From the
Prostaglandin (PG) F
These data provide a new insight into
regulatory mechanisms of Ras-MAP kinase pathway through heterotrimeric
G-protein-mediated pathways.
Prostaglandin (PG)
Mitogen-activated protein (MAP) kinases (MAPKs) are activated during
differentiation and cell cycle transition triggered by a variety of
stimuli
(5) , thereby playing a key role in the kinase cascade
originating from receptor activation
(6) . MAPK seems to
transmit mitogenic signals by phosphorylating downstream components
such as transcription factors (c- myc (7) ,
c-jun (8) , and p62
Several subtypes of
We report here that in NIH-3T3 cells,
PGF
Each plasmid DNA was
transfected into NIH-3T3 cells by DEAE-dextran method, as described
(31) or using Transfectam, as described in the manual provided
by the supplier (Sepracor, Marlborough, MA). Typically, 2 µg of
plasmid DNA was transfected into 5
The immune complex MAPK
assay was carried out essentially as described
(32) . MAPK was
immunoprecipitated with anti-MAPK (erk2) monoclonal antibody, with the
aid of Pansorbin, washed with lysis buffer, and resuspended in the same
buffer.
In the transfection experiments, MAPK was partially purified
by batch treatment with Q-Sepharose; the cell lysate was mixed with 0.5
volume of Q-Sepharose beads equilibrated with lysis buffer containing
0.12
M NaCl for 30 min at 4 °C and then briefly
centrifuged (3,000
The
sample for MAPK assay was incubated with MBP (1 mg/ml) in 25 µl of
kinase buffer (20 m
M Tris-HCl, pH 7.5, 10 m
M MgCl
In the kinase
detection assay in the MBP-containing gel (gel kinase assay), the
supernatant of the cell lysate was electrophoresed onto an
SDS-polyacylamide gel containing 1 mg/ml MBP. Proteins were denatured
in 6
M guanidine-HCl and renatured as described previously
(33) . Phosphorylation of MBP was carried out in 5 ml of kinase
buffer containing 25 µCi of [
Immunoprecipitation and Western blot Analysis- Quiescent
cultures of NIH-3T3 cells on a 60-mm
The addition of 1 µ
M PGF
In NIH-3T3 cells, PGF
In the present study,
we found that PGF
MAPKs
are a family comprising enzymes with molecular mass between 40 and 58
kDa
(48) and are activated by phosphorylation of their
intrinsic tyrosine and threonine residues
(34, 49) .
Based on the results from a gel MAPK assay of the supernatant of cell
lysate (Fig. 3 A) and that of anti-42-kDa MAPK
immunoprecipitates (data not shown), a 42-kDa MAPK seems to be the main
MAPK activated by PGs in NIH-3T3 cells. We also noted tyrosine
phosphorylation of a MAPK with a molecular mass around 42 kDa by
Western blots with anti-42-, -43-, and -44-kDa MAPKs monoclonal
antibody of immunoprecipitates of cell lysates with
anti-phosphotyrosine Sepharose (Fig. 4).
Two pathways have
been proposed for MAPK activation: via MEKK activation and via Ras-Raf
activation. The pathway via MEKK and that via Ras-Raf may be linked to
tyrosine kinase receptors and G-protein-coupled receptors, respectively
(11) . However, activation of MAPK by various agonists via p21
and p74 in an IAP-sensitive fashion was reported
(15, 16, 17, 18, 19, 20) .
In the present study, we noted a small, but significant Ras activation
induced by PGF
With regard to the
regulatory mechanisms of MAPK activity, the activation of MAPK was
independent of extracellular Ca
It is important to question the role of
PGF
Our
results, taken together, suggest 1) that PGF
A typical result
from three independent experiments with similar results. Each datum is
the mean of duplicate samples. The radioactivity in the no addition
control sample (100%) was 1707 cpm.
Each datum is the
mean ± S.D. ( n = 3).
We thank Drs. Y. Gotoh and E. Nishida (Institute of
Virus Research, Kyoto University) for providing rMAPK, and M. Ohara for
helpful comments.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
activated
mitogen-activated protein (MAP) kinase and MAP kinase kinase in NIH-3T3
cells by a mechanism that was completely inhibited by protein kinase
inhibitors, staurosporine (20 n
M) or H-7 (20 µ
M),
but was insensitive to pretreatment with islet-activating protein (100
ng/ml; 24 h) or 12- O-tetradecanoylphorbol 13-acetate (2.5
µ
M; 24 h). PGF
stimulation also led to a
significant increase in Ras
GTP complex. Transfection of a cDNA
encoding a constitutively active mutant of G
-subunit
(Q209L) mimicked PGF
-induced MAP kinase activation,
increase in Ras
GTP complex, and DNA synthesis in these cells,
suggesting that activation of G
mediates the
PGF
-activation of Ras-MAP kinase pathway and
mitogenesis in NIH-3T3 cells.
(
)
F
stimulates cell proliferation in NIH-3T3 cells
(1, 2) . Activation of phospholipase C is to date the
only known biochemical signal via the G
-coupled
PGF
receptor
(2) . Such being the case, this
G
-coupled pathway is likely to be linked to the mitogenic
response, and NIH-3T3 cells may be a useful model system to examine
G-protein-mediated intracellular mechanisms linked to mitogenic
responses. Possible intracellular mechanisms that may explain
PGF
effects are the activation of specific
serine/threonine and/or tyrosine protein kinases (PKs). Activation of
phospholipase C leads to elevation of intracellular Ca
([Ca
]
) and/or
formation of diacylglycerol, which in turn leads to activation of
[Ca
]
/calmodulin-dependent
PKs and/or PKC, a family of multipotent serine/threonine kinases that
elicits cellular responses, including mitogenesis
(3) ,
respectively. We recently found that PGF
receptor-mediated,
[Ca
]
-dependent
tyrosine phosphorylation of cellular components including p125
correlated well with PGF
-induced mitogenesis
(4) . However, the entire spectra of intracellular PK cascades
and their target cellular proteins remained to be determined.
(9) ). A pathway leading from the tyrosine kinase receptor
to MAP kinase activation has been elucidated; ligand-receptor
interaction causes formation of the Ras
GTP complex, which in turn
activates a kinase cascade comprising p74
(10) , MAP kinase
kinase (MAPKK), and MAPK. However, another protein with MAP kinase
kinase kinase activity (MEKK) has been cloned
(11) . As
overexpressed MEKK can activate MAPKK without activating p74, p74 and
MEKK may converge on MAPKK. Recent studies revealed that MAPK is also
activated through heterotrimeric G-protein-mediated mechanisms
(12, 13) . It was suggested that receptor tyrosine
kinase may activate MAPKK via p21 and p72, while G-protein coupled
receptors may be linked to MEKK
(11) . This hypothesis is
supported by our recent observation that transfected
platelet-activating factor receptor cDNA into Chinese hamster ovary
cells mediates platelet-activating factor-induced activation of MAPK
and MAPKK without detectable increase in GTP form of Ras
(14) .
However, lysophosphatidic acid
(15, 16, 17) ,
thrombin
(16, 18) ,
2 adrenergic
(19) , and
M2 muscarinic
(20) agonists stimulate formation of the GTP form
of Ras and MAPK activity via an islet-activating protein
(IAP)-sensitive pathway. Moreover, MAPK activation by lysophosphatidic
acid can be blocked by dominant negative p21 or p74
(15) .
Therefore, MAPK can be activated by an IAP-sensitive G-protein-coupled
pathway that requires both p21 and p74. Even though MAPK activation
induced by endothelin via an IAP-insensitive G-protein has been
reported
(21, 22) , much less is known of the
involvement of p21 in MAPK activation dependent upon an IAP-insensitive
G-protein.
-subunit of IAP-insensitive
G-proteins have shown to link to phospholipase C-
; these include
-subunit of G
(G
) family
(G
, G11
, G14
, and G16
)
(23, 24, 25, 26) . Subtypes of
phospholipase C; phospholipase C-
1 and phospholipase C-
2, are
known to be stimulated by specific types of G
family.
Phospholipase C-
2 is also activated by
dimers of
G-protein
(26) . The activity of G
is regulated by the
exchange of GDP and GTP and by intrinsic GTPase activity. Agonist
binding to cell surface receptors stimulate G
by enhancing GDP-GTP
exchange, and the intrinsic GTPase activity reverses this state to
inactivate G
. It was reported that substitution of arginine 183 or
glutamine 209 of G
with cysteine or leucine,
respectively, constitutively activates G
by inhibiting
intrinsic GTP hydrolysis
(27, 28) . It was also reported
that NIH-3T3 cells stably expressing G
with a mutation
of conserved glutamine residue or overexpressing the wild-type of
G
exhibited transformation of the cells
(29, 30) . These recent advances have led us to direct
examination of the interrelationship between activation of
G
and PGF
-induced cellular responses
in NIH-3T3 cells.
stimulates MAPKK and MAPK by a mechanism that is
inhibited by staurosporine and H-7 but is independent of classical
TPA-sensitive PKC or Ca
/calmodulin-dependent PKs and
that G
and Ras may mediate this MAPK activation.
Materials
Materials were obtained from the
following sources: [-
P]ATP (specific
activity, >5,000 Ci/mmol) from Amersham Corp.;
[
H]thymidine (specific activity, 20.1 Ci/mmol)
and myo-[
H]inositol (specific activity,
45.1 Ci/mmol) from DuPont NEN;
P-labeled carrier-free
P
from ICN; myelin basic protein (MBP),
12- O-tetradecanoylphorbol 13-acetate (TPA), epidermal growth
factor (EGF), insulin, forskolin, staurosporine, H-7, W-7, and KN-62
from Sigma; IAP from Funakoshi Biochemicals (Tokyo);
1,2-bis(2-aminophenoxy)ethane- N, N, N`, N`-tetraacetic
acid tetraacetoxymethyl ester (BAPTA-AM) from Dojin (Kumamoto, Japan);
protein G-Sepharose and Q-Sepharose Fast Flow from Pharmacia LKB
Biotechnology Inc; anti-v-H-ras monoclonal antibody Y13-259 from
Oncogene Science; anti-MAPK (erk2) monoclonal antibody against murine
recombinant p42, anti-rat MAPK R2 (erk1-CT), and
anti-phosphotyrosine-conjugated Sepharose from Upstate Biotechnology
(Lake Placid, NY); Pansorbin from Calbiochem; Transfectam from Sepracor
(Marlborough, MA); P-81 phosphocellulose papers from Whatman;
polyethyleneimine-cellulose plates from Merck. PGF
,
PGE
, and PGD
were donated by Ono
Pharmaceuticals (Osaka, Japan). A cDNA encoding guinea pig
G
and that encoding a GTPase deficient mutant, in
which glutamine 209 was replaced with leucine
(27, 28) (M6 mutant), were subcloned into an expression vector
(pCDNA-1: Invitrogen Corp., San Diego, CA). All other chemicals were of
analytical grade. Reagents for cell culture were from Nissui (Tokyo)
and Life Technologies, Inc.
Cells and Transfection
NIH-3T3 cells were cultured
in Dulbecco's modified Eagle's medium (DMEM) containing 10%
fetal calf serum (FCS), under the conditions described elsewhere
(2) . The cells were washed 3 times with FCS-free DMEM and
cultured for 24 h before the assays of MAPKK and MAPK, analysis of
GTP-bound Ras, or Western blot analysis.
10
cells
(counted 1 day before transfection)/60-mm
dish. One day
after transfection, cells from one 60-mm
dish were seeded
onto two wells on a six-well coaster for the measurement of
[
H]thymidine incorporation and phosphoinositide
breakdown. Three days after transfection, the cells were washed 3 times
with DMEM without FCS and cultured in FCS-free DMEM for 24 h prior to
the measurement of [
H]thymidine incorporation,
phosphoinositide breakdown, MAPK assay, and analysis of Ras-bound GTP
and GDP.
MAPK Assay
Quiescent Cells were washed twice with
Tyrode buffer containing 20 m
M HEPES pH 7.4 and 1 m
M CaCl(HEPES-Tyrode) and then stimulated with agonists
in HEPES-Tyrode for the indicated times. After a wash with ice-cold
phosphate-buffered saline, the cells were lysed in ice-cold lysis
buffer (20 m
M Tris-HCl, pH 7.5, 25 m
M
-glycerophosphate, 2 m
M EGTA, 1 m
M phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 2 m
M dithiothereitol, and 1 m
M sodium orthovanadate) and
centrifuged at 10,000
g for 10 min. The supernatant
was used as the source of MAPK and MAPKK.
g for 5 min). The resultant pellet
was washed twice with the same buffer and incubated with the original
volume of lysis buffer containing 0.3
M NaCl for 30 min at 4
°C. MAPK was recovered in the supernatant by centrifugation.
, 1 m
M MnCl
, and 40 m
M ATP) containing 1 µCi of [
-
P]ATP
for 25 min at 25 °C. A 12-µl aliquot was spotted onto P-81
phosphocellulose paper and extensively washed with 0.5% phosphoric
acid. The paper was dried, and
P incorporation into MBP
was measured by Cerenkov counting
(14) .
-
P]ATP,
and the gel was extensively washed with 7% acetic acid. The dried gel
was subjected to an image analyzing system using FUJI BAS 2000.
MAPKK Assay
MAPKK activity was assayed by
measuring P incorporation into a kinase-negative mutant of
a recombinant Xenopus MAPK (rMAPK)
(34, 35) ,
which was kindly provided by Drs. Y. Gotoh and E. Nishida of Kyoto
University. MAPKK was partially purified by batch treatment with
Q-Sepharose; the cell lysate was mixed with 0.5 volume of Q-Sepharose
beads equilibrated with lysis buffer containing 0.12
M NaCl
and briefly centrifuged. Under these conditions, MAPKK activity was
recovered in the supernatant. The supernatant was incubated with rMAPK
(final concentration, 50 µg/ml) in 12.5 µl of the kinase buffer
containing 1 µCi of [
-
P]ATP for 20 min
at 25 °C. The sample was subjected to SDS-polyacrylamide gel
electrophoresis, and
P incorporation into the rMAPK band
was measured using a Fuji image analyzer (FUJI BAS 2000).
culture dish (3
10
) were washed twice with HEPES-Tyrode, treated
with each agonist in HEPES-Tyrode at 37 °C for 3 min and then
frozen in liquid nitrogen. The cells were lysed on ice in 1 ml of
solution containing 10 m
M Tris-HCl, pH 7.6, 5 m
M EDTA, 50 m
M NaCl, 30 m
M sodium pyrophosphate, 50
m
M NaF, 100 µ
M sodium orthovanadate, and 1%
Triton X-100 (immunoprecipitaion buffer). Cell lysates were centrifuged
at 15,000 rpm for 10 min. Resultant supernatants were precleared by
incubation with agarose at 4 °C for 1 h. After removal of agarose
by centrifugation at 15,000 rpm for 5 min, the supernatants were
incubated with 100 µl of anti-phosphotyrosine conjugated to agarose
at 4 °C for 4 h and then cleared by centrifugation at 15,000 rpm
for 5 min. Immunoprecipitates were washed 3 times with 1 ml of
immunoprecipitaion buffer and then treated with 30 µl of
Laemmli's sample buffer. After proteins were separated by 8%
SDS-polyacrylamide gel electrophoresis, Western blot analysis was
performed using 2 µg/ml of anti-Rat MAPK (erk1-CT) as the first
antibody and goat anti-rabbit IgG conjugated to horseradish peroxidase
(200-fold dilution) as the second antibody as described
(36) .
Analysis of Ras-bound GTP and GDP
Quiescent cells
were labeled with 0.1 mCi/ml P-labeled carrier-free
P
in phosphate-free DMEM medium for 4 h, and then
stimulated with agonists for 3 min, unless otherwise stated. Next, the
cells were lysed in Triton X-114 buffer (50 m
M HEPES-NaOH, pH
7.4, 1% Triton X-114, 100 m
M NaCl, 5 m
M MgCl
, 1 mg/ml bovine serum albumin, 1 m
M phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 100
µ
M GTP, 100 µ
M GDP, and 1 m
M ATP)
supplemented with phosphatase inhibitors (1 m
M sodium
pyrophosphate and 1 m
M sodium orthovanadate). Membrane-bound
Ras was recovered by detergent phase splitting, as described
(37) and immunoprecipitated with a monoclonal antibody
Y13-259 with the aid of protein G-Sepharose
(38) . The
immune complex was extensively washed with washing buffer (50 m
M HEPES-NaOH, pH 7.4, 0.1% Triton X-100, 0.05
M NaCl, 5
m
M MgCl
, and 1 mg/ml bovine serum albumin)
(37) supplemented with phosphatase inhibitors (1 m
M sodium pyrophosphate and 1 m
M sodium orthovanadate).
Guanine nucleotides bound to Ras were eluted and analyzed by thin-layer
chromatography on a polyethyleneimine-cellulose plate. The GTP/(GTP
+ GDP) ratio was measured using an image analyzer (FUJI BAS 2000).
Assessment of Phosphoinositide Breakdown
Formation
of inositol phosphates (IPs) for 1 min in the presence or absence of
PGFwas examined as described
(2) using the
methods of Bijsterbosch et al. (39) .
Assay for DNA
Synthesis
[H]Thymidine incorporation into
DNA was measured by the method of Nakamura et al. (40) , with slight modifications. Quiescent cells (6-well
coaster) were washed twice with DMEM at 37 °C and then incubated
for 24 h in 1 ml of DMEM in the presence or absence of
PGF
. One µCi of [
H]thymidine
was added to each dish 6 h before harvest.
[
H]Thymidine incorporation into trichloroacetic
acid-insoluble materials was determined.
Miscellaneous
Protein concentration was measured
by Bio-Rad protein assay kits using bovine serum albumin as the
standard. Statistical analyses were made by the procedure of analysis
of variance.
caused
a transient activation of MAPK and MAPKK with a peak at around 3 min,
while 1 µ
M TPA induced a time-dependent increase of MAPK
activity up to 30 min and EGF/insulin activated MAPK with a peak value
at around 5 min and a sustained phase within 60 min (Fig. 1). Dose
dependence of PGF
on MAPK and MAPKK activation with
ED
of around 10
M was similar
to that for elevation of
[Ca
]
, formation of
IPs, and [
H]thymidine incorporation, as described
previously
(2) (Fig. 2). Pretreatment of the cells with
IAP did not affect the dose-dependence of PGF
on MAPKK
activation, even under conditions that are assumed to ADP ribosylate
almost all of the IAP substrate, as described
(2) (Fig. 2). A gel MAPK assay of the supernatant of cell
lysate (see ``Experimental Procedures'') (Fig. 3 A)
and that of anti-42-kDa MAPK immunoprecipitates (data not shown) showed
that a 42-kDa MAPK was the main MAPK activated by PGs in the NIH-3T3
cells. We also found that MAPK and MAPKK were activated by PGs (1
µ
M); PGF
> PGE
,
PGD
, which correlates with the order of potencies of these
PGs to activate the elevation of
[Ca
]
, formation of
IPs, and [
H]thymidine incorporation, as described
previously
(2) (Fig. 3, A and B). These
two kinases (MAPK and MAPKK) were also activated by EGF (100
ng/ml)/insulin (1 µ
M)
TPA (1 µ
M) >
ionomycin (100 n
M), but not by forskolin (10 µ
M)
(Fig. 3, A and B). Western blot analysis with
anti-rat MAPK (erk1-CT) of immunoprecipitates with
antiphosphotyrosine-conjugated Sepharose of cellular lysates
demonstrated that a MAPK with a molecular mass around 42-44 kDa
was tyrosine phosphorylated (Fig. 4). The
PGF
-induced activation of MAPK was almost completely
inhibited by staurosporine (20 n
M) and by H-7 (20
µ
M) at concentrations that inhibit PKC
(41, 42) , but not by Ca
/calmodulin
kinase inhibitors; W-7 or KN-62, even at concentrations that inhibit
calmodulin-dependent kinases (20 µ
M)
(43, 44) ; nor by removing extracellular
Ca
. The
[Ca
]
chelator, BAPTA,
only partially attenuated PGF
-induced activation of
MAPK activity, even under conditions that almost completely prevented
the PGF
-induced elevation of
[Ca
]
(50
µ
M, 15 min)
(4) (Table I). Pretreatment of the
cells with TPA (2.5 µ
M, 24 h), which is assumed to
down-regulate classical PKC, did not affect the
PGF
-induced activation of MAPK ().
Figure 2:
Dose-response of PGFon
MAPK and MAPKK activities. Quiescent NIH-3T3 cells (3
10
/60-mm diameter culture dish) in HEPES-Tyrode stimulated
with the indicated concentrations of PGF
at 37 °C
for 3 min, and then immersed in liquid nitrogen. MAPK activity was
measured by immune complex assay (
) and MAPKK assay in control
cells (
) or in cells pretreated with IAP (100 ng/ml; 18 h)
(
) were performed as described under ``Experimental
Procedures.'' Each symbol represents the mean of duplicate
samples.
Figure 3:
Effects of various agents on MAPK
( A) and MAPKK ( B) activities. Quiescent NIH-3T3 cells
(3 10
/60-mm diameter culture dish) in HEPES-Tyrode
were stimulated with various agonists at 37 °C for 3 min. MAPK
activity of the supernatant of cell lysate measured by gel kinase
assay, and MAPKK assays were performed as described under
``Experimental Procedures.'' One µ
M of
PGF
, PGD
, PGE
, U46619,
iloprost or TPA, a combined use of 100 ng/ml EGF and 1 µ
M insulin ( EGF/Ins.), 100 n
M ionomycin
( Iono.), or 10 µ
M forskolin ( Forsk.) was
used as an agonist. MAPKK assay was done in the presence (+rMAPK)
or absence (-rMAPK) of rMAPK, as described under
``Experimental Procedures.'' Results typical of experiments
repeated at least three times are depicted.
Figure 4:
Western blot analysis with anti-rat MAPK
monoclonal antibody (erk-1 CT) of anti-phosphotyrosine
immunoprecipitates of NIH-3T3 cell lysates. Quiescent NIH-3T3 cells (3
10
/60-mm diameter culture dish) in HEPES-Tyrode
buffer were stimulated with 1 µ
M PGF
or
the combined use of 100 ng/ml EGF and 1 µ
M insulin at 37
°C for 3 min. Immunoprecipitation of cell lysates with
Sepharose-conjugated anti-phosphotyrosine (100 µl gel/dish) and
Western blot analysis with anti-rat MAPK (erk1-CT) (2 µg/ml) were
done as described under ``Experimental
Procedures.''
The
ratio of GTP/GTP + GDP bound to p21 was also significantly
increased by PGFor PGE
stimulation by
about 30% compared with the basal level, while that ratio was increased
by 78% with the combined use of EGF and insulin (Fig. 5).
Figure 5:
Effects of PGs and EGF/insulin on GTP form
of Ras in NIH-3T3 cells. Quiescent NIH-3T3 cells (3
10
/60-mm diameter culture dish) labeled with
P
carrier-free P
were stimulated with vehicle
( control), 1 µ
M PGF
, 1
µ
M PGE
, or the combined use of 100 ng/ml EGF
and 1 µ
M insulin ( EGF/Ins.) at 37 °C for 3
min. Labeling of the cells and analysis of Ras-bound GDP and GTP were
done as described under ``Experimental Procedures.''
A, a representative thin layer chromatogram from five
independent experiments, visualized using a Fuji image analyzer (FUJI
BAS 2000). B, the ratio of radioactive GTP/GTP + GDP
bound to p21 measured using an image analyzer system (FUJI BAS 2000).
Each column and bar represents the mean ± S.E.
( n = 6). Statistical analyses for differences between
agonist-stimulated groups versus control were made using the
procedure of analysis of variance. *, p < 0.05; **, p < 0.01.
In
order to test the involvement of G activation, we
performed transfection study using the M6 mutant cDNA, wild-type
G
cDNA, and the vector pCDNA-1.
[
H]Thymidine incorporation, IPs formation, MAPK
activity, and the ratio of GTP/GDP + GTP bound to p21 at the basal
levels ( open columns in Fig. 6, A-D,
respectively) were significantly higher in transfectants of the M6
mutant cDNA than in those of the vector control (172, 154, 122, and
155% versus the control, respectively).
PGF
-induced stimulation of
[
H]thymidine incorporation, IPs formation, and
Ras
GTP complex were not significant in transfectants of the M6
mutant, and PGF
-stimulation of MAPK in those
transfectants was less significant compared to the control cells
(stimulation by 21% ( p < 0.05) versus 42% ( p < 0.01)) (Fig. 6 B). There were no significant
differences in IPs formation, DNA synthesis, and MAPK activity between
transfectants of the wild-type G
cDNA and the vector
control.
Figure 6:
[H]Thymidine
incorporation ( A), the formation of IPs ( B), MAPK
activity (C), and the ratio of GTP/GDP+GTP bound to p21
( D) in NIH-3T3 cells transfected with the wild-type
G
cDNA (G
), those with the constitutively
active G
mutant cDNA (M6) and those with the vector:
pCDNA-1 ( control). Transfectants were treated with 1
µ
M PGF
( closed columns)
or vehicle ( open columns) in FCS-free DMEM
( A), HEPES-Tyrode containing 10 m
M LiCl ( B),
HEPES-Tyrode ( C), or FCS- and phosphate-free DMEM
( D), and each assay was done as described under
``Experimental Procedures.'' MAPK activity was partially
purified by batch treatment with Q-Sepharose ( C). The sample
numbers are three for each group in A and B, six for
each group in C, and eight for each group in D. Each
column and bar represent the mean ± S.E. Statistical differences
between the two groups were determined by the procedure of analysis of
variance. *, p < 0.05; **, p < 0.01; ***, p < 0.001. N.S., not
significant.
, PGD
, and
PGE
were reported to activate phospholipase C via a
G
-coupled receptor(s), which is assumed to be responsible
for cellular responses evoked by these PGs, such as mitogenesis
(2) . One mechanism that may participate in intracellular
signaling leading to mitogenesis is activation of the PK cascade
comprising specific tyrosine and/or serine/threonine PKs, an event that
occurs following receptor tyrosine kinase activation
(5) , even
though the precise intracellular signaling mechanisms triggering by
phospholipase C activation leading to mitogenesis remain to be
elucidated. The activation of phospholipase C may lead to PKC
activation by liberation of diacylglycerol from cleaved
phosphatidylinositol. Concomitantly, the creation of inositol
1,4,5-trisphosphates may elevate
[Ca
]
and subsequently
activate Ca
/calmodulin-dependent PKs. Tyrosine
phosphorylation of cellular components was also reported to be induced
by phospholipase C activating agonists such as vasoactive peptides or
by neuropeptides
(45) . We have recently demonstrated that
PG-evoked
[Ca
]
-dependent
tyrosine phosphorylation of cellular components via a
G
-coupled receptor pathway correlated with PG-induced DNA
synthesis
(4) . Thus, tyrosine phosphorylation of cellular
components may participate in signaling pathways to mitogenesis.
However, the identification of kinases and their target proteins has
not been made, except that p125
and/or p130 were seen to
function as substrates for tyrosine phosphorylated by bombesin,
vasopressin
(45) , or PGF
(4) in mouse
3T3 cells. MAPKs are considered to play key roles in kinase cascade
triggered by stimulation of receptor tyrosine kinases leading to cell
cycle transition and to differentiation
(5, 6, 22, 46, 47) . MAPKs are
also activated through heterotrimeric G-protein-mediated mechanisms
(15, 16, 17, 18, 19, 20) .
However, the effect of PG stimulation on MAPK cascade in the NIH-3T3
cell system has apparently not been reported.
, PGD
, and PGE
activate MAPK and the direct upstream activator, MAPKK, in
NIH-3T3 cells. Potencies of these PGs to activate MAPK and MAPKK
correlate with those that stimulate phospholipase C in NIH-3T3 cells
(2) (Fig. 3, A and B). Dose dependence
and sensitivity to IAP of PGF
on MAPKK and MAPK
activation are much the same as those for phospholipase C activation
(Fig. 2), supporting our conclusion that this activation of MAPK
is mediated via a G
-coupled receptor (see below).
and PGE
. The potencies of
PGF
and EGF/insulin to increase the GTP form of Ras
(30 and 78% versus the basal level, respectively) are
comparative to those that activate MAPKK activity (2.56- and 7.55-fold,
respectively). 1) Because PGF
did not inhibit
forskolin-induced cAMP formation,
(
)
neglecting a
possibility that the PGF
receptor is linked to G
in NIH-3T3 cells and 2) because IAP-pretreatment of the cells did
not affect PGF
-induced activation of MAPK activation,
even under conditions that all of the 41 kDa IAP-substrate
(G
) is assumed to be ADP-ribosylated (100 ng/ml; 24 h)
(2) , this PGF
-induced activation of Ras-MAPK
pathway may be mediated by an IAP-insensitive G-protein, G
,
instead of G
. This hypothesis was further supported by
transfection experiments; transfection of cDNA encoding an active
mutant of G
(27, 28) , but not that of
the wild G
, into NIH-3T3 cells led to constitutional
activation of MAPK activity and increase in GTP form of Ras, thereby
providing direct evidence of the G
-mediated activation
of the Ras-MAPK pathway. However, while no further increase in IPs
formation, Ras
GTP complex and DNA synthesis were observed with
PGF
in the M6 transfectants, the MAPK response appears
to show a PGF
-dependent increase in the M6
transfectants. Thus, it is still possible that other G-proteins may be
partly involved in this response. Neverthless, to our knowledge, this
is the first direct demonstration of Ras and MAPK activation by an
IAP-insensitive G-protein-dependent pathway.
and
Ca
/calmodulin kinases, but sensitive to PK inhibitors
staurosporine and H-7 (). These characteristics are similar
to those of the tyrosine phosphorylation of cellular components
including p125
and [
H]thymidine
incorporation, both induced by PGF
and PGE
(4) . However, the PGF
-induced tyrosine
phosphorylation is completely blocked by BAPTA under the same condition
as we used in the present study (50 µ
M, 15 min) and is
mimicked by ionomycin (0.1 µ
M)
(4) , while the
PGF
-induced MAPK activation was only partially
dependent upon [Ca
]
.
These results suggest that the tyrosine phosphorylation of major
cellular proteins and the activation of MAPK both induced by
PGF
stimulation are regulated by independent
mechanisms. PGF
-induced MAPK activation was not
affected by down-regulation of classical PKC with TPA-pretreatment of
the cells but completely inhibited by staurosporine and H-7 at
concentrations that inhibit PKC
(41, 42) . In relation
to these results, an isozyme of PKC, which was stimulated by acidic
phospholipids and phosphatidylinositol 3,4,5-trisphosphate, but not by
Ca
, TPA, or diacylglycerol, was purified
(PKC
)
(50) . In addition, a staurosporine-sensitive PK was
proposed for lysophosphatidic acid-induced Ras activation through an
IAP-sensitive G-protein in Rat-1 fibroblasts
(17) . Thus, it is
possible that PKC
or an unidentified staurosporine-sensitive PK
may participate in PGF
-induced activation of MAPK in
NIH-3T3 cells. This PK will need to be identified if intracellular
mechanisms of G-protein-mediated Ras-MAPK activation are to be
understood.
-MAPK cascade on PGF
-induced cell
proliferation. In the present work, we found that the
PGF
-induced MAPK activation has characteristics
similar to the PGF
-induced DNA synthesis as the
PGF
-induced tyrosine phosphorylation of major cellular
proteins including p125
(4) . At present, we
cannot completely answer what pathway is the main one leading to
mitogenesis. In relation to this question, however, transient
transfection of a cDNA encoding an active mutant of G
(27, 28) leads to a constitutional activation of
phospholipase C, Ras-MAPK pathway, and
[
H]thymidine incorporation, and attenuates
further enhancement of these parameters by PGF
(Fig. 6). Therefore, the PGF
-induced
activation of Ras-MAPK pathway may, at least in part, play some role in
PGF
-induced cellular responses such as cell
proliferation. Recent reports demonstrated that expression of a mutant
G
or over-expression of the wild G
in
NIH-3T3 cells induced transformation in the presence of FCS
(29, 30) . We used NIH-3T3 cells in quiescent culture
under FCS-free conditions to assess PGF
effects on
mitogenesis of NIH-3T3 cells. It is, therefore, suggested that the
over-expression of the wild G
per se has no
apparent effect on DNA synthesis in the absence of agonists
(Fig. 6 A), but it did have transforming effects with
continuous exposure to mitogens. We also compared the contents of
phosphotyrosyl cellular proteins in transfectants of the M6 mutant cDNA
and those of the vector, but we found no significant differences
between these two types of transfectants, regardless of whether the
cells were stimulated with PGF
(data not shown).
Therefore, PGF
-induced
[Ca
]
-dependent
tyrosine phosphorylation may not be mediated through G
activation, and may be independent of cell proliferation. However, it
is also possible that PGF
-induced tyrosine
phosphorylation may be mediated through the action of free
dimers, as shown in case of phospholipase C-
2 activation
(51) , and may lead to DNA synthesis in this cell line. Other
studies using stable transformants of the M6 mutant, those of a
dominant inhibitory mutant of G
, or those of specific types
of
and
subunits
(51) need to be tested.
activates
MAPKK and MAPK by a mechanism that depends partially on
[Ca
]
and totally on a
PK(s) sensitive to staurosporine and to H-7, but not on classical PKC
nor calmodulin-dependent PKs, 2) that G
and Ras may be
involved in this PGF
-induced MAPK activation, and 3)
that this PGF
receptor-Ras-MAPK cascade may be
responsible for the effects of PGF
in NIH-3T3 cells.
Table:
Effects of various inhibitors on
PGF-induced activation of MAPK
Table:
Effects of PKC down-regulation on
PGF-induced activation of MAPK
,
-subunit of
G-protein; IPs, inositol phosphates; DMEM, Dulbecco's modified
Eagle's medium.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.