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J Biol Chem, Vol. 274, Issue 26, 18675-18685, June 25, 1999
From the Laboratories of Although muscarinic acetylcholine receptors
(mAChR) regulate the activity of smooth muscle myosin, the effects of
mAChR activation on cytoplasmic myosin have not been characterized. We
found that activation of transfected human M3 mAChR
induces the phosphorylation of myosin light chains (MLC) and the
formation of myosin-containing stress fibers in Chinese hamster ovary
(CHO-m3) cells. Direct activation of protein kinase C (PKC) with
phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain
phosphorylation and myosin reorganization in CHO-m3 cells. Conventional
( Muscarinic acetylcholine receptors
(mAChR)1 are heterotrimeric G
protein-coupled receptors that regulate contraction of smooth muscle.
Multiple subtypes of mAChR that transduce different intracellular signals are often co-expressed in smooth muscle tissues (reviewed in
Ref. 1). The M1, M3, and M5 mAChR
subtypes activate protein kinase C (PKC) by elevating intracellular
Ca2+ and diacylglycerol. In contrast, the M2
and M4 mAChR subtypes inhibit protein kinase A (PKA) by
diminishing adenylyl cyclase activity (reviewed in Refs. 2 and 3).
Pharmacological studies indicate that M3 mAChR activation
induces smooth muscle contraction, although activation of other
co-expressed mAChR subtypes may modulate this response (reviewed in
Ref. 1). Dissection of the biochemical pathways involved in
M3 mAChR-mediated contraction is complicated by the
co-expression of multiple mAChR subtypes in many smooth muscle tissues.
Contractile processes in non-muscle cells mimic those occurring in
smooth muscle tissues, indicating the value of using non-muscle cells
to investigate the biochemical pathways that regulate contraction (4-8). Phosphorylation of myosin light chains (MLC) in non-muscle cells causes the formation of myosin-containing stress fibers, which
are contractile bundles of actin filaments associated with myosin II
(4-8). Phosphorylation of MLC similarly increases actin-myosin interactions in smooth muscle cells, resulting in smooth muscle contraction (reviewed in Refs. 9-12). In addition to smooth muscle contraction, many fundamental cellular processes such as adhesion, migration, and division depend upon the interaction of myosin with
actin in contractile filaments (reviewed in Ref. 12). Activation of
mAChR may affect these fundamental processes by altering myosin activity in non-muscle cells as it does in smooth muscle. Although mAChR activation induces MLC phosphorylation and subsequent contraction in smooth muscle cells (13-17), the ability of mAChR to regulate MLC
phosphorylation and myosin organization in non-muscle cells has not
been reported.
We investigated the ability of transfected human mAChR subtypes to
regulate myosin organization in Chinese hamster ovary (CHO) cells.
Activation of transfected M3 mAChR induces MLC
phosphorylation and causes myosin-containing stress fibers to form in
CHO cells. The involvement of PKC in these events is indicated by our
findings that 1) direct activation of PKC with phorbol esters induces
MLC phosphorylation and myosin reorganization in CHO cells, 2) specific PKC antagonists inhibit M3 mAChR-mediated myosin
reorganization, and 3) activation of transfected M1 but not
M2 mAChR subtypes also induces the formation of
myosin-containing stress fibers, demonstrating that only mAChR subtypes
that stimulate PKC activity induce myosin reorganization. The
participation of myosin light chain kinase (MLCK) and RhoA in
mAChR-mediated myosin reorganization was also investigated, since these
proteins regulate contractile processes in other systems (reviewed in
Refs. 9-12). We found that MLCK antagonists inhibit mAChR-mediated
myosin reorganization but only at antagonist concentrations that may
affect PKC. Stable expression of dominant negative
RhoAAsn-19 or inactivation of Rho with C3 exoenzyme lessens
mAChR-mediated myosin reorganization but does not abolish it.
This study demonstrates that M3 mAChR activation
significantly affects myosin organization in non-muscle cells. Our
findings indicate that M3 mAChR activation induces
cytoplasmic myosin reorganization by both PKC- and
Rho-dependent mechanisms. These receptor-mediated changes
in cytoplasmic myosin may contribute to the unique ability of
M3 mAChR to regulate the adhesion (18-20) and morphology
(21, 22) of non-muscle cells.
Reagents--
The SA-2 human IgM monoclonal autoantibody that
reacts specifically with myosin heavy chains was obtained from cultures
of a thymic B lymphocyte clone transformed with Epstein-Barr virus, as
described previously (23, 24). Mouse monoclonal antibody to
hemagglutinin (HA) was purchased from Babco (Berkeley, CA), and mouse
monoclonal antibodies to PKC isoforms were purchased from Transduction
Laboratories (Lexington, KY). Fluorescein-labeled goat anti-human IgM
was obtained from Fisher. Horseradish peroxidase-labeled antibody to
mouse immunoglobulins and enhanced chemiluminescence (ECL) reagents
were obtained from Amersham Pharmacia Biotech. Reagents for
LipofectAMINE-mediated transfection were purchased from Life
Technologies, Inc. Carbachol and phorbol 12-myristate 13-acetate (PMA)
were purchased from Sigma. The kinase inhibitors calphostin C, Go6976,
chelerythrine, KT5926, ML-7, and ML-9 were obtained from Calbiochem,
and KN-62 was bought from Research Biochemicals, Inc. (Natick, MA).
Ham's F-12 medium was purchased from Mediatech (Herndon, VA); fetal
calf serum (FCS) was obtained from Biofluids (Rockville, MD), and
zeocin was bought from Invitrogen (Carlsbad, CA). Other reagents were
obtained from Sigma or from sources listed in the text.
Cell Lines--
CHO-K1 sublines stably transfected with the
M1, M2, or M3 subtypes of human
mAChR are referred to as CHO-m1, CHO-m2, and CHO-m3, respectively.
These sublines, as well as untransfected CHO-K1 cells, were generously
provided by Dr. Mark Brann (University of Vermont). Cells were cultured
in complete medium consisting of Ham's F-12 medium, heat-inactivated
FCS (5%), glutamine (0.3 mg/ml), penicillin (20 units/ml), and
streptomycin sulfate (20 µg/ml). Cells were maintained at 37 °C in
a humidified atmosphere of 5% CO2, 95% air at densities
that promoted exponential proliferation.
Establishment and Characterization of CHO-m3 Cell Lines Stably
Expressing Wild-type or Mutant RhoA--
The pEF-BOS-HA-RhoA and
pEF-BOS-HA-RhoAVal-14 plasmids coding for hemagglutinin
(HA)-tagged wild-type RhoA or constitutively active RhoAVal-14, respectively, were a generous gift from Dr.
Shinya Kuroda (Nara Institute of Science and Technology, Nara, Japan).
The pEF-BOS-HA-RhoAAsn-19 plasmid coding for HA-tagged
dominant negative RhoAAsn-19 was generated by site-directed
mutagenesis of the pEF-BOS-HA-RhoA construct using the GeneEditor
system from Promega (Madison, WI). Sequencing of the
pEF-BOS-HA-RhoAAsn-19 plasmid indicated that the threonine
at codon 19 of wild-type RhoA was successfully mutated to asparagine,
with no other mutations. CHO-m3 cells were transfected with the
pEF-BOS-HA-RhoA, pEF-BOS-HA-RhoAVal-14, or
pEF-BOS-HA-RhoAAsn-19 plasmid and simultaneously
co-transfected with the pZeoSV2 plasmid coding for zeocin resistance
(Invitrogen, Carlsbad, CA), using LipofectAMINE according to the
manufacturer's instructions (Life Technologies, Inc.). After
transfection, the cells were cultured in complete medium for 3 days and
then passaged every 3 days in complete medium containing zeocin (500 µg/ml). Cell lines expressing HA-tagged wild-type or mutant RhoA were
detected by ECL Western blotting using HA antibody and cloned by
limiting dilution. Clonal lines were considered to be stably
transfected if they demonstrated expression of HA-tagged wild-type or
mutant RhoA in three independent ECL Western blots conducted over a
period of more than 1 month. The stably transfected clonal lines were
subjected to DNA sequencing to ensure that the cells expressed the
appropriate HA-tagged wild-type or mutant RhoA with the correct DNA
sequence. The levels of M3 mAChR expressed by the cells
were determined by measuring the binding of
[3H]N-methylscopolamine on the surface of live
cells and in total cell lysates, as described previously (25, 26).
Carbachol-induced Ca2+ mobilization in the cells was
determined by previously described methods using the fluorescent
Ca2+-binding reagent, Fura-2AM (27).
Measurement of Myosin-containing Stress Fiber Formation--
The
SA-2 human monoclonal autoantibody to the myosin heavy chain was used
as described previously to determine the intracellular distribution of
myosin (24). Cells were cultured on glass coverslips for 2 days in
complete medium and then incubated in complete medium in the presence
or absence of kinase inhibitors for 30 min before exposure to
carbachol, PMA, or no drug. After incubation for the designated times,
the cells were fixed by immersion in ice-cold acetone and incubated (30 min, 24 °C) in phosphate-buffered saline (PBS) containing 1% bovine
serum albumin (BSA) to block nonspecific antibody-binding sites. The
fixed cells were incubated (1 h, 24 °C) with the SA-2 antibody,
washed in PBS, and incubated (1 h, 24 °C) with fluorescein-labeled
anti-human IgM antibody diluted 1:100 in PBS, 1% BSA. In some assays,
nuclei were fluorescently labeled by propidium iodide (5 µg/ml) that
was present during the incubation with the secondary antibody. After
washing, the cells were mounted in PBS containing 90% glycerol and
0.1% p-phenylenediamine and examined by fluorescence
microscopy using a Nikon Optiphot fluorescence microscope.
The samples were scored for the presence of stress fibers by two
investigators without knowledge of the identity nor treatment of the
cell lines being examined. The investigators independently assigned
scores to the cells in 10 different microscope fields in each sample,
using the following values based on the presence of stress fibers in
the cells: 0 = no detectable stress fibers, 1 = few,
moderately defined stress fibers, 2 = well defined stress fibers,
and 3 = abundant, strongly defined stress fibers. The calculated
mean of the scores was used as a measurement of myosin-containing stress fiber formation.
Immunofluorescent Localization of PKC Isoforms--
Cells
cultured on glass coverslips in complete medium were incubated in the
absence or presence of drugs for the indicated times and fixed by
incubating for 10 min in 3.7% formaldehyde diluted in PBS containing
0.2% Triton X-100. After nonspecific antibody-binding sites were
blocked as described above, the cells were incubated (1 h, 24 °C)
with mouse monoclonal antibodies to PKC isoforms diluted in PBS, 1%
BSA. The cells were washed and incubated (1 h, 24 °C) with
fluorescein-labeled anti-mouse IgG antibody diluted in PBS, 1% BSA.
After washing, the cells were mounted and examined by fluorescence
microscopy as described above.
ECL Western Blotting--
Cells were lysed by periodic agitation
for 15 min in ice-cold lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 2.5 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40, pH 7.4) containing protease inhibitors (200 µM phenylmethylsulfonyl fluoride and 5 µg/ml leupeptin). The lysate was centrifuged (16,000 × g, 10 min, 4 °C), and the resulting supernatant was
boiled 5 min with sample buffer and subjected to SDS-polyacrylamide gel
electrophoresis using a 5% stacking gel and 10% separating gel.
Proteins in the gels were electrophoretically transferred to PVDF
membranes. The PVDF membranes were incubated overnight in blocking
buffer (10 mM Tris, 150 mM NaCl, 0.1% Tween
20, 10% dried milk powder, pH 7.6), placed in a 25-channel miniblotter
(Immunetics Inc., Cambridge, MA), and incubated (1.5 h, 4 °C) with
antibodies diluted in blocking buffer. After washing twice in wash
buffer (10 mM Tris, 150 mM NaCl, 0.1% Tween
20, pH 7.6), the PVDF membranes were incubated (1 h, 4 °C) with
horseradish peroxidase-labeled anti-mouse immunoglobulins diluted
1:4000 in wash buffer. The PVDF membranes were washed three times in
wash buffer, and bound antibody was visualized by ECL.
Measurement of MLC Phosphorylation--
The phosphorylation of
MLC in Triton X-100-insoluble lysates was determined as described
previously (28). Briefly, CHO-m3 cells were incubated for 16 h
with 5 µCi/ml inorganic 32P in phosphate-free Dulbecco's
modified Eagle's medium containing 1% heat-inactivated FCS. After
incubating with drugs for the appropriate times, the cells were lysed
in Triton X-100 lysis buffer (50 mM Tris-HCl, pH 7.4, 1%
Triton X-100, 200 µM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 10 mM sodium fluoride, 1 mM
sodium orthovanadate, 0.2 mM sodium PPi, and 10 mM Preparation and Use of Recombinant C3 Exoenzyme--
Recombinant
C3 exoenzyme was purified from bacteria transformed with the pGEX2T-C3
exoenzyme expression vector, as described previously (29). CHO-m3 cells
were electroporated in the presence or absence of recombinant C3
exoenzyme (15 µg/ml) by subjecting them to three electric pulses
(capacitance, 0.25 microfarads; voltage, 0.5 kV; duration, 100 µs)
using a Bio-Rad Gene Pulser. Electroporated cells were cultured in
complete medium for 2 days before being used in the different assays.
Measurements of [32P]ADP-ribosylation of endogenous Rho
by recombinant C3 exoenzyme were performed as described previously
(29). Briefly, lysates from cells electroporated in the presence or
absence of recombinant C3 exoenzyme were incubated (30 min, 37 °C)
with ribosylation buffer (100 mM Tris-HCl, pH 8.0, 20 mM nicotinamide, 10 mM thymidine, 10 mM dithiothreitol, 10 µM
[32P]NAD, 5 mM MgCl2, 5 ng of
recombinant C3 exoenzyme). The samples were mixed with 100 µl of 2×
sample buffer (125 mM Tris, 4% SDS, 20% glycerol, 10%
2-mercaptoethanol, 0.01% bromphenol blue, pH 6.8), boiled for 5 min,
and subjected to SDS-polyacrylamide gel electrophoresis using a 5%
stacking gel and 15% separating gel. The proteins were
electrophoretically transferred to PVDF membranes and subjected to ECL
Western blotting and autoradiography.
Myosin Reorganizes into Stress Fibers upon Activation of
M1 or M3 mAChR--
We tested the ability of
transfected M3 mAChR to regulate myosin organization in CHO
cells. Treatment with carbachol, which is an agonist for all mAChR
subtypes, induces myosin reorganization in the CHO-m3 subline
transfected with human M3 mAChR (Fig.
1). Myosin is diffusely distributed in
untreated CHO-m3 cells (Fig. 1, panel A) and becomes
peripherally localized within 15 min of carbachol exposure (Fig. 1,
panel B). The close proximity of the cells to one another
after treatment with carbachol for 15 min reflects cell-cell adhesion
induced by mAChR activation (20). Myosin-containing stress fibers
appear within 30 min of mAChR activation and remain prominent for
several hours in the presence of carbachol (Fig. 1, panel
C). The carbachol-treated cells initially exhibit increased cell
spreading but become elongated with less detectable myosin-containing
stress fibers after prolonged exposure to the agonist (Fig. 1,
panel D). These findings indicate that M3 mAChR
activation profoundly affects myosin organization.
Carbachol also induces myosin reorganization in CHO-m1 cells
transfected with human M1 mAChR but does not alter myosin
organization in untransfected CHO cells nor in CHO-m2 cells transfected
with the human M2 mAChR subtype (Fig.
2). These results indicate that only
mAChR subtypes which activate PKC induce myosin reorganization. Direct
activation of PKC with PMA induces myosin reorganization in all CHO
sublines (Fig. 2), indicating that PKC activation reorganizes myosin.
Stimulation of M1 or M3 mAChR induces the
formation of myosin-containing stress fibers to a greater extent than
does treatment with PMA (Fig. 2).
Phosphorylation of MLC alters myosin organization in a variety of cell
types (4-8,13-17). MLC phosphorylation also occurs in CHO-m3 cells
incubated with carbachol or PMA (Fig. 3).
Densitometry analysis indicates that MLC phosphorylation is increased
by 319 ± 80% in carbachol-treated cells and 199 ± 57% in
PMA-treated cells, compared with untreated cells (p < 0.05, n = 3). MLC phosphorylation is more effectively
induced by carbachol than by PMA in CHO-m3 cells (Fig. 3), consistent
with greater stress fiber formation induced by carbachol than by PMA
(Fig. 2).
Conventional PKC Isoforms Are Activated by M3 mAChR
Stimulation and Are Required for mAChR-mediated Myosin
Reorganization--
CHO-m3 cells were immunofluorescently stained with
antibodies to different PKC isoforms to determine whether mAChR
agonists or PMA induce PKC translocation, which indicates PKC
activation (30). Immunofluorescent staining of PKC-
Activation of PKC results in degradation of the enzyme; this effect is
most evident when PKC is activated by phorbol esters (31, 32), but it
can also occur when PKC is activated by Ca2+ and
diacylglycerol (33). Degradation of PKC isoforms was measured to
characterize further which isoforms are activated by PMA or M3 mAChR stimulation (Fig. 4, b and
c). PKC-
The effects of specific PKC antagonists on myosin reorganization were
investigated to determine the participation of different PKC isoforms
in carbachol-induced cytoskeletal reorganization (Fig.
5). Previous in vitro studies
using purified PKC isoforms indicate that conventional and novel PKC
isoforms are inhibited by calphostin C (IC50 = 0.05 µM) and chelerythrine (IC50 = 0.66 µM) (34, 35), whereas only the conventional PKC isoforms
are inhibited by Go6976 (IC50 = 0.006 µM)
(36). These PKC antagonists also inhibit other purified kinases such as
PKA when the antagonist concentrations exceed 50-170 µM
(34-36). Carbachol-mediated stress fiber formation in CHO-m3 cells is
inhibited by calphostin C (IC50 = 2.4 ± 0.3 µM), chelerythrine (IC50 = 8.0 ± 0.5 µM), and Go6976 (IC50 = 0.4 ± 0.02 µM) (Fig. 5). Calphostin C and chelerythrine inhibit both
cell spreading and stress fiber formation (Fig. 5a, panels C
and D), whereas Go6976 inhibits stress fiber formation but
not cell spreading (Fig. 5a, panel E). The
ability of Go6976 to inhibit mAChR-mediated stress fiber formation
indicates that conventional PKC isoforms are required for
mAChR-mediated stress fiber formation.
Intracellular Ca2+ mobilized by M3 mAChR
stimulation may enhance activation of conventional PKC isoforms, which
differ from other PKC isoforms by being sensitive to Ca2+
(reviewed in Ref. 30). Other Ca2+-dependent
kinases, such as Ca2+/ calmodulin-dependent
protein kinase II (CaMKII) or MLCK, may also participate in myosin
reorganization. The role of CaMKII in mAChR-mediated stress fiber
formation was tested using the antagonists KN-62 and KT5926. KN-62
specifically inhibits the in vitro activity of CaMKII
(IC50 = 0.90 µM) (37), whereas KT5926 inhibits the in vitro activities of several kinases,
including CaMKII (IC50 = 0.004 µM), MLCK
(IC50 = 0.018 µM), and PKC (IC50 = 0.72 µM) (38, 39). Myosin reorganization induced by
M3 mAChR activation is not affected by KN-62 at
concentrations up to 20 µM, but it is inhibited by KT5926
(IC50 = 0.6 ± 0.04 µM) (Fig. 5).
Interestingly, KT5926 induces the same morphological effects as the PKC
antagonist Go6976; the cells are well spread but lack detectable stress
fibers after M3 mAChR activation (Fig. 5a, panel F).
The effects of the MLCK antagonists ML-7 and ML-9 on mAChR-mediated
stress fiber formation were also determined (Fig. 5). ML-7 and ML-9
were previously found to inhibit MLCK activity (IC50 = 0.3 and 3.8 µM, respectively) as well as PKC activity
(IC50 = 42 and 54 µM, respectively) (40-42).
We found that mAChR-mediated stress fiber formation is inhibited by
ML-7 (IC50 = 75 ± 6.9 µM) and by ML-9
(IC50 = 82 ± 5.8 µM) (Fig. 5). Cells
treated with 40 µM ML-7 continue to exhibit
carbachol-induced stress fibers even though cell spreading is
diminished (Fig. 5a, panel G). Carbachol-induced stress
fibers are lost only when the cells are treated with high enough
concentrations of ML-7 to cause the cells to detach from the substratum
(Fig. 5a, panel H). Similar morphological effects were
induced by comparable concentrations of the MLCK antagonist ML-9 (data
not shown).
Inactivation of RhoA Diminishes but Does Not Abolish Stress Fiber
Formation Induced by M3 mAChR Activation--
The
involvement of RhoA in myosin organization was investigated using
CHO-m3 cells stably transfected with HA-tagged wild-type or mutant
RhoA. Two independent clonal CHO-m3 cell lines expressing HA-RhoA were
generated and named m3WTRho-1 and m3WTRho-11. Two independent clonal
CHO-m3 cell lines expressing constitutively active
HA-RhoAVal-14 were named m3CARho-1 and m3CARho-4, and three
independent clonal lines expressing dominant negative
HA-RhoAAsn-19 were named m3DNRho-2, m3DNRho-4, and
m3DNRho-6. These cell lines express similar levels of HA-tagged
wild-type or mutant RhoA (Fig. 6a). Two independent clonal
CHO-m3 cell lines stably transfected with only the pZeoSV2 plasmid,
named m3Zeo-1 and m3Zeo-2, do not express HA (Fig. 6a).
Similar levels of M3 mAChR are expressed by these cell
lines and parental CHO-m3 cells, as indicated by [3H]N-methylscopolamine binding (data not
shown). Proximal signal transduction by M3 mAChR is also
similar in these cell lines, as indicated by carbachol-induced
Ca2+ mobilization (Fig. 6b).
Expression of constitutively active RhoAVal-14 induces the
formation of myosin-containing stress fibers (Fig.
7a, panel D, and
b). Carbachol increases stress fiber formation in all the
sublines (Fig. 7). However, carbachol-induced stress fiber formation is diminished in cells expressing dominant negative
RhoAAsn-19, compared with the other cell lines (Fig.
7a, panel G, and b).
Since expression of dominant negative RhoAAsn-19 diminishes
carbachol-induced stress fiber formation (Fig. 7), we investigated the effects of expressing dominant negative RhoAAsn-19 on
PMA-induced stress fiber formation (Fig.
8). Interestingly, PMA treatment induces
similar increases in stress fiber formation in cells expressing
dominant negative RhoAAsn-19 and in the control cell lines
(Fig. 8). These results indicate that expressing dominant negative
RhoAAsn-19 does not alter PMA-induced stress fiber
formation.
Some reports indicate that ADP-ribosylation of Rho by C3 exoenzyme more
effectively produces an altered phenotype than expression of dominant
negative RhoAAsn-19 (43, 44). CHO-m3 cells were
electroporated with C3 exoenzyme to determine the effects of
inactivating Rho by ADP-ribosylation. The ability of C3 exoenzyme to
ribosylate Rho in CHO-m3 cells was tested in
[32P]ADP-ribosylation assays (Fig. 6c).
Ribosylation by C3 exoenzyme slows the migration of RhoA in Western
blots (lanes 1 and 2, Western blot,
Fig. 6c). Addition of C3 exoenzyme to lysates of untreated CHO-m3 cells causes [32P]ADP-ribosylation of a protein
(lane 1, Autoradiograph, Fig. 6c)
which co-migrates with the slower migrating form of RhoA (lane 1, Western blot, Fig. 6c). In contrast,
[32P]ADP-ribosylation is greatly diminished if the cells
are treated with C3 exoenzyme in vivo prior to adding C3
exoenzyme to the cell lysates (lane 2, Autoradiograph, Fig. 6c). These results indicate
that RhoA in cells treated with C3 exoenzyme is ADP-ribosylated in situ and subsequently cannot be
[32P]ADP-ribosylated by C3 exoenzyme in
vitro.
Inactivation of Rho with C3 exoenzyme inhibits CHO-m3 cytokinesis,
resulting in multinucleate cells (Fig. 9,
panel B). Although these cells are abnormally spread and
elongated, they exhibit a diffuse cytosolic distribution of myosin
similar to cells electroporated in the absence of C3 exoenzyme (Fig.
9). Activation of M3 mAChR induces the formation of stress
fibers in cells electroporated with C3 exoenzyme (Fig. 9, panel
D), although stress fiber formation is somewhat diminished
compared with untreated cells. These results indicate that myosin
reorganization induced by M3 mAChR activation is reduced
but not abolished by Rho inactivation.
This study demonstrates that M1 or M3
mAChR activation profoundly alters myosin organization in CHO cells.
Conventional PKC isoforms and Rho proteins participate in the
mAChR-mediated formation of myosin stress fibers, as depicted in the
model shown in Fig. 10. Similar
pathways of mAChR-mediated myosin reorganization occur in CHO cells and
smooth muscle cells, although some differences exist. Comparing these
pathways provides insight into the mAChR-mediated mechanisms
controlling myosin organization.
Role of PKC in mAChR-mediated Myosin Reorganization--
PKC
participates in mAChR-mediated myosin reorganization in CHO cells. This
conclusion is supported by our finding that M3 mAChR
stimulation or PMA treatment activates several PKC isoforms and induces
myosin stress fiber formation in these cells. Our finding that myosin
reorganization is induced by the M1 but not the
M2 mAChR subtype also supports this conclusion, since the M1 but not the M2 mAChR subtype activates PKC
(2, 3).
The effects of the PKC antagonists Go6976, calphostin C, and
chelerythrine provide compelling evidence that conventional PKC isoforms are required for myosin reorganization. Both mAChR-mediated stress fiber formation and cell spreading are inhibited by calphostin C
or chelerythrine, which antagonize conventional and novel PKC isoforms
(34, 35). In contrast, only mAChR-mediated myosin reorganization is
inhibited by Go6976, which specifically antagonizes conventional PKC
isoforms (36). It is generally believed that PKC activation is required
for cell spreading, and other signals are required for
cytoskeletal reorganization (45-51). Our findings suggest that
while the novel PKC isoforms are needed for cell spreading, the
conventional PKC isoforms are needed for myosin reorganization.
The concentrations of Go6976, calphostin C, or chelerythrine which
inhibit myosin reorganization in CHO cells are approximately 10-70-fold higher than those that inhibit PKC in vitro
(34-36). Higher concentrations of these drugs may be needed in
vivo for the antagonists to cross the cell membrane and gain
access to intracellular PKC, in contrast to in vitro studies
in which the antagonists interact directly with purified PKC. Despite
this requirement for higher drug concentrations in vivo,
these antagonists still inhibit myosin reorganization more effectively
in vivo than they inhibit other kinases such as PKA in
vitro. This finding indicates that these antagonists inhibit
myosin reorganization by inactivating PKC.
The ability of PKC to regulate myosin organization in CHO cells is
consistent with the effects of PKC activation on smooth muscle myosin.
Activation of PKC with phorbol esters enhances MLC phosphorylation in
smooth muscle cells and induces sustained contraction of different
smooth muscle tissues (reviewed in Ref. 10). Phosphorylation of MLC
increases actin-myosin interactions by inducing the formation of
bipolar myosin filaments and exposing actin-binding sites on myosin
(reviewed in Refs. 9-12). PKC can directly phosphorylate MLC (52, 53)
or enhance MLC phosphorylation by inhibiting MLC phosphatase (54). It
is believed that smooth muscle contraction induced by phorbol esters
involves the PKC-dependent inhibition of MLC phosphatase,
rather than direct phosphorylation of MLC by PKC (reviewed in Refs.
9-11). PKC activation may similarly enhance MLC phosphorylation in CHO
cells by inhibiting MLC dephosphorylation, as depicted in Fig. 10.
Although PKC is required for carbachol-induced myosin reorganization in
CHO-m3 cells, the role of PKC in the mAChR-mediated regulation of
smooth muscle myosin is less clear. PKC antagonists inhibit
carbachol-induced contractions in some types of smooth muscle (55) but
not in others (56). These variable responses may be due to the
expression of different mAChR subtypes or PKC isoforms by different
types of smooth muscle.
It was previously shown that PKC activation induces or enhances the
formation of actin-containing stress fibers in some types of non-muscle
cells (45, 47, 57) but not in others (58). These results indicate that
PKC activation has cell type-specific effects on stress fiber
formation. These specific effects may be due to altered PKC isoform
expression or dissimilarities in PKC-mediated signaling pathways among
different cell types.
Activation of M3 mAChR induces greater MLC phosphorylation
and myosin reorganization than does treatment with PMA in CHO cells. We
found that PKC- Role of RhoA in mAChR-mediated Myosin
Reorganization--
Increased stress fiber formation in cells
expressing constitutively active RhoAVal-14 indicates that
active RhoA contributes to myosin reorganization in CHO-m3 cells.
However, active RhoA is not essential for mAChR-mediated myosin
reorganization in CHO-m3 cells. This conclusion is supported by our
finding that carbachol-induced stress fiber formation is diminished but
not abolished in CHO-m3 cells expressing dominant negative
RhoAAsn-19 or treated with C3 exoenzyme. Expression of
dominant negative RhoAAsn-19 may specifically inhibit RhoA
activity because dominant negative RhoAAsn-19 competitively
interacts with RhoA regulatory proteins. In contrast, C3 exoenzyme can
ADP-ribosylate several forms of Rho, including RhoA and RhoB (59),
resulting in potentially greater Rho inactivation than that produced by
expressing dominant negative RhoAAsn-19. Consistent with
these possibilities, we found that the morphology of CHO-m3 cells is
altered more drastically by C3 exoenzyme than by expression of dominant
negative RhoAAsn-19.
Treatment with C3 exoenzyme causes CHO-m3 cells to become
multi-nucleate, which is an indication of Rho inactivation (60-62). If
Rho is inactive in dividing cells, the actomyosin contractile ring at
the cleavage furrow does not function properly and cytokinesis is
inhibited, resulting in multi-nucleate cells (61, 62). The ability of
carbachol to induce stress fiber formation in multi-nucleate, C3
exoenzyme-treated CHO-m3 cells provides strong evidence that mAChR-mediated stress fiber formation still occurs even when Rho is inactive.
Although Rho is apparently not essential for stress fiber formation
induced by mAChR activation, Rho may be required for stress fiber
formation induced by other agonists. Rho must be active for bombesin or
lysophosphatidic acid (LPA) to induce stress fiber formation in
serum-starved Swiss 3T3 cells (58, 63). Bombesin- or
LPA-dependent stress fiber formation is significantly
diminished by 27 µM genistein and is completely abolished
by 110 µM genistein, indicating that a tyrosine kinase is
required for stress fiber formation induced by these agonists (58). In
contrast, we found that genistein concentrations up to 180 µM do not alter mAChR-mediated stress fiber formation
(data not shown). These findings indicate that different Rho-mediated
signaling pathways leading to stress fiber formation are induced by
bombesin or LPA in serum-starved Swiss 3T3 cells and by mAChR
activation in exponentially proliferating CHO-m3 cells.
Rho inactivation diminishes the agonist-induced contraction of smooth
muscle (17, 64-68). Carbachol-induced contraction of tracheal (66),
ileal (68), and longitudinal intestinal (17, 67) smooth muscle strips
is diminished by treatment with C3 exoenzyme. Phosphorylation of MLC
induced by carbachol (17) or other agonists (reviewed in Ref. 10) is
also diminished by inactivating Rho in smooth muscle. These and other
findings support the model that Rho participates in contraction by
enhancing MLC phosphorylation (reviewed in Refs. 10-12). Active Rho
proteins can enhance MLC phosphorylation by inhibiting MLC phosphatase (69) or by activating Rho kinase, which directly phosphorylates MLC
(70, 71). We are investigating the possibility that Rho proteins
similarly regulate myosin organization in CHO cells by altering MLC
phosphorylation, as depicted in Fig. 10. This possibility is supported
by studies demonstrating that Rho inactivation diminishes MLC
phosphorylation in non-muscle cells (8, 72).
Rho inactivation in CHO-m3 cells does not diminish PMA-induced myosin
reorganization, even though it diminishes mAChR-mediated myosin
reorganization. These findings are consistent with a report that Rho
inactivation does not affect phorbol ester-induced contraction of
cerebrovascular smooth muscle but inhibits serotonin-induced contraction of the same tissue (64). These results may occur because
Rho and PKC participate in separate signaling pathways to regulate
myosin organization, as depicted in Fig. 10. According to this model,
PKC induces myosin reorganization independently of Rho. This model
explains why Rho inactivation does not affect PMA-induced myosin
reorganization but diminishes mAChR-mediated myosin reorganization.
This model also explains why Rho inactivation does not completely
abolish mAChR-mediated stress fiber formation; mAChR-mediated
activation of PKC induces myosin reorganization even when Rho is inactive.
We found that PKC antagonists completely abolish mAChR-mediated stress
fiber formation. This finding indicates that PKC inactivation inhibits
both Rho- and PKC-dependent myosin reorganization induced by mAChR stimulation. This result may occur because PKC inactivation inhibits mAChR-mediated signaling to Rho. PKC may act in parallel with
several other effectors to activate Rho following mAChR stimulation. If
this possibility is correct, then PKC antagonists should inhibit mAChR-mediated activation of Rho and subsequent
Rho-dependent myosin reorganization. This model does not
predict that PKC activation can independently activate Rho, because
other mAChR-mediated signals in addition to PKC activation may be
needed to stimulate Rho activity.
Role of MLCK in mAChR-mediated Myosin Reorganization--
Many
studies indicate that MLCK plays a central role in regulating myosin
activity in muscle and non-muscle cells (reviewed in Refs. 9-12).
Intracellular Ca2+ mobilized by M1 or
M3 mAChR stimulation can activate MLCK, resulting in MLC
phosphorylation and subsequent contraction of smooth muscle (13-16).
Activation of MLCK in non-muscle cells similarly affects myosin
activity (5, 6, 73). Thus, it is reasonable to assume that MLCK
participates in mAChR-mediated myosin reorganization in CHO-m3 cells.
However, the effects of the MLCK antagonists do not support the
assumption that MLCK contributes to mAChR-mediated myosin
reorganization in CHO-m3 cells. Very high concentrations of the MLCK
antagonists ML-7 and ML-9 are needed to inhibit mAChR-mediated stress
fiber formation in CHO-m3 cells. Carbachol-induced stress fiber
formation in CHO-m3 cells is half-maximally inhibited by 75 µM ML-7 or 82 µM ML-9 and maximally
inhibited by 100 µM ML-7 or 120 µM ML-9.
These concentrations exceed those needed to inhibit MLCK, PKA, or PKC
activity in vitro (40-42). The high antagonist concentrations needed to inhibit CHO-m3 microfilament formation do not
simply reflect an inability of the drugs to enter the cells. Other
studies demonstrated that concentrations as low as 3 µM ML-7 or 5 µM ML-9 can significantly affect in
vivo processes (74). We found that 40 µM ML-7
diminishes mAChR-mediated cell spreading (Fig. 5a, panel G),
indicating that effective intracellular concentrations of ML-7 and ML-9
accumulate in CHO-m3 cells even when the cells are exposed to
relatively low antagonist concentrations. Thus, the higher ML-7 and
ML-9 concentrations needed to inhibit mAChR-mediated stress fiber
formation (100 and 120 µM, respectively) indicate that
these MLCK antagonists may affect myosin organization by inactivating
other kinases, such as PKC.
Incubation of CHO-m3 cells with 2 µM KT5926 abolishes
mAChR-mediated stress fiber formation but not cell spreading (Fig.
5a, panel F). The effects of KT5926 are probably not
mediated by CaMKII, since the CaMKII antagonist KN-62 does not affect
mAChR-mediated myosin reorganization. Instead, KT5926 probably inhibits
myosin reorganization by diminishing MLCK or PKC activity. Choi and
colleagues (73) concluded that KT5926 inactivates both PKC and MLCK,
based on their finding that 1 µM KT5926 inhibits both
PKC-dependent and MLCK-dependent
phosphorylation of MLC in rat basophilic RBL-2H3 cells. This result is
consistent with our observation that KT5926 induces the same effects as
the PKC antagonist Go6976 in CHO-m3 cells (Fig. 5a, panels E
and F). Thus, the effects of KT5926 on mAChR-mediated myosin
reorganization may involve inactivation of PKC.
Although MLCK can be activated by mAChR-mediated increases in
intracellular Ca2+, it is possible that other kinases
diminish MLCK activity following mAChR stimulation. CaMKII can
phosphorylate MLCK and reduce the affinity of MLCK for
Ca2+/calmodulin, diminishing MLCK activity (reviewed in
Refs. 9-11). Thus, it is conceivable that mAChR stimulation does not
significantly increase MLCK activity in CHO-m3 cells, due to
concomitant mAChR-mediated activation of CaMKII. Previous studies
demonstrated that mAChR or PKC stimulation can induce smooth muscle
contraction in the absence of MLCK activity, indicating that MLCK
activity is not absolutely required for mAChR- or
PKC-dependent activation of myosin (75, 76).
The potential lack of MLCK participation in mAChR-mediated myosin
reorganization may explain why myosin-containing stress fibers form in
CHO-m3 cells only after prolonged exposure to carbachol. If mAChR
stimulation does not activate MLCK, MLC phosphorylation may only slowly
increase due to a PKC- and Rho-mediated inhibition of MLC phosphatase,
resulting in a gradual increase in myosin-containing stress fibers.
This possibility is consistent with reports that much slower smooth
muscle contractions are induced by carbachol or PMA when MLCK is
inactive, compared when MLCK is active (75, 76).
Conclusions--
This study provides the first evidence that
M1 or M3 mAChR activation induces myosin
reorganization in non-muscle cells. Both PKC and Rho participate in
mAChR-mediated myosin reorganization in CHO-m3 cells. PKC and Rho also
participate in the mAChR-mediated regulation of smooth muscle myosin.
These findings indicate that mAChR regulate myosin activity by similar
mechanisms in CHO cells and smooth muscle. The CHO sublines transfected
with mAChR subtypes provide a useful system for elucidating
receptor-mediated signals affecting myosin activity in both muscle and
non-muscle cells.
The M1 and M3 mAChR subtypes have a
unique ability to modulate the morphology (21, 22), adhesion (18-20),
and proliferation (25, 77-80) of non-muscle cells. Changes in myosin
activity also significantly alter the morphology, adhesion, and
division of non-muscle cells (reviewed in Ref. 12). The ability of
M1 or M3 mAChR to regulate non-muscle myosin
suggests that some of the unique effects of activating these receptors
may involve changes in cytoplasmic myosin activity.
We thank Dr. Shinya Kuroda (Nara Institute of
Science and Technology, Nara, Japan) for the generous gift of the
pEF-BOS-HA-RhoA and pEF-BOS-HA-RhoAVal-14 plasmids and Jeff
Mattison and Thomas Shay for excellent technical assistance.
*
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: Molecular Pharmacology
Laboratory, Guthrie Research Institute, Sayre, PA 18840. Tel.:
570-882-4650; Fax: 570-882-5151; E-mail:
cwilliam{at}inet.guthrie.org.
The abbreviations used are:
mAChR, muscarinic
acetylcholine receptor;
BSA, bovine serum albumin;
CaMKII, Ca2+/calmodulin-dependent protein kinase II;
CHO, Chinese hamster ovary;
FCS, fetal calf serum;
HA, hemagglutinin;
LPA, lysophosphatidic acid;
MLC, myosin light chain;
MLCK, myosin light
chain kinase;
PKA, protein kinase A;
PKC, protein kinase C;
PMA, phorbol 12-myristate 13-acetate;
PBS, phosphate-buffered saline;
PVDF, polyvinylidene difluoride.
M3 Muscarinic Acetylcholine Receptors Regulate
Cytoplasmic Myosin by a Process Involving RhoA and Requiring
Conventional Protein Kinase C Isoforms*
,,,
, and**
Molecular Pharmacology,
§ Neurobiology, and Molecular Biology,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), novel (
), and atypical (
) PKC isoforms are activated by
mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC
translocation or degradation. mAChR-mediated myosin reorganization is
abolished by inhibiting conventional PKC isoforms with Go6976
(IC50 = 0.4 µM), calphostin C
(IC50 = 2.4 µM), or chelerythrine
(IC50 = 8.0 µM). Stable expression of
dominant negative RhoAAsn-19 diminishes, but does not
abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells.
Similarly, mAChR-mediated myosin reorganization is diminished, but not
abolished, in CHO-m3 cells which are multi-nucleate due to inactivation
of Rho with C3 exoenzyme. Expression of dominant negative
RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does
not affect PMA-induced myosin reorganization. These findings indicate
that the PKC-mediated pathway of myosin reorganization (induced either
by M3 mAChR activation or PMA treatment) can continue to
operate even when RhoA activity is diminished in CHO-m3 cells.
Conventional PKC isoforms and RhoA may participate in separate but
parallel pathways induced by M3 mAChR activation to
regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by
M3 mAChR activation may contribute to the unique ability of
these receptors to regulate cell morphology, adhesion, and proliferation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate). The resulting insoluble
cytoskeletal pellets were washed twice with Triton X-100 lysis buffer
and dissolved by boiling in sample buffer for 30 min. The samples were
subjected to ECL Western blotting using a mouse monoclonal antibody to
the 20-kDa myosin light chain (Sigma), followed by autoradiography.
Densitometry was performed using a Storm PhosphorImager (Molecular
Dynamics, Inc., Sunnyvale, CA).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activation of M3 mAChR induces
myosin reorganization in CHO-m3 cells. CHO-m3 cells were incubated
in the absence (A) or presence of 10 µM
carbachol for 15 min (B), 90 min (C), or 24 h (D) and were immunofluorescently labeled with the SA-2
antibody to myosin. Bar represents 15 µm.
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Fig. 2.
Myosin reorganization is induced by carbachol
in CHO-m1 and CHO-m3 cells and by PMA in all CHO sublines.
a, untransfected CHO cells (A-C), and CHO-m1
(D-F), CHO-m2 (G-I), and
CHO-m3 (J-L) cells were examined. The cells were incubated
with no drug (A, D, G, and J), 10 µM carbachol (B, E, H, and K), or
10 nM PMA (C, F, I, and L) for 90 min
and immunofluorescently labeled with the SA-2 myosin antibody.
Bar represents 15 µm. b, the indicated CHO
sublines were incubated with 10 µM carbachol, 10 nM PMA, or no drug for 90 min and immunofluorescently
labeled with the SA-2 myosin antibody. The presence of
myosin-containing stress fibers in the cells was scored by two
independent investigators without knowledge of the identity nor
treatment of the cells. Results are the means ± 1 S.E. calculated
from three independent experiments.
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Fig. 3.
Phosphorylation of MLC is enhanced by
treatment with carbachol or PMA. CHO-m3 cells labeled with
inorganic 32P were incubated with 10 µM
carbachol, 10 nM PMA, or no drug for 60 min and lysed in
1% Triton X-100. The phosphorylated 20-kDa MLC in the Triton
X-100-insoluble pellet from cells treated with no drug (lane
1), carbachol (lane 2), or PMA (lane 3) was
identified by anti-MLC antibody in Western blots (a) and by
autoradiography (b). The Western blot has smaller lane
widths than the autoradiograph because the channels in which the
antibodies were applied in the Western blot were a smaller width than
the wells in which proteins were applied to the polyacrylamide gel.
Results shown are representative of three independent experiments which
produced similar results.
, -
, -
, and
- in CHO-m3 cells was undetectable, and immunofluorescent staining of PKC- and -µ produced a diffuse cytosolic pattern that was not
detectably altered by PMA or carbachol treatment (data not shown). In
contrast, PKC- is diffusely distributed in the cytosol of untreated
CHO-m3 cells (Fig. 4a, panel
A), and localizes to cell-cell junctions within 15 min of exposure
to carbachol (Fig. 4a, panel B). PKC- remains at
cell-cell junctions for over 24 h in the continuous presence of
carbachol (Fig. 4a, panel D). Treatment with PMA increases
PKC- at cell-cell junctions and at regions of membrane ruffling
(Fig. 4a, panel E). However, PKC- is
undetectable at cell-cell junctions or membrane ruffles after 24 h
of PMA treatment (Fig. 4a, panel F).
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Fig. 4.
Translocation and degradation of PKC isoforms
occurs in CHO-m3 cells treated with carbachol or PMA. a,
CHO-m3 cells were untreated (A) or incubated with 10 µM carbachol for 15 min (B), 45 min
(C), or 24 h (D) or incubated with 10 nM PMA for 15 min (E) or 24 h
(F). All cells were immunofluorescently labeled with an
antibody to PKC- . Bar represents 15 µm. b,
CHO-m3 cells were untreated, or incubated with 1 mM
carbachol or 1 µM PMA for 24 h and lysed. The
lysates were probed by ECL Western blotting using PKC isoform-specific
antibodies. Densitometry of the ECL Western blots was performed to
determine the percent of PKC isoform levels remaining in the
drug-treated cells compared with untreated control cells. Results are
the means ± 1 S.E. from three independent experiments.
c, representative ECL Western blots from the experiments
described in b.
, -, -, and -µ were detected by Western
blotting of CHO-m3 cell lysates. Levels of PKC- are significantly
diminished by PMA but only minimally affected by carbachol (Fig. 4,
b and c). This finding is consistent with the
sustained presence of translocated PKC- at cell membranes in
carbachol-treated CHO-m3 cells (Fig. 4a, panel D) and the
loss of translocated PKC- in PMA-treated cells (Fig. 4a, panel
F). Prolonged exposure to PMA or carbachol significantly
diminishes the levels of PKC- and - but does not diminish PKC-µ
(Fig. 4, b and c). These results indicate that
conventional (), novel (), and atypical () PKC isoforms are
activated by PMA or carbachol in CHO-m3 cells.
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Fig. 5.
Kinase antagonists inhibit the formation of
myosin-containing stress fibers in CHO-m3 cells. a, CHO-m3
cells were untreated (A) or incubated with 10 µM carbachol (B-H) for 90 min. Before being
exposed to carbachol, the cells were preincubated for 30 min with 4 µM calphostin C (C), 10 µM
chelerythrine (D), 2 µM Go6976 (E),
2 µM KT5926 (F), 40 µM ML-7
(G), or 100 µM ML-7 (H). All cells
were immunofluorescently labeled with the SA-2 antibody to myosin.
Bar represents 15 µm. b, CHO-m3
cells were preincubated for 30 min with no antagonist (control cells)
or with antagonists selective for PKC (A), CaMKII
(B), or MLCK (C). The cells were then incubated
with 10 µM carbachol for 90 min and immunofluorescently
labeled with the SA-2 antibody to myosin. The presence of
myosin-containing stress fibers in the cells was scored by two
independent investigators without knowledge of the cell treatments.
These scores were used to calculate the percent of carbachol-induced
stress fiber formation in cells exposed to antagonist, compared with
cells that were not exposed to antagonists. Results are the means ± 1 S.E. calculated from three to six independent experiments.
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Fig. 6.
Characterization of CHO-m3 cells stably
expressing HA-tagged wild-type or mutant RhoA or electroporated with C3
exoenzyme. a, equal amounts of lysates from the indicated
cell lines were probed with antibody to HA in ECL Western blots. A
representative ECL Western blot is shown. Similar results were obtained
in four other independent experiments. b, calcium
mobilization induced by 10 µM carbachol (applied at
30 s) is similar in parental CHO-m3, m3Zeo-2, and m3DNRho-4 cells.
Carbachol similarly mobilized Ca2+ in the other CHO-m3
sublines (data not shown). c, C3 exoenzyme ADP-ribosylates
RhoA in CHO-m3 cells. Lysates were prepared from parental CHO-m3 cells
cultured in vivo in the absence (lanes 1 and
3) or presence (lane 2) of C3 exoenzyme. The
lysates were incubated in vitro with [32P]NAD
in the absence (lane 3) or presence (lane 1 and
2) of C3 exoenzyme. The samples were probed with RhoA
antibody (Western blot) and subjected to autoradiography
(Autoradiograph). The striped and open
rectangles indicate the positions of RhoA that migrated slower and
faster, respectively.
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Fig. 7.
Stable expression of mutant RhoA alters
myosin organization in the CHO-m3 sublines. a, m3Zeo-2
(A and E), m3WTRho-1 (B and
F), m3DNRho-4 (C and G), and m3CARho-4
(D and H) cells were incubated in the absence
(A-D) or presence (E-H) of carbachol (10 µM, 90 min). The cells were immunofluorescently labeled
with the SA-2 myosin antibody. Bar represents 15 µm.
b, control cell lines (solid bars) and cell lines
expressing wild-type RhoA (open bars), constitutively active
RhoAVal-14 (dotted bars), or dominant negative
RhoAAsn-19 (striped bars) were incubated in the
absence or presence of 10 µM carbachol for 90 min. The
cells were immunofluorescently labeled with the SA-2 antibody to
myosin. The presence of myosin-containing stress fibers in the cells
was scored by two independent investigators without knowledge of the
identity nor treatment of the cells. Results are the means ± 1 S.E. calculated from two to six independent experiments.
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Fig. 8.
Expression of dominant negative
RhoAAsn-19 does not alter PMA-induced myosin
reorganization. a, m3Zeo-2 (A and B)
and m3DNRho-4 (C and D) cells were incubated in
the absence (A and C) or presence (B
and D) of PMA (10 nM, 90 min). The cells were
immunofluorescently labeled with the SA-2 myosin antibody.
Bar represents 15 µm. b, control cell lines
(solid bars) and cell lines expressing dominant negative
RhoAAsn-19 (striped bars) were incubated in the
absence or presence of 10 nM PMA for 90 min and
immunofluorescently labeled with the SA-2 myosin antibody. The presence
of myosin-containing stress fibers in the cells was scored by two
independent investigators without knowledge of the identity nor
treatment of the cells. Results are the means ± 1 S.E. calculated
from three independent experiments.
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Fig. 9.
Rho inactivation with C3 exoenzyme causes
CHO-m3 cells to become multi-nucleate, but does not abolish carbachol-
or PMA-induced myosin reorganization. CHO-m3 cells were
electroporated in the absence (A, C, and
E) or presence (B, D, and F) of
recombinant C3 exoenzyme (15 µg/ml) and cultured for 2 days. The
cells were incubated for 90 min with no drug (A and
B), 10 µM carbachol (C and
D), or 10 nM PMA (E and
F). Myosin was labeled with the SA-2 antibody, and nuclei
were labeled with propidium iodide. The left side of each
panel shows myosin and nuclei, and the right side of each
panel shows only nuclei. Bar represents 15 µm.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
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Fig. 10.
A model depicting the involvement of PKC and
Rho in myosin reorganization induced by M3 mAChR
activation. PKC and Rho may participate in independent but
parallel pathways to induce myosin reorganization following
M3 mAChR activation. We found that Rho inactivation
diminishes but does not abolish mAChR-mediated myosin reorganization.
This finding indicates that the PKC-mediated pathway of myosin
reorganization can continue to operate when Rho is inactive. In
contrast, PKC inactivation completely abolishes mAChR-mediated myosin
reorganization. This result may occur because PKC activity is required
for mAChR-mediated signaling to Rho. The depicted regulation of MLC
phosphorylation by PKC and Rho and the effects of MLC phosphorylation
on stress fiber formation are based on previous studies (reviewed in
Refs. 9-12).
remains at the junctions of carbachol-treated cells
longer than it does in PMA-treated cells, indicating that mAChR
stimulation and PMA treatment have different effects on PKC-. These
differences may contribute to the greater stress fiber formation
induced by carbachol compared with PMA, as well as the sustained
elongation of CHO-m3 cells which occurs during prolonged carbachol
exposure (Fig. 1, panel D, and Fig. 4a, panel D)
(21, 22).
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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D. Strassheim, R. A. Porter, S. H. Phelps, and C. L. Williams Unique in Vivo Associations with SmgGDS and RhoGDI and Different Guanine Nucleotide Exchange Activities Exhibited by RhoA, Dominant Negative RhoAAsn-19, and Activated RhoAVal-14 J. Biol. Chem., March 15, 2000; 275(10): 6699 - 6702. [Abstract] [Full Text] [PDF]
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 A. P Somlyo and A. V Somlyo Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II J. Physiol., January 15, 2000; 522(2): 177 - 185. [Abstract] [Full Text] [PDF]
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 N. Pausawasdi, S. Ramamoorthy, V. Stepan, J. del Valle, and A. Todisco Regulation and function of p38 protein kinase in isolated canine gastric parietal cells Am J Physiol Gastrointest Liver Physiol, January 1, 2000; 278(1): G24 - G31. [Abstract] [Full Text] [PDF]
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D. Strassheim and C. L. Williams P2Y2 Purinergic and M3 Muscarinic Acetylcholine Receptors Activate Different Phospholipase C-beta Isoforms That Are Uniquely Susceptible to Protein Kinase C-dependent Phosphorylation and Inactivation J. Biol. Chem., December 8, 2000; 275(50): 39767 - 39772. [Abstract] [Full Text] [PDF]
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