F.,
Mayerhofer, A.,
Ruth, P.,
Fassler, R.,
and Hofmann, F.
(1998)
EMBO J.
17,
3045-3051[CrossRef][Medline]
[Order article via Infotrieve]
19.
Sauzeau, V.,
Jeune, H. L.,
Cario-Toumaniantz, C.,
Smolenski, A.,
Lohmann, S. M.,
Bertoglio, J.,
Chardin, P.,
Pacaud, P.,
and Loirand, G.
(2000)
J. Biol. Chem.
275,
21722-21729 20.
Sandu, O. A.,
Ito, M.,
and Begum, N.
(2001)
J. Appl. Physiol.
91,
1475-1482 21.
Gohla, A.,
Schultz, G.,
and Offermanns, S.
(2000)
Circ. Res.
87,
221-227 22.
Sawada, N.,
Itoh, H.,
Yamashita, J.,
Doi, K.,
Inoue, M.,
Masatsugu, K.,
Fukunaga, Y.,
Sakaguchi, S.,
Sone, M.,
Yamahara, K.,
and Yurugi, T.
(2001)
Biochem. Biophys. Res. Commun.
280,
798-805[CrossRef][Medline]
[Order article via Infotrieve]
23.
Eigenthaler, M.,
Lohmann, S. M.,
Walter, U.,
and Pilz, R. B.
(1998)
Rev. Physiol. Biochem. Pharmacol.
135,
174-209
24.
Filippov, G.,
Bloch, D. B.,
and Bloch, K. D.
(1997)
J. Clin. Invest.
100,
942-948[Medline]
[Order article via Infotrieve]
25.
Gudi, T.,
Huvar, I.,
Meinecke, M.,
Lohmann, S. M.,
Boss, G. R.,
and Pilz, R. B.
(1996)
J. Biol. Chem.
271,
4597-4600 26.
Gudi, T.,
Lohmann, S. M.,
and Pilz, R. B.
(1997)
Mol. Cell. Biol.
17,
5244-5254[Abstract]
27.
Gudi, T.,
Casteel, D. E.,
Vinson, C.,
Boss, G. R.,
and Pilz, R. B.
(2000)
Oncogene
19,
6324-6333[CrossRef][Medline]
[Order article via Infotrieve]
28.
Ren, X.-D.,
Kiosses, W. B.,
and Schwartz, M. A.
(1999)
EMBO J.
18,
578-585[CrossRef][Medline]
[Order article via Infotrieve]
29.
Scheele, J. S.,
Rhee, J. M.,
and Boss, G. R.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
1097-1100 30.
Sharma, P. M.,
Egawa, K.,
Huang, Y.,
Martin, J. L.,
Huvar, I.,
Boss, G. R.,
and Olefsky, J. M.
(1998)
J. Biol. Chem.
273,
18528-18537 31.
Grussenmeyer, T.,
Scheidtmann, K. H.,
Hutchinson, M. A.,
Eckhart, W.,
and Walter, G.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
7952-7954 32.
Althoefer, H.,
Eversole-Cire, P.,
and Simon, M. I.
(1997)
J. Biol. Chem.
272,
24380-24386 33.
Majumdar, M.,
Seasholtz, T. M.,
Buckmaster, C.,
Toksoz, D.,
and Brown, J. H.
(1999)
J. Biol. Chem.
274,
26815-26821 34.
Sah, V. P.,
Minamisawa, S.,
Tam, S. P., Wu, T.,
Dorn, G. W.,
Ross, J.,
Chien, K. R.,
and Brown, J. H.
(1999)
J. Clin. Invest.
103,
1627-1634[Medline]
[Order article via Infotrieve]
35.
Amano, M.,
Chihara, K.,
Kimura, K.,
Fukata, Y.,
Nakamura, N.,
Matsuura, Y.,
and Kaibuchi, K.
(1997)
Science
275,
1308-1311 36.
Dong, L. Q.,
Landa, L. R.,
Wick, M. J.,
Zhu, L.,
Mukai, H.,
Ono, Y.,
and Liu, F.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5089-5094 37.
Vincent, S.,
and Settleman, J.
(1997)
Mol. Cell. Biol.
17,
2247-2256[Abstract]
38.
Moiseyeva, E. P.,
Weller, P. A.,
Zhidkova, N. I.,
Corben, E. B.,
Patel, B.,
Jasinska, I.,
Koteliansky, V. E.,
and Critchley, D. R.
(1993)
J. Biol. Chem.
268,
4318-4325 39.
Rohrer, D. K.,
Hartong, R.,
and Dillmann, W. H.
(1991)
J. Biol. Chem.
266,
8638-8646 40.
Rothman, A.,
Kulik, T. J.,
Taubman, M. B.,
Berk, B. C.,
Smith, W. J.,
and Nadal-Ginard, B.
(1992)
Circulation
86,
1977-1986 41.
Gudi, T.,
Hong, G. K. P.,
Vaandrager, A. B.,
Lohmann, S. M.,
and Pilz, R. B.
(1999)
FASEB J.
13,
2143-2152 42.
von Lintig, F. C.,
Pilz, R. B.,
and Boss, G. R.
(2000)
Oncogene
19,
4029-4034[CrossRef][Medline]
[Order article via Infotrieve]
43.
Suhasini, M., Li, H.,
Lohmann, S. M.,
Boss, G. R.,
and Pilz, R. B.
(1998)
Mol. Cell. Biol.
18,
6983-6994 44.
Clancy, R. M.,
Rediske, J.,
Tang, X.,
Nijher, N.,
Frenkel, S.,
Philips, M.,
and Abramson, S. B.
(1997)
J. Clin. Invest.
100,
1789-1796[Medline]
[Order article via Infotrieve]
45.
Gratacap, M.-P.,
Payrastre, B.,
Nieswandt, B.,
and Offermanns, S.
(2001)
J. Biol. Chem.
276,
47906-47913 46.
Wang, Y.,
Falasca, M.,
Schlessinger, J.,
Malstrom, S.,
Tsichlis, P.,
Settleman, J., Hu, W.,
Lim, B.,
and Prywes, R.
(1998)
Cell Growth Differ.
9,
513-522[Abstract]
47.
Chrisman, T. D.,
and Garbers, D. L.
(1999)
J. Biol. Chem.
274,
4293-4299 48.
Yu, S.-M.,
Hung, L.-M.,
and Lin, C.-C.
(1997)
Circulation
5,
1269-1277
49.
Komalavilas, P.,
Shah, P. K., Jo, H.,
and Lincoln, T. M.
(1999)
J. Biol. Chem.
274,
34301-34309 50.
Silberbach, M.,
Gorenc, T.,
Hershberger, R. E.,
Stork, P. J.,
Steyger, P. S.,
and Roberts, C. T., Jr.
(1999)
J. Biol. Chem.
274,
24858-24864 51.
Gu, M.,
Lynch, J.,
and Brecher, P.
(2000)
J. Biol. Chem.
275,
11389-11396 52.
Kim, S. O., Xu, Y.,
Katz, S.,
and Pelech, S.
(2000)
Biochim. Biophys Acta
1496,
277-284[Medline]
[Order article via Infotrieve]
53.
Fromm, C.,
Coso, O. A.,
Montaner, S., Xu, N.,
and Gutkind, J. S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
91,
10098-10103
54.
Mao, J.,
Yuan, H.,
Xie, W.,
and Wu, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12973-12976 55.
Wollert, K. C.,
Fiedler, B.,
Gambaryan, S.,
Smolenski, A.,
Heineke, J.,
Butt, E.,
Trautwein, C.,
Lohmann, S. M.,
and Drexler, H.
(2002)
Hypertension
39,
87-92 56.
Chihara, K.,
Amano, M.,
Nakamura, N.,
Yano, T.,
Shibata, M.,
Tokui, T.,
Ichikawa, H.,
Ikebe, R.,
Ikebe, M.,
and Kaibuchi, K.
(1997)
J. Biol. Chem.
272,
25121-25127 57.
Dong, J.-M.,
Leung, T.,
Manser, E.,
and Lim, L.
(1998)
J. Biol. Chem.
273,
22554-22562 58.
Majumdar, M.,
Seasholtz, T. M.,
Goldstein, D.,
Lanerolle, P.,
and Brown, J. H.
(1998)
J. Biol. Chem.
273,
10099-10106 59.
Kranenburg, O.,
Poland, M.,
van Horck, F. P.,
Drechsel, D.,
Hall, A.,
and Moolenaar, W. H.
(1999)
Mol. Biol. Cell
10,
1851-1857 60.
Ramakers, G. J.,
and Moolenaar, W. H.
(1998)
Exp. Cell Res.
245,
252-262[CrossRef][Medline]
[Order article via Infotrieve]
61.
Morissette, M. R.,
Sah, V. P.,
Glembotski, C. C.,
and Brown, H.
(2000)
Am. J. Physiol.
278,
H1769-H1774
62.
Quilliam, L. A.,
Lambert, Q. T.,
Mickelson-Young, L. A.,
Westwick, J. K.,
Sparks, A. B.,
Kay, B. K.,
Jenkins, N. A.,
Gilbert, D. J.,
Copeland, N. G.,
and Der, C. J.
(1996)
J. Biol. Chem.
271,
28772-28776 63.
Reynaud, C.,
Fabre, S.,
and Jalinot, P.
(2000)
J. Biol. Chem
275,
33962-33968 64.
Beqaj, S.,
Jakkaraju, S.,
Mattingly, R. R.,
Pan, D.,
and Schuger, L.
(2002)
J. Cell Biol.
156,
893-903 65.
Ingram, A. J.,
James, L.,
Cai, L.,
Thai, K., Ly, H.,
and Scholey, J. W.
(2000)
J. Biol. Chem.
275,
40301-40306 66.
Yao, J.,
Schoecklmann, H. O.,
Prols, F.,
Gauer, S.,
and Sterzel, R. B.
(1998)
Kidney Int.
53,
598-608[CrossRef][Medline]
[Order article via Infotrieve]
67.
Laudanna, C.,
Campbell, J. J.,
and Butcher, E. C.
(1997)
J. Biol. Chem.
272,
24141-24144 68.
Kreisberg, J. I.,
Ghosh-Choudhury, N.,
Radnik, R. A.,
and Schwartz, M. A.
(1997)
Am. J. Physiol.
273,
F283-F288[Medline]
[Order article via Infotrieve]
69.
Tigyi, G.,
Fischer, D. J.,
Sebok, A.,
Marshall, F.,
Dyer, D. L.,
and Miledi, R.
(1996)
J. Neurochem.
66,
549-558[Medline]
[Order article via Infotrieve]
70.
Lang, P.,
Gesbert, F.,
Delespine-Carmagnat, M.,
Stancou, R.,
Pouchelet, M.,
and Bertoglio, J.
(1996)
EMBO J.
15,
510-519[Medline]
[Order article via Infotrieve]
71.
Essler, M.,
Staddon, J. M.,
Weber, P. C.,
and Aepfelbacher, M.
(2000)
J. Immunol.
164,
6543-6549 72.
O'Connor, K. L.,
Nguyen, B.-K.,
and Mercurio, A. M.
(2000)
J. Cell Biol.
148,
253-258 73.
Gorodeski, G. I.
(2000)
Am. J. Physiol.
278,
C942-C952
74.
Reinhard, M.,
Jarchau, T.,
and Walter, U.
(2001)
Trends Biochem. Sci.
26,
243-249[CrossRef][Medline]
[Order article via Infotrieve]
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