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INTRODUCTION |
Phospholipase D (PLD)1
enzymes belong to a newly identified enzyme family known to exist in
plant, bacteria, yeast, and mammalian sources. Stimulation of PLD has
been described in many cellular systems in response to a large variety
of agonist-activated tyrosine kinase receptors and receptors coupled to
heterotrimeric G proteins and is apparently involved in various
signaling processes (1-3). Specifically, PLD and its immediate
reaction product, phosphatidic acid, have been reported to regulate
diverse cellular events, such as vesicular trafficking, actin stress
fiber formation, activation of Raf-1 kinase, and phosphatidylinositol
4-phosphate (PtdIns4P) 5-kinase isoforms, to name but a few (4-9).
The two mammalian PLD isoforms identified thus far, PLD1 (with the two
splice variants PLD1a and PLD1b) and PLD2, differ greatly in their
regulatory properties. PLD2 is thought to be solely stimulated by the
phosphoinositide phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2; Refs. 10 and 11), although recent reports on cloned PLD2 enzymes suggest that this PLD isoform may also be
activated, but very modestly, by ADP ribosylation factor (ARF), a
member of the low molecular weight GTPase superfamily (12, 13). On the
other hand, PLD1 enzymes are strongly stimulated by
PtdIns(4,5)P2 and ARF and, in addition, by some protein
kinase C (PKC) isoforms and by Rho GTPases (14, 15). Specifically, the
Rho family GTPases RhoA, Rac1, and Cdc42, which are activated by the
stable GTP analog guanosine 5'-O-(3-thio)triphosphate
(GTP
S), have been shown to stimulate purified recombinant PLD1
enzymes, apparently by a direct interaction of PLD1 with these GTPases (16-19). Furthermore, studies performed with toxins inactivating Rho
GTPases indicated that these GTPases are also involved in PLD
stimulation by G protein-coupled and growth factor receptors in intact
cells (20-23). However, stimulation of endogenous PLD by Rho GTPases
seems to be rather complex. Whereas RhoA stimulation of ARF-sensitive
PLD has been reported in some cell-free systems (24-26), it was
without effect in others or could even be resolved from the
ARF-stimulated PLD (15, 26, 27). Rho GTPases may also indirectly
stimulate PLD enzymes by increasing the synthesis of
PtdIns(4,5)P2 by PtdIns4P 5-kinases (28), which has been demonstrated in various cellular systems to be of crucial importance for signaling to PLD (2, 3, 7, 29, 30).
We have recently reported that in HEK-293 cells stably expressing the G
protein-coupled m3 muscarinic acetylcholine receptor (mAChR), PLD
activity depends on PtdIns(4,5)P2 and that PLD stimulation by phorbol ester-activated PKC involves the Ras-related Ral proteins, whereas m3 mAChR signaling to PLD is mediated by members of the ARF and
Rho GTPase families (20, 30-34). The aim of the present study was to
identify the mechanism of PLD stimulation by Rho proteins in HEK-293
cells. We demonstrate here that the PLD stimulatory effect of
recombinant RhoA in HEK-293 cell membranes is
phosphorylation-dependent. In the search for the putative
kinase, we studied the effect of the RhoA-stimulated serine/threonine
kinase Rho-kinase (35), which has also been termed ROK
(36) and
ROCK-II (37), on PLD regulation. We found that overexpression of
Rho-kinase greatly increases m3 mAChR-mediated but not PKC-mediated PLD
stimulation in intact cells. Furthermore, we show that HA-1077, a
Rho-kinase inhibitor, specifically suppresses receptor-mediated PLD
stimulation and that recombinant Rho-kinase mimics the stimulatory
effect of RhoA on PLD activity in HEK-293 cell membranes. These
findings strongly suggest that Rho-kinase is involved in Rho-controlled PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells.
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EXPERIMENTAL PROCEDURES |
Materials--
[3H]Oleic acid (10 Ci/mmol) and
1-palmitoyl-2-[3H]palmitoyl-glycerophosphocholine
([3H]PtdCho; 37.5 Ci/mmol) were from New England Nuclear.
HA-1077 was from Calbiochem, and glutathione-Sepharose was from
Amersham Pharmacia Biotech. Unlabeled PtdCho, phorbol 12-myristate
13-acetate (PMA), and TNM-FH insect medium were from Sigma, and
PtdIns(4,5)P2 and GTP
S were from Roche Molecular
Biochemicals. Antibodies against RhoA and Rho-kinase were purchased
from Santa Cruz Biotechnology.
Plasmids--
DNA encoding human RhoA was subcloned into pRK5
expression vector. DNA encoding myc-tagged C3 transferase subcloned in
pEF (38) was a kind gift of Dr. A. Hall. DNAs encoding myc-tagged wild-type Rho-kinase, the catalytic domain of Rho-kinase,
Rho-kinase-CAT (amino acids 6-553), and the kinase-deficient mutant of
Rho-kinase-CAT, Rho-kinase-CAT-KD (Rho-kinase-CAT K121G), were
subcloned into pEF (39). For expression in Sf9 cells, DNA
encoding RhoA was subcloned into a pAcGHLT baculovirus transfer vector
(PharMingen), and DNA encoding Rho-kinase-CAT was subcloned into a
pAcGLT transfer vector (39).
Cell Culture and Transfection--
Culture conditions of HEK-293
cells stably expressing the m3 mAChR were as reported previously (31).
For experiments, cells subcultured in Dulbecco's modified Eagle's
medium/F-12 medium were grown to near confluence (145-mm culture
dishes) and transfected with either the indicated concentrations of DNA
encoding RhoA, myc-tagged C3 transferase, Rho-kinase, Rho-kinase-CAT or
Rho-kinase-CAT-KD, or the corresponding vectors using the calcium
phosphate method (40). Transfection efficiency of HEK-293 cells, which
ranged from 50% to 80%, was determined by in situ staining
for
-galactosidase activity of the cells cotransfected with the
constitutively active pSV
-gal (Promega). All assays were performed
48 h after transfection. Transient overexpression of the proteins
was verified by immunoblotting of cell lysates using specific
antibodies. Transient overexpression of C3 transferase was detected by
the mobility shift of ADP-ribosylated endogenously expressed RhoA (41).
Morphological changes induced by overexpression of RhoA and Rho-kinase
were visualized by phase-contrast microscopy (Nikon TMS).
Assay of PLD Activity in Intact Cells--
For measurement of
PLD activity in intact HEK-293 cells, the cells were replated 24 h
after transfection on 145-mm culture dishes. Cellular phospholipids
were labeled by incubating monolayers for 20-24 h with
[3H]oleic acid (2 µCi/ml) in growth medium. Thereafter,
cells were detached from the dishes, washed twice in Hank's balanced
salt solution containing 118 mM NaCl, 5 mM KCl,
1 mM CaCl2, 1 mM MgCl2, and 5 mM D-glucose buffered at pH 7.4 with 15 mM HEPES, and resuspended at a cell concentration of 1 × 107 cells/ml. PLD activity was measured for 60 min at
37 °C in a total volume of 200 µl containing 100 µl of cell
suspension (1 × 106 cells), 400 mM
ethanol, and the indicated stimulatory agents. The reaction was
stopped, and labeled phospholipids, including the specific PLD product
[3H]phosphatidylethanol ([3H]PtdEtOH), were
isolated as described previously (31). The formation of
[3H]PtdEtOH is expressed as a percentage of the total
amount of labeled phospholipids. Data shown are the mean ± S.D.
from one experiment performed in triplicate and repeated as indicated
in the figure legends.
Assay of PLD Activity in Membranes--
To measure PLD activity
in HEK-293 cell membranes prepared as described previously (30),
[3H] PtdCho was mixed with PtdIns(4,5)P2 in
a molar ratio of 8:1, dried, and resuspended in 50 mM
HEPES, pH 7.5, 3 mM EGTA, 80 mM KCl, and 1 mM dithiothreitol, followed by sonication on ice. PLD activity was determined as described previously (30) with
[3H]PtdCho/PtdIns(4,5)P2 (200 µM/25 µM) as substrate vesicles and 200 µg of membrane protein for 60 min at 37 °C or for 15 min at 30 °C.
Purification of Recombinant Proteins--
Sf9 cells
(1 × 106 cells/ml) cultured at 25 °C in TNM-FH
insect medium containing 10% fetal calf serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin were infected with pAcGHLT containing RhoA or pAcGLT containing Rho-kinase-CAT baculovirus transfer vectors
(multiplicity of infection = 5) for 48 h at 25 °C.
Thereafter, the cells were centrifuged, resuspended in Buffer A (50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 10 µM phenylmethylsulfonyl
fluoride, and 10 mM Tris-HCl, pH 7.5), and homogenized by
sonication on ice. The lysates were centrifuged for 1 h at
20,000 × g. The supernatant, which contained GST-RhoA
or GST-Rho-kinase-CAT, was incubated with glutathione-Sepharose beads
for 30 min at 4 °C. Thereafter, the beads were washed three times
with Buffer A to remove unbound proteins. RhoA and Rho-kinase-CAT were
released from the parent GST-fusion proteins bound to the beads by
incubation with thrombin (PharMingen; 10 units) overnight at 4 °C in
a buffer containing 150 mM NaCl, 5 mM
MgCl2, 2.5 mM CaCl2, 1 mM dithiothreitol, and 50 mM Tris-HCl, pH 8.0. The beads were removed by centrifugation, and the excess thrombin was
removed by the addition of p-aminobenzamidine beads. The
homogeneity of the recombinant RhoA and Rho-kinase-CAT proteins was
analyzed by SDS-polyacrylamide gel electrophoresis.
Immunoblot Analysis--
For immunoblot analysis, an aliquot of
the homogenates was subjected to SDS-polyacrylamide gel electrophoresis
on 10% acrylamide gels to separate the proteins. After a transfer to
nitrocellulose membranes and a 1-h incubation with anti-RhoA (1:500
dilution) or anti-Rho-kinase (1:200 dilution) antibodies, the proteins
were visualized by enhanced chemiluminescence.
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RESULTS |
PLD Stimulation by RhoA in HEK-293 Cell Membranes Is
Phosphorylation-dependent--
We have reported previously
that PLD stimulation by the m3 mAChR, which activates endogenous RhoA
in HEK-293 cells (42), is potently inhibited by the inactivation of Rho
family GTPases with Clostridium difficile toxin B (20).
Furthermore, it has been shown that toxin B and the Rho-specific C3
transferase (41) decrease the cellular level of
PtdIns(4,5)P2 and that PtdIns(4,5)P2 regulates
PLD activity in HEK-293 cell membranes (30, 32, 43). Thus, to study PLD
stimulation by Rho proteins in HEK-293 cells, we measured PLD
activities in the membranes of HEK-293 cells in the presence of
PtdIns(4,5)P2. Several previous studies have demonstrated
that activated RhoA stimulates purified recombinant PLD1 enzymes under
this condition, apparently by a direct RhoA-PLD1 interaction (16-19).
In HEK-293 cell membranes, the addition of GTP
S (100 µM) alone caused about a 2-fold increase in PLD activity, which is probably due to the activation of endogenous
membrane-associated ARF proteins (30, 32). Surprisingly, however, the
addition of purified recombinant RhoA (10 µM) in the
presence of GTP
S (100 µM) had no effect on PLD
activity (Fig. 1). Similar data were
obtained in the membranes of HEK-293 cells pretreated with toxin B,
causing the inactivation of endogenous Rho proteins (data not shown).
In contrast, under the same assay conditions, GTP
S-activated recombinant RhoA stimulated PLD activity in the membranes of human PLD1a-expressing Sf9 cells about 20-fold, from 50 ± 10 to
1070 ± 80 pmol × h
1 × mg
protein
1 (mean ± SD; n = 5 experiments).

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Fig. 1.
Phosphorylation-dependent PLD
stimulation by RhoA. PLD activity was measured in HEK-293 cell
membranes as described under "Experimental Procedures" with
[3H]PtdCho/PtdIns(4,5)P2 substrate vesicles
in the absence (Basal) and presence of 100 µM
GTP S or 10 µM RhoA plus 100 µM GTP S,
added alone (No MgATP) or with 1 mM MgATP, for
60 min at 37 °C. Data are representative of three experiments.
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Besides PtdIns4P 5-kinases, Rho GTPases can stimulate various protein
kinases (for reviews, see Refs. 28 and 44). Therefore, we studied
whether a phosphorylation reaction is involved in RhoA stimulation of
HEK-293 cell PLD activity. For this study, PLD activity was measured in
the presence of 1 mM MgATP. Under this condition, the
addition of RhoA in the presence of GTP
S markedly increased PLD
activity in HEK-293 cell membranes (Fig. 1). Although a permissive
effect of MgATP on GTP
S binding by RhoA cannot be excluded, we first
considered the possibility that a RhoA-dependent lipid or
protein kinase is involved in PLD stimulation by RhoA. The involvement
of a RhoA-stimulated PtdIns4P 5-kinase was unlikely for the following
reasons: (a) PtdIns(4,5)P2 was added at a
maximally effective concentration of 25 µM (30);
(b) although the added PtdIns(4,5)P2 was
degraded at the end of the incubation period in the absence of MgATP to
10 µM, this PtdIns(4,5)P2 concentration was
virtually identical to that (12 µM) used by many others
to demonstrate RhoA stimulation of PLD in vitro (16-19,
45); and (c) in the presence of MgATP, which by itself
prevented (by ~50%) the degradation of added
PtdIns(4,5)P2, the addition of RhoA had a very modest
protective effect on the PtdIns(4,5)P2 level (data not
shown), which was quite distinct from the marked PLD stimulation by
RhoA under this condition. Thus, the
phosphorylation-dependent PLD stimulation by RhoA in
HEK-293 cell membranes may involve a protein kinase rather than
PtdIns4P 5-kinase.
Rho-Kinase Stimulates PLD in HEK-293 Cells--
Recently,
several direct RhoA target proteins have been identified,
including the two Rho-stimulated serine/threonine kinases: (a) Rho-kinase, also termed ROK
or ROCK-II, and
(b) p160ROCK, also termed ROCK-I (35-37, 46). To test the
hypothesis that Rho-induced PLD stimulation involves Rho-kinase, we
studied the effects of HEK-293 cell transfection with RhoA and
different Rho-kinase constructs on cell morphology and PLD activity.
Expression of RhoA and the myc-tagged Rho-kinases was verified by
immunofluorescence and immunoblotting (data not shown). Overexpression
of RhoA caused drastic changes in HEK-293 cell morphology, as
demonstrated by the occurrence of a high number of rounded cells (Fig.
2). As reported previously by others (36,
44, 47), similar morphology changes were observed in HEK-293 cells
overexpressing Rho-kinase-CAT, which lacks the regulatory Rho-binding
and PH domains and is constitutively active, and although less
pronounced, in wild-type Rho-kinase-overexpressing cells. In contrast,
overexpression of a kinase-deficient mutant of Rho-kinase-CAT,
Rho-kinase-CAT-KD, did not cause rounding of HEK-293 cells.

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Fig. 2.
Cell morphology changes induced by
overexpressed RhoA and Rho-kinase. HEK-293 cells were transfected
without (Control) and with 100 µg of DNA of pEF/pRK5, RhoA
in pRK5 (RhoA), wild-type Rho-kinase in pEF
(Rho-kinase), Rho-kinase-CAT in pEF
(Rho-kinase-CAT), or Rho-kinase-CAT-KD in pEF
(Rho-kinase-CAT-KD). After 48 h, cell morphology was
assessed by phase-contrast microscopy.
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We then determined the effects of the same proteins on PLD activity in
HEK-293 cells. As illustrated in Fig. 3,
overexpression of RhoA and either wild-type Rho-kinase or
Rho-kinase-CAT markedly increased m3 mAChR-mediated PLD stimulation
without significantly altering basal PLD activity. The PLD stimulatory
effects were most pronounced in cells overexpressing RhoA and
Rho-kinase-CAT. For example, the transfection of HEK-293 cells with 100 µg of RhoA DNA and Rho-kinase-CAT DNA increased PLD stimulation by
carbachol (1 mM) by about 150% and 250%, respectively
(Fig. 3, A and C). A significant but less
pronounced increase in m3 mAChR-mediated PLD stimulation was observed
in HEK-293 cells overexpressing wild-type Rho-kinase (Fig.
3B). In contrast, the transfection of HEK-293 cells with up
to 150 µg of kinase-deficient Rho-kinase-CAT-KD DNA did not change
PLD stimulation by carbachol (Fig. 3D). The potentiating
effect of Rho-kinase and Rho-kinase-CAT overexpression on m3
mAChR-mediated PLD stimulation was even more evident at low agonist
concentrations. For example, at 3 µM, carbachol had only
a very small effect on PLD activity in control cells, but it increased
the PLD activity in Rho-kinase-CAT-expressing cells to the same extent
as 1 mM carbachol in control cells (Fig.
4). Finally, the effects of wild-type
Rho-kinase and Rho-kinase-CAT overexpression on PLD stimulation by
phorbol ester-activated PKC were studied. In contrast to m3
mAChR-mediated PLD stimulation, stimulation of PLD by PMA (100 nM) was not altered by the transfection of HEK-293 cells
with either wild-type Rho-kinase or Rho-kinase-CAT (Fig.
5).

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Fig. 3.
Potentiation of m3 mAChR-mediated PLD
stimulation by RhoA and Rho-kinase. m3 mAChR-expressing HEK-293
cells were transfected with the indicated concentrations of RhoA in
pRK5 (A), wild-type Rho-kinase DNA in pEF (B),
Rho-kinase-CAT DNA in pEF (C), or Rho-kinase-CAT-KD DNA in
pEF (D). As a control (0), cells received the
highest concentration of the corresponding vector DNA. After 48 h,
[3H]PtdEtOH formation was determined in
[3H]oleic acid-labeled cells in the absence
(Basal) and presence of 1 mM carbachol as
described under "Experimental Procedures." Data are representative
of two to four similar experiments.
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Fig. 4.
Sensitization of m3 mAChR-mediated PLD
stimulation by Rho-kinase. m3 mAChR-expressing HEK-293 cells were
transfected with 100 µg of pEF DNA (Control), wild-type
Rho-kinase DNA in pEF (Rho-kinase), or Rho-kinase-CAT in pEF
(Rho-kinase-CAT). After 48 h, [3H]PtdEtOH
formation was determined in [3H]oleic acid-labeled cells
in the absence (0) and presence of 3 or 1000 µM carbachol. Data are representative of four similar
experiments.
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Fig. 5.
Lack of effect of Rho-kinase on PMA-induced
PLD stimulation. HEK-293 cells were transfected with 100 µg of
pEF DNA (pEF), wild-type Rho-kinase DNA in pEF
(Rho-kinase), or Rho-kinase-CAT DNA in pEF
(Rho-kinase-CAT). After 48 h, [3H]PtdEtOH
formation was determined in [3H]oleic acid-labeled cells
in the absence (0) and presence of 100 nM PMA as
indicated. Data are representative of three similar experiments.
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To study the involvement of Rho in the potentiating effect of
Rho-kinase on m3 mAChR-mediated PLD stimulation, cotransfection experiments with C3 transferase were performed. Overexpressed C3
transferase reduced the carbachol (1 mM)-stimulated PLD
activity in control cells and fully prevented the stimulatory effect of coexpressed wild-type Rho-kinase (Fig.
6A). In contrast,
cotransfection with C3 transferase did not inhibit the potentiating
effect of Rho-kinase-CAT on m3 mAChR-mediated PLD stimulation (Fig.
6B).

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Fig. 6.
Rho mediation of Rho-kinase-induced PLD
stimulation. m3 mAChR-expressing HEK-293 cells were transfected
with 100 µg of pEF DNA (pEF) and (A) with 100 µg of wild-type Rho-kinase DNA in pEF (Rho-kinase), C3
transferase in pEF (C3), or Rho-kinase DNA plus C3
transferase DNA (Both) or (B) with 100 µg of
Rho-kinase-CAT DNA in pEF (Rho-kinase-CAT), C3 transferase
in pEF (C3), or Rho-kinase-CAT DNA plus C3 transferase DNA
(Both). After 48 h, [3H] PtdEtOH
formation was determined in [3H]oleic acid-labeled cells
in the absence (Basal) and presence of 1 mM
carbachol. Data are characteristic of three similar experiments.
Inset, immunoblot detection of RhoA and ADP-ribosylated RhoA
(ADPR-RhoA) with the anti-RhoA antibody in lysates of
HEK-293 cells transfected with pEF DNA (pEF) or C3
transferase DNA in pEF (C3).
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Rho-Kinase Stimulates PLD in Vitro--
To study whether
Rho-kinase activates PLD in vitro, the effects of purified
recombinant Rho-kinase-CAT were measured on PLD activity in HEK-293
cell membranes. In the absence of MgATP, Rho-kinase-CAT had no effect
on PLD activity in either the absence or presence of GTP
S (data not
shown). However, in the presence of MgATP (1 mM), the
addition of Rho-kinase-CAT (2 µM) markedly increased PLD
activity (Fig. 7). The net increase in
activity was similar in both the absence and presence of GTP
S (100 µM), which enhanced PLD activity by itself about 2-fold.
In contrast, in the presence of RhoA (10 µM) and GTP
S
(100 µM), further addition of Rho-kinase-CAT had no
effect on enzyme activity. These results indicate that Rho-kinase can
stimulate HEK-293 cell PLD activity in vitro almost as
efficiently as GTP
S-activated RhoA.

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Fig. 7.
Stimulation of PLD activity in HEK-293 cell
membranes by Rho-kinase. PLD activity was measured in HEK-293 cell
membranes in the presence of 1 mM MgATP without
(Basal) and with 100 µM GTP S or 10 µM RhoA plus 100 µM GTP S, added alone
(Control) or with 2 µM purified
Rho-kinase-CAT, for 15 min at 30 °C. Data are representative of two
experiments.
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PLD Stimulation Is Inhibited by a Rho-Kinase Inhibitor--
To
examine whether endogenously expressed Rho-kinase is involved in the m3
mAChR-induced and Rho-mediated PLD stimulation in HEK-293 cells, we
studied the effects of HA-1077, a membrane-permeable Rho-kinase
inhibitor (Ki = 0.33 µM) (48, 49), on PLD stimulation. Pretreatment of intact HEK-293 cells with this inhibitor caused a concentration-dependent inhibition of
carbachol (1 mM)-stimulated PLD activity without affecting
the basal activity (Fig. 8A).
Half-maximal inhibition was observed with 0.3 µM HA-1077, and at 10 µM, HA-1077 suppressed carbachol-induced PLD
stimulation by 78 ± 5% (n = 4). In contrast,
HA-1077 (up to 10 µM) had no effect on PLD stimulation by
PMA-activated PKC (Fig. 8B). Previous studies excluded the
involvement of cAMP-dependent protein kinase, which is also
rather potently inhibited by HA-1077 (Ki = 1 µM) (48), in m3 mAChR-induced PLD stimulation (Ref. 31; data not shown). Finally, the effect of HA-1077 on PLD stimulation by
RhoA in HEK-293 cell membranes was examined. Pretreatment of the
membranes with HA-1077 (10 µM) had no effect or only
minor effects on basal and GTP
S-stimulated activities in the absence of RhoA but largely suppressed PLD stimulation by GTP
S-activated RhoA (Fig. 9).

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Fig. 8.
Inhibition of m3 mAChR-induced PLD
stimulation by a Rho-kinase inhibitor. m3 mAChR-expressing HEK-293
cells prelabeled with [3H]oleic acid were treated with
HA-1077 at the indicated concentrations for 15 min, followed by PLD
activity measurements (A) in the absence (Basal,
) and presence of 1 mM carbachol ( ) or (B)
in the absence (Basal, ) and presence of 100 nM PMA ( ). Data are representative of three to five
similar experiments.
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Fig. 9.
Inhibition of Rho-induced PLD stimulation by
a Rho-kinase inhibitor. PLD activities were measured in HEK-293
cell membranes pretreated or not pretreated for 15 min at 4 °C with
10 µM HA-1077 in the absence (Basal) and
presence of 1 mM MgATP plus 100 µM GTP S,
10 µM recombinant RhoA, or the indicated combinations for
60 min at 37 °C. Data are representative of four similar
experiments.
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DISCUSSION |
In the present study, we provide evidence that the Rho-stimulated
serine/threonine kinase Rho-kinase participates in PLD stimulation by
the G protein-coupled m3 mAChR in HEK-293 cells. The evidence is based
on the following findings: (a) stimulation of PLD activity in HEK-293 cell membranes measured in the presence of
PtdIns(4,5)P2 by purified GTP
S-activated RhoA was fully
MgATP-dependent; (b) overexpression of wild-type
Rho-kinase and particularly of Rho-kinase-CAT but not of
Rho-kinase-CAT-KD potentiated and sensitized m3 mAChR-mediated PLD
stimulation in intact HEK-293 cells, similar to overexpression of RhoA;
(c) inactivation of Rho by coexpressed C3 transferase blocked the stimulatory effect of wild-type Rho-kinase but not of
Rho-kinase-CAT, indicating that Rho mediates the potentiation of m3
mAChR-induced PLD stimulation by Rho-kinase; (d) recombinant Rho-kinase-CAT mimicked the phosphorylation-dependent
stimulatory effect of GTP
S-activated RhoA on PLD stimulation in
HEK-293 cell membranes; and (e) the Rho-kinase inhibitor
HA-1077 largely suppressed PLD stimulation by RhoA in HEK-293 cell
membranes as well as by the m3 mAChR in intact cells.
We reported previously that the inactivation of Rho family GTPases with
C. difficile toxin B potently inhibits m3 mAChR-mediated PLD
stimulation in HEK-293 cells (20). Furthermore, we showed that toxin B
and Rho-specific C3 transferase reduce the cellular level of
PtdIns(4,5)P2 and that PtdIns(4,5)P2 stimulates
PLD activity in HEK-293 cell membranes (30, 43). Therefore, we examined the effect of activated RhoA on PLD activity in HEK-293 cell membranes in the presence of PtdIns(4,5)P2. Previous studies on
various crude cell-free preparations (24-26) and with purified
recombinant PLD1 enzymes (16-19) using similar in vitro PLD
assay conditions reported that activated RhoA stimulates PLD activity.
Furthermore, the data obtained with purified PLD1 enzymes strongly
suggested that activated RhoA directly interacts with and stimulates
PLD1 activity. Using membranes of human PLD1a-expressing Sf9
cells, we also observed that activated RhoA potently stimulates PLD
activity. However, there are also reports on the failing effects of
RhoA on PLD activity. For example, Vinggaard et al. (27)
reported that GTP
S-activated RhoA stimulates PLD activity in crude
membranes of the human placenta, but that this stimulation is lost
after partial PLD purification, in contrast to the stimulatory effect of ARF3 that was maintained with the purified enzyme preparation. Furthermore, Park et al. (15) showed that activated ARF3 but not activated RhoA stimulates PLD activity in native COS-7 cell membranes, whereas in the membranes of COS-7 cells, overexpressing rat
PLD1 enzyme RhoA stimulated PLD activity. Thus, stimulation of PLD by
RhoA can apparently occur by both direct and indirect mechanisms.
In contrast to human PLD1a-expressing Sf9 cells, activated RhoA
did not stimulate PLD activity in the membranes of HEK-293 cells. The
ineffectiveness of RhoA was not due to saturation of the membranes with
endogenous Rho, because RhoA was similarly ineffective in the membranes
of HEK-293 cells in which the endogenous Rho proteins had been
inactivated with toxin B. However, when MgATP was added, RhoA strongly
stimulated PLD activity, thus suggesting that a phosphorylation
reaction is involved. Because the PLD assay was performed in the
presence of a saturating concentration of PtdIns(4,5)P2
(30) and because the addition of RhoA in the presence of MgATP had only
a very small effect on the PtdIns(4,5)P2 level in contrast
to the marked PLD stimulation, we considered that a protein kinase
rather than a PtdIns4P 5-kinase is primarily responsible for the
phosphorylation-dependent PLD stimulation by RhoA. Here we
provide evidence that the Rho-stimulated serine/threonine kinase,
Rho-kinase, is mediating Rho-controlled PLD stimulation in HEK-293
cells. Overexpression of wild-type Rho-kinase and of constitutively
active Rho-kinase-CAT but not of the kinase-deficient Rho-kinase-CAT-KD
potentiated PLD stimulation by the m3 mAChR, similar to the
overexpression of RhoA. In contrast, PLD stimulation by PMA-activated
PKC, which is less sensitive to the inactivation of Rho GTPases by
toxin B than the receptor response (20, 32, 33), was not affected. C3
transferase fully blocked the stimulatory effect of wild-type
Rho-kinase, indicating that even the overexpressed Rho-kinase is under
tight control by endogenous Rho proteins. Finally, purified
Rho-kinase-CAT increased PLD activity in HEK-293 cell membranes,
similar to activated RhoA and in a non-additive manner, suggesting a
common pathway for PLD stimulation by these two proteins.
Overexpressed RhoA and wild-type Rho-kinase increased the
receptor-induced PLD stimulation but had no effect on the basal PLD
activity. Strikingly, the constitutively active Rho-kinase-CAT, which
stimulated PLD activity in HEK-293 cell membranes, also had no effect
on basal PLD activity in intact cells, but it required the input by the
activated receptor, and this effect was C3-insensitive. These findings
suggest that for stimulation of PLD by Rho and Rho-kinase in intact
HEK-293 cells, an additional component or signal is necessary, which is
apparently provided by the receptor and may sensitize PLD for
stimulation by Rho-kinase. The second signal required for PLD
stimulation could be ARF activated by the receptor (34). A similar
bifurcating pathway involving both Rho and ARF proteins has been
described for PLD stimulation by the formyl peptide receptor in human
neutrophils (23). Alternatively, the receptor may inactivate or cause
the redistribution of PLD inhibitory proteins, which are mainly
identified in and purified from brain cytosol (50-53), which may
prevent PLD stimulation by Rho-kinase-CAT in intact cells in the
absence of receptor stimulation. In contrast, in membrane preparations,
Rho-kinase-CAT may have unrestricted access to stimulate PLD. Among the
PLD inhibitory proteins, synucleins have recently been identified as
selective inhibitors of PLD2 enzymes (53). Furthermore, evidence has
recently been provided suggesting that PLD2 enzymes can be activated by epidermal growth factor and insulin receptors (54, 55). However, which
of the PLD isoforms endogenously expressed in HEK-293 cells represents
the enzyme stimulated by the m3 mAChR is presently unknown.
Furthermore, although very attractive, in preliminary experiments with
membranes of HEK-293 cells overexpressing human PLD1a or mPLD2 and
purified Rho-kinase-CAT, no clear evidence for phosphorylation of the
expressed PLD proteins has been obtained thus far (data not shown).
Finally, it has to be noted that, similar to recombinant PLD enzymes,
HEK-293 cell PLD activity is regulated by PtdIns(4,5)P2 and
that inactivation of Rho GTPases by toxins decreases the cellular
PtdIns(4,5)P2 level (30, 43). Although the in
vitro data in HEK-293 cell membranes did not support the hypothesis that PtdIns4P 5-kinase plays an important role in
RhoA-induced PLD stimulation, this does not exclude the involvement of
regulation of PtdIns(4,5)P2 synthesis in Rho-mediated PLD
stimulation in intact cells, where the concentrations of the various
PLD regulatory factors and their interactions are most likely quite
distinct from the in vitro situation.
Rho-controlled and m3 mAChR-induced signaling to PLD in HEK-293 cells
seems to involve endogenously expressed Rho-kinase. Treatment with the
Rho-kinase inhibitor HA-1077 greatly reduced but did not completely
abolish PLD stimulation by activated RhoA in membranes as well as the
receptor response in intact cells. The observed IC50 value
of HA-1077 (0.3 µM) to inhibit m3 mAChR-induced PLD
stimulation fits perfectly with the Ki value
reported for this compound to inhibit Rho-kinase (0.33 µM) (48). Thus, m3 mAChR not only activates endogenous
RhoA in HEK-293 cells (42) but, in consequence, Rho-kinase is also
apparently activated. Although a direct PLD stimulatory effect of RhoA
in intact HEK-293 cells is not excluded by our findings, they strongly
suggest that RhoA-activated Rho-kinase mediates the m3 mAChR-induced
PLD stimulation to a major extent in these cells.
In conclusion, this study identifies Rho-kinase as a novel and
essential component of the Rho-dependent signaling pathway leading to PLD stimulation by a G protein-coupled receptor. Thus, for
the first time, a role for Rho-kinase in phospholipid signaling is demonstrated.