Biphasic and Synergistic
Activation
of p44mapk (ERKI) by Growth Factors:
Correlation
between Late Phase
Activation
and Mitogenicity
Sylvain Meloche*,
JacquesPouyssegur
Klaus
Seuwen,
Gilles
Pages,
and
Centre de Biochime-CNRS
Universiti, de Nice
06108 Nice Gdex 2, France
G protein-coupled
receptors are thought to converge
at the level of serine/threonine
kinases and to initiate a
complex network of protein phosphorylation
events (1).
Protein kinase cascades involved in the mitogenic stimulation of ribosomal protein S6 phosphorylation
have
been clearly demonstrated
(2-5). The two best candidates for cytoplasmic serine/threonine
kinase integrators are Raf-1 (6, 7) and mitogen-activated
protein
kinases (mapk).
MAP kinases, also described as microtubule-associated protein 2 (MAP2) kinases, myelin basic protein
(MBP) kinases, or extracellular signal-regulated
kinases
(ERK), are a family of serine/threonine
kinases that are
rapidly activated in response to all mitogens tested
(reviewed in Refs. 2, 5). Similar protein kinase activities
with specificity for MBP are also activated during oocyte
maturation (8-10). The notion of a family of MAP kinases was clearly demonstrated
by the recent cloning
of cDNAs coding for three related mammalian MAP
kinases, ~44”“~” (ERKl), ~42”“~~ (ERK2), and ERK3
(11-l 4). The sequence of a MAP kinase homolog from
Xenopus oocytes, Xp42, was also reported (10). Sequence analysis has revealed that MAP kinases are
closely related to the yeast kinases KSSl (15) and
FUS3 (16) which are involved in the regulation of pheromone-induced
G, arrest, and to ~34”~“‘.
Dual phosphorylation
of ~42”~~~ on both tyrosine and
threonine residues was shown to be required for full
activity of the enzyme (17). The sites of phosphorylation
of ~42”“~” were identified and found to reside on a
single phosphopeptide
separated by only one residue
(18). Despite these findings, the exact mechanism of
activation of MAP kinases is still unclear. Two groups
have reported that recombinant
~42”~~~ and ~44~~~“,
purified from fscherichia
co/i, display basal intramolecular autophosphorylation
on both tyrosine and threonine residues (19, 20). However, the rate and extent of
phosphorylation
and activation are very low as compared to in vivo situations, suggesting the requirement
for upstream activators. On the other hand, Gomez and
Cohen (21) have recently identified NGF-stimulated
MAP kinases which are dependent on serine/threonine
We have examined the phosphorylation
and protein
kinase activity of p44 mitogen-activated
protein kinase (~44~~~~) in growth factor-stimulated
hamster
fibroblasts
using a specific antiserum.
The activity
of p44mapk was stimulated
both by receptor tyrosine
kinases and G protein-coupled
receptors.
Detailed
kinetics revealed that cu-thrombin induces a biphasic
activation of p44 mapk in CCL39 cells: a rapid phase
appearing
at 5-10 min was followed by a late and
sustained phase still elevated after 4 h. Inactivation
of cr-thrombin with hirudin after 30 set, which prevented DNA synthesis, did not alter the early ~44~“~“
response but completely
abolished
the late phase.
Pretreatment
of the cells with pertussis toxin, which
inhibits by more than 95% a-thrombin-induced
mitogenicity,
resulted
in the complete
loss of late
phase activity, while the early peak was partially
attenuated.
Treatment
of CCL39 cells with basic
fibroblast growth factor also induced a strong actiwhich is not a mitogen
vation of ~44”~~~. Serotonin,
by its own, had no effect on late phase ~44~‘~~
activity, but synergized
with basic fibroblast
growth
factor to induce late kinase response and DNA synthesis. Both early and late phase activation
of
p44”apk were accompanied
by tyrosine phosphorylation of the enzyme. Together,
the results indicate
that there is a very close correlation
between
the
ability of a growth factor to induce late and sustained
p44map’ activation and its mitogenic potential. Therefore, we propose that sustained
~44~~~~ activation
is an obligatory event for growth factor-induced
cell
cycle progression.
(Molecular
Endocrinology
6: 845
854, 1992)
INTRODUCTION
The multiple signaling pathways initiated by growth
factors interacting with receptor tyrosine kinases and
0888-8809/92/0845-0854$03
00/o
Molecular
Endocmology
CopyrIght 0 1992 by The Endocrine Society
845
MOL END0.1992
Vol6
No. 5
846
phosphorylation for their activity. Most interestingly,
these kinases were shown to promote the phosphorylation of both serinelthreonine and tyrosine residues
on MAP kinases.
Although the exact functions of MAP kinases in cellular regulation are not known, several arguments suggest that ~42~~~~ and ~44~~~~ are likely to be critical
elements for cell cycle progression in mammalian cells.
First, they are rapidly activated in response to all mitogens. Second, they were shown to phosphorylate and
activate in vitro phosphatase-inactivated S6 kinase (22,
23) and S6 peptide kinase activity (24). Furthermore,
Chung et a/. (25) have recently identified two kinases,
analogous to p42mapkand p44mapk,capable of phosphorylating and partially activating pp90’Sk S6 kinase in
mouse fibroblasts. Third, they are related in term of
structure and regulation to ~34’~” and yeast KSSl and
FUS3, protein kinases involved in cell cycle control.
In this study, we specifically examined the phosphorylation and enzymatic activation ~44~~~~in growth factor-stimulated hamster fibroblasts. Our results show
that mitogenic signals originating from distinct signaling
pathways lead to a biphasic and sustained activation
of p44mapk. We demonstrate that there is a strong
correlation between late and sustained activation of
p44ma+ and DNA synthesis.
RESULTS
kD
97
66
PM
P 42
45
31
Fig.
1. Specific lmmunoprecipitation
of ~44~“~
with Antibody
837
Confluent
CCL39
cells were lysed in Triton X-100 lysis
buffer. Lysates
were incubated
overnight
at 4 C with 10 ~1
antiserum
837 preadsorbed
to protein A-Sepharose.
Immune
complexes
were washed as described
in Materials
and Metbods and resuspended
in Laemmli sample buffer. Proteins from
cell lysates and immunoprecipitates
were separated
on 10%
acrylamide/6
M urea gels
and transferred
to nitrocellulose
membranes.
Membranes
were probed with antibody
837 and
the proteins visualized
using a horseradish
peroxydase
detection system.
Lanes 1 and 3, immunoprecipitates from 800 pl
and 400 pl cell lysate, respectively;
lanes 2 and 4, 60 ~1 total
cell lysate. Molecular
weight standards
are shown on the right.
Arrows indicate the position of ~44”“~
and ~42”‘“.
Specific Assay for ~44~‘~~ Activity
Thrombin induces Biphasic Activation of p44mapk
Chromatographic evidence (12, 26-29), as well as immunological data (29) indicate that at least two enzymes, with molecular masses in the range of 41-42
kilodalton (kDa) and 43-45 KDa, contribute to the observed stimulation of MAPyMBP kinase activity in response to growth factors. We have examined the relative abundance of these two kinases, referred to as
p42”“pk (ERK2) and p44mapk (ERKl), in the Chinese
hamster fibroblast cell line CCL39 using antiserum 837
(29). This polyclonal antibody, raised against a C-terminal peptide from the predicted ERKl sequence, recognizes both the p42 and p44 MAP kinases in immunoblotting experiments. Figure 1 (lanes 2 and 4) shows
that both kinases are expressed in CCL39 cells, with
the predominant protein being ~44”“~~. In order to specifically investigate the regulation of ~44~~~~activation,
we developed an immune-complex kinase assay using
antibody 837 to specifically immunoprecipitate ~44”“~~
from cell lysates. The validation of the assay is shown
in Fig. 1. Western blot analysis of CCL39 cell lysate
after immunoprecipitation with antibody 837 showed
the presence of a single ~44~~~~band with no detectable 42-kDa band. These results are consistent with
previous observations on the specificity of the antibody
(2%
We first examined the effect of human a-thrombin, a
very strong mitogen for CCL39 cells (30) on the activation state of p44”“pk. CCL39 cells were made quiescent by serum deprivation, stimulated with cy-thrombin,
lysed, and incubated with antibody 837. MBP kinase
activity was then assayed on the immunoprecipitate.
Detailed kinetic analysis showed that stimulation with
10 nM cu-thrombin invariably resulted in a biphasic activation of p44mapk(Fig. 2A). The first phase reached a
maximum between 5-10 min, decreased to a minimum
value around 60 min, and was followed by a sustained
phase reaching a peak at 2 h and slowly declining
thereafter. The MBP kinase activity was still elevated
(2- to 4-fold) over basal level at 4 h. The stimulatory
effect of a-thrombin was dose-dependent, resulting in
up to 8-fold activation of ~44~~~~ at 10 min (Fig. 28).
Half-maximal stimulation was observed between 0.1
and 1 nM.
To determine whether the activation of ~44~~~” correlated with increased phosphorylation of the protein,
32P-labeled quiescent CCL39 cells were stimulated with
cY-thrombin, lysed, and ~44”“~~ was immunoprecipitated
with antibody 837. Little 32P labeling of the protein was
detected in immunoprecipitates from untreated cells
(Fig. 3A, lane 1). Treatment of the cells with 10 nM (Ythrombin caused a large increase in the phosphorylation
Sustained
Activation
of ~44~~~”
by Growth
a47
Factors
3
2
1
0
I
I
I
0
1
2
;ME
3
I
I
(h;
and most probably serinelthreonine
phosphorylation
of
the protein.
a-Thrombin-induced
early signaling events and DNA
synthesis have been shown to require the continual
presence of catalytically active a-thrombin for 4 h or
more (31, 32). We therefore tested the dependence of
the p44 mapk response on the presence of active LYthrombin by using hirudin, a high affinity inhibitor that
rapidly inactivates the protease (33, 34). Addition of a
1 O-fold excess neutralizing
concentration
of hirudin
after 30 set cy-thrombin stimulation did not attenuate
the early ~44”~~~ response but completely abolished
the late sustained activation of the kinase (Fig. 4).
Hirudin had no effect on fibroblast growth factor (FGF)stimulated MAP kinase activity (not shown).
Pertussis Toxin Inhibits Both Phases of
a-Thrombin-Induced
~44~~~~ Activation
B
l -----•
‘L1
l
1
i
/
/
0 -IHA
0
d ,17--0
12
11
10
9
-log [THR]
8
(M)
Fig. 2. Biphasic Activation
of ~44”“~”
by a-Thrombin
Quiescent
CCL39 cells were stimulated
with a-thrombin
for
the indicated times, washed rapidly, and lysed in Triton X-l 00
lysis buffer. Cell lysates were incubated
for 2 h at 4 C with
antibody
837 preadsorbed
to protein A-Sepharose.
lmmuno
complexes
were washed,
and ~44”~p” activity was assayed
directly by incubation
with [r3’P]ATP
and MBP as described
in Materials
and Methods.
Samples
were resolved
on 12%
acrylamide
gels, and phosphate
incorporation
was quantified
by counting the radioactivity
of excised substrate
bands. Protein kinase assays were linear over the time of the assay, and
less than 20% variation
were found in kinase activity
from
independent
immunoprecipitation
and independent
wells. A,
Time course
of a-thrombin
(10 nM) stimulation
of ~44”“~~
activity. B, Dose-response
curve of ~44”“~” activation
after 10
min stimulation
with a-thrombin.
Similar results were obtained
in four separate
experiments.
of p44m=pk, which was observed both at 10 min (early
phase) and 120 min (late phase) of stimulation (Fig. 3A,
lanes 2 and 4). To evaluate if this phosphorylation
included tyrosine residues, the gels were treated with
1 M KOH before autoradiography.
Although not quantitative,
this treatment resulted in a reduction of total
32P labeling and revealed the presence of an alkalistable ~44~~~~ band both at 10 min and 120 min of (Ythrombin stimulation (Fig. 38, lanes 2 and 4). These
results indicate that biphasic activation of ~44”“~~ by (Ythrombin is accompanied
by tyrosine phosphorylation
We have previously shown that pertussis toxin was
capable to inhibit up to 95% of cY-thrombin-induced
reinitiation of DNA synthesis in CCL39 cells (30). Therefore, it was of interest to investigate the effect of the
toxin on both phases of ~44~~~ activation. Quiescent
CCL39 cells were treated with a saturating concentration of pertussis toxin before stimulation with 10 nM (Ythrombin for 2 h and for 10 min in the continuous
presence of the toxin. Figure 5A shows that pertussis
toxin completely
inhibited the late phase of ~44”‘~~~
activation, while the early phase was only partially
attenuated.
The same toxin treatment
reduced (Ythrombin-induced
DNA synthesis by over 95% (Fig. 58).
We also determined whether the inhibition of kinase
activity was paralleled by a reduction in the phosphorylation state of the ~44”~~~ protein. Under the same
conditions,
pertussis toxin partially reduced the LYthrombin-stimulated
32P labeling of ~44~~~~ at 10 min,
while the phosphorylation
of the protein was totally
prevented at 120 min (Fig. 3A). The same inhibition was
reflected on the phosphotyrosine
content of the kinase
(Fig. 38). These results strongly suggest the involvement of a member of the Cl/G0 family of pertussis toxinsensitive G proteins in the mechanism of activation of
p44”ap” by cu-thrombin.
Serotonin Potentiates
Basic FGF
the Activation
of ~44~~~~ by
Basic FGF is a strong mitogen for CCL39 cells. Serotonin, on the other hand, does not stimulate DNA
synthesis alone, but strongly potentiates
basic FGF
mitogenicity.
This effect is mediated through 5-HTlb
receptors negatively coupled to adenylyl cyclase (35).
We thus examined the relative effects of basic FGF and
serotonin on p44mapk activity to investigate whether
similar synergism could be observed on the kinase
response. Figure 6 shows that addition of basic FGF to
quiescent CCL39 cells caused a strong dose-dependent stimulation of p44”apk activity with a half-maximal
effect observed between 3-10 rig/ml. Serotonin alone
MOL ENDO. 1992
848
Vol6 No. 5
a - Thrombin Stimulates Phosphorylation
on Tyrosine
--+-+
of p44mapk
PTX
;:
,-
:
..\,
\_
_ 2.
\,
:*,,i‘ib
\
kDa
- 97.4
- 66.2
-.
+ p44m*pk -)
C
-II
--
10 min 120 min
10 min 120 min
Thrombin
C
Thrombin
Fig. 3. a-Thrombin Stimulates the Tyrosine Phosphorylation of ~44”‘~~
Quiescent CCL39 cells were labeled with 32Pi and stimulated or not (C) with 10 nM cu-thrombin for 10 min or 120 min. When
present, pertussis toxin was added for the labeling period. The cells were lysed with Triton X-l 00 lysis buffer, and the lysates were
incubated overnight at 4 C with antibody 837 preadsorbed to protein A-Sepharose. Immune complexes were washed, and the
proteins were analyzed by SDS-gel electrophoresis on 10% acrylamide gels. A, Autoradiogram of total 32P-labeled proteins after 1
day exposure. B, Autoradiogram of a duplicate gels pretreated with 1 M KOH after 4 days exposure. Molecular weight standards
are shown on the left. The arrow indicates the position of p44”@. Similar results were obtained in two separate experiments.
Fig. 4. Effect of Hirudin on a-Thrombin Stimulation of ~44”“~
Activity
Quiescent CCL39 cells were stimulated with 10 nM 01thrombin for 30 set, at which time the medium was rapidly
aspirated and replaced with new medium containing 100 nM
hirudin. The cells were incubated for the remainder of the times
indicated. ~44”“” was immunoprecipitated from the cell lysates, and MBP kinase activity was measured as described in
the legend of Fig. 2.
weakly increasedkinase activity (-e-fold), but a more
than additive effect was observed in the presence of
30 rig/ml basic FGF. When lower concentrations of
basic FGF were used, a marked synergy between the
two agents was detected (Fig. 7). This effect was
mostly evident after prolonged times of stimulation.At
120 min, no effect of serotoninalonecould be observed
on p44”“pkactivity, while the hormonesignificantly potentiated the stimulatory effect of basic FGF.
Beside 5-HT,,, receptors, CCL39 cells also express
5-HT2receptors coupledto phospholipaseC activation.
In order to identify the receptor(s) and the subsequent
signalingpathway(s) involved in the stimulatory effect
of serotonin on p44mapk
activity, we tested the effect of
the 5-HT2 receptor antagonist ketanserin(36). Saturating concentration of ketanserinalone did not influence
kinaseactivity but inhibitedthe early phaseof serotonin
stimulation by 70% (Fig. 7). The synergistic effect of
serotonin and basic FGF was inhibited by 40-50%.
These results indicate that the stimulator-yeffects of
serotonin on p44mapk
are mediated both by the 5-HT2
and 5-HT,Breceptors.
We also determinedwhether the synergisticeffect of
serotonin and suboptimalconcentration of basic FGF
Sustained
Activation
of p44mapk by Growth
Factors
849
A
B
--_0
--__
0
10 mln
C
C
120 min
Thrombln
1
mrombin
Fig. 5. Inhibition of ~44”“~” Activation
by Pertussis
Toxin
A, Quiescent
CCL39 cells were pretreated
with (R) or without (U) pertussis
toxin before stimulation
with medium (C) or 10 nM
cu-thrombin
for 10 min or 120 min. P44mapk was immunoprecipitated
from the cell lysates, and MBP kinase activity was measured
as described
in the legend of Fig. 2. Similar results were obtained
in three separate
experiments.
B, Quiescent
CCL39 cells were
pretreated
(Bl) for 4 h with pet-tussis toxin before stimulation
with 10 nM a-thrombin
for 24 h. [3H]Thymidine
incorporation
was
assayed as described
in Materials
and Methods.
1
dI
0
I
/
0.3
I
3
I
30
I
100
Fig. 7. Synergistic
Activation
of Late Phase ~44”“~~ by Serotonin and Basic FGF
Quiescent
CCL39 cells were stimulated
with the indicated
agents for 10 min or 120 min. C, medium;
K, ketanserin
(1Om6
M); 5HT, serotonin
(1 OW6 M); FGF, basic FGF (3 rig/ml). P44mapk
was immunoprecipitated
from the cell lysates, and MBP kinase
activity was measured
as described
in the legend of Fig. 2.
Similar results were obtained
in three separate
experiments.
[FGF] (rig/ml)
B
.-I\
+FGF
.
~J+-----O’“-o
--1-m
0
9
a
7
6
-log [5HT]
5
(M)
Fig. 6. Effects of Basic FGF and Serotonin
on Early Phase
p44mapk Activity
A, CCL39 cells were stimulated
for 10 min with indicated
concentrations
of basic FGF. B, Quiescent
CCL39 cells were
stimulated
for 10 min with indicated
concentrations
of serotonin in the presence
(0) or absence (0) of 30 rig/ml basic
FGF. p44”@
was immunoprecipitated
from the cell lysates,
and MBP kinase activity was measured
as described
in the
legend of Fig. 2. Similar results were obtained
in two separate
experiments.
on p44mapk activation
correlates
with the phosphorylation state of the protein. In good agreement with the
data on kinase activity, serotonin alone weakly stimulated the phosphorylation
of ~44”“~~ at 10 min, but not
at 120 min (Fig. 8A). However, serotonin potentiated
the stimulatory effect of a low concentration
of basic
FGF on the phosphorylation
of ~44”“~~, both at the
early and late phases of kinase activation. As expected,
the synergistic effect of serotonin and basic FGF was
clearly observed on the tyrosine phosphorylation
of the
kinase (Fig. 8B).
DISCUSSION
Although a large number
activation of MAP2/MBP
of studies have reported the
kinases in response to a va-
MOL ENDO. 1992
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Vol6 No. 5
10 min
120 min
10 min
120 min
A
97.4
-
66.2 -
97.4
-
66.2 45 7
mapk
P44
45
P44
r
-
mapk
Fig. 8. Synergistic Tyrosine Phosphorylation of ~44”“” by Serotonin and Basic FGF
Quiescent CCL39 cells were labeled with 32Pi and stimulated with the indicated agents for 10 min or 120 min. C, medium; 5-HT,
serotonin (1 O-@M); FGF, basic FGF (3 rig/ml); 5-HT/FGF, serotnin (1 Ow6M) and basic FGF (3 rig/ml). P44”@ was immunoprecipitated
from cell lysates and analyzed by SDS-gel electrophoresis on 10% acrylamide gels as described in the legend of Fig. 3. A,
Autoradicgram of total 32P-labeled proteins after 3 days exposure. 8, Autoradiogram of a duplicate gel pretreated with 1 M KOH
after 6 days exposure. Molecular weight standards are shown on the left. The arrow indicates the position of ~44”““. Similar
results were obtained in two separate experiments.
riety of mitogenic agents, very few studies have looked
at the specific contribution of individual members of the
MAP kinase family to this activity. In particular, very
little is known on the regulation of p44mapk (ERKl). In
this study, we have examined the phosphorylation
and
protein kinase activity of ~44”~~~ in growth factor-stimulated hamster fibroblasts using an antiserum that specifically immunoprecipitate
p44”apk from cell lysates.
Our results demonstrate that: 1) ~44”“~~ is activated by
both G protein-coupled
receptors (cr-thrombin) and receptor tyrosine kinases (basic FGF) in hamster fibroblasts; 2) mitogenic activation of ~44”~~” is biphasic and
sustained in quiescent cells; 3) activation of ~44”“~~ is
accompanied
by tyrosine phosphorylation
of the enzyme; 4) pertussis toxin inhibits both cY-thrombin-induced phosphorylation
and enzymatic
activation of
~44”~~~ at both phases of activation; 5) the synergistic
effect of serotonin and basic FGF on DNA synthesis is
preceded by the synergistic activation of ~44”“~~; and
6) there is a strong correlation
between sustained
p44”@ activation and mitogenicity.
In a very recent study, L’Allemain et a/. (37) have
reported that a-thrombin
and basic FGF were able to
induce the tyrosine phosphorylation
and enzymatic ac-
tivation of p42 mapkin CCL39 cells. Here we show that
these two growth factors, which use distinct early
signaling pathways, also strongly stimulate the tyrosine
phosphorylation
and activation of ~44~~~~ in the same
cellular system. It was previously shown that p44”“pk
was phosphorylated
on serinelthreonine
and tyrosine
residues and activated upon stimulation with epidermal
growth factor (EGF) (26) and nerve growth factor (NGF)
(12). We extend these studies by demonstrating
that
p44mapk is also regulated by mitogens interacting with
G protein-coupled
receptors, confirming its central role
in the integration of growth factor signaling pathways.
The most interesting
finding of this study is the
observation
that potent growth factors such as athrombin induce a biphasic and sustained activation of
p44”apk in quiescent fibroblast cells. A rapid phase
which reached a maximum between 5-10 min is followed by a second broad phase reaching a peak at 2 h
and slowly declining thereafter (Fig. 2A). The enzyme
activity is still elevated (2- to 4-fold) at 4 h relative to
control cells. Biphasic stimulation of MAP kinase activity
has been observed after treatment of PC12 cells with
NGF (38, 39). The time course of activation was different from what we report here, with the second peak
Sustained
Activation
of ~44”“~~
by Growth
Factors
being attained in 30 min and declining more rapidly.
However, the specific enzymes contributing to the stimulation of MAP kinase activity were not identified, although the phosphorylation
of a 42-kDa protein that
coeluted with enzymatic activity was reported. The
kinetic of ~44”~~~ activation is also reminiscent of the
stimulation of the S6 kinases pp70-S6K (40; Kakan, C.,
K. Seuwen, S. Meloche, and J. Pouyssegur, submitted)
and pp90’sk (41) and the generation of 1,2-diacylglycerol (32, 42) which all exhibit a biphasic behavior in
response to mitogenic signals. Although the precise
role of p44”apk in the mitogenic response is yet to be
established, the sustained activation observed in the
present study suggests that the enzyme might be involved in both the Go to G, transition and the G, to S
progression. The close homology of ~44”~~~ with yeast
KSSl and FUS3 (11) two protein kinases involved in
the regulation of G, arrest upon pheromone binding, is
consistent with such a role. P44”apk could promote G,
progression by either phosphorylating
critical G,-regulatory proteins or, alternatively, by transmitting the relay
to other serine/threonine
kinases. In this context, ClarkLewis and coworkers (43) have recently defined a consensus sequence for substrate recognition by p44mapk,
a sea star homolog of mammalian ~44”~~~, using synthetic peptides. Computer search analysis has revealed
that this potential ~44”~~~ phosphorylation
site is present in over 2000 proteins, among which are 40 distinct
protein kinases. Thus, it is conceivable
that ~44”~~~
could phosphorylate
various substrates, including protein kinases, important for cell cycle progression. Interestingly, consensus phosphorylation
sites were found
in the two forms of S6 kinase, pp90’“” and pp70 S6K,
the retinoblastoma
protein, Raf-1, and the product of
S. pombe cell cycle control gene weel, proteins which
are known to be implicated in cell cycle regulation.
Indeed, MAP kinase is known to phosphorylate
the 90kDa S6 kinase (22, 23) and Raf-1 (44) in vitro. Obviously, further experiments with synchronized cell cultures will be important to explore the precise role of
p44”ap” in growth factor-induced
Go to G, and G, to S
transitions, as well as in DNA synthesis. Most importantly, the identification of the physiological substrates
of ~44”“~~ will be crucial to understanding
its functions.
In CCL39 cells, cy-thrombin is known to activate
phospholipase
C by interacting with pertussis toxinsensitive and -insensitive G proteins and to inhibit adenylyl cyclase through a pertussis toxin-sensitive
Gi
protein (45, 46). In this study, we found that pertussis
toxin also inhibits cy-thrombin-induced
tyrosine phosphorylation and activation of ~44”“~~ (Figs. 3 and 5).
These results constitute the first demonstration
of the
involvement of a G protein in the activation pathway of
p44”@. L’Allemain et al. (37) have recently reported
that pertussis toxin was able to inhibit by 7585%
the
activation of ~42”~~~. Likewise, fluoroaluminate,
a direct
activator of G proteins, was shown to stimulate the
activity of ~42”~~~ (47). Hence, it appears that both MAP
kinase isoforms can be regulated by one or several G
proteins. Interestingly, our results indicate that the G
851
proteins involved contribute differentially to the two
phases of ~44”“~~ response. The first peak is only
partially sensitive to the toxin, whereas stimulation of
the second peak is completely abolished. Similar effects
of pertussis toxin were observed on the potentiating
effect of serotonin on basic FGF-induced ~44”~~~ activation (data not shown). We believe that both G, and a
pertussis toxin-insensitive
G protein coupled to phospholipase C, of the G, class, for example, contribute to
the first peak, while the G, pathway prevails late after
stimulation.
The strong correlation observed between the phosphorylation, and most particularly the tyrosine phosphorylation, of ~44”“~~ and its enzymatic activity suggests that both the early and late phase of ~44”~~~
activation are regulated by phosphorylation.
Previous
studies have shown that dual phosphorylation
of
~42”~~~ and ~44”~p~ on tyrosine and serine/threonine
is
required for full activity of the enzymes (17, 27,29, 48).
Gomez and Cohen (21) have recently identified MAP
kinase kinases in PC12 cells which are activated by
NGF and are dependent on serinelthreonine
phosphorylation for their activity. The nature of the kinase(s) and
the signaling mechanisms responsible for the cY-thrombin-induced
biphasic activation
of ~44”“~~ are not
known. However, results obtained with hirudin, together with the inhibition studies with pertussis toxin,
strongly suggest the existence of differential regulatory
pathways involved in the early (O-l h) and sustained
(l->4
h) activation of ~44”~~~. The early increase in
enzyme activity can be triggered by a brief (30 set)
exposure to Lu-thrombin, while the late phase requires
the continuous presence of the hormone (Fig. 4). Analogous results have been described in llC9 hamster
fibroblasts where a-thrombin stimulates a biphasic increase in 1 ,Bdiacylglycerol(32).
Early inactivation of (Ythrombin was shown to eliminate the sustained (l-4 h)
increase in 1,2-diacylglycerol
and to inhibit the release
of choline metabolites. We have recently found that (Ythrombin stimulates the sustained hydrolysis of phosphatidylcholine
through activation of specific-phospholipase C (McKenzie, F., K. Seuwen, and J. Pouyssegur,
manuscript in preparation). Thus, it is conceivable that
the sustained formation of 1,2-diacylglycerol
could regulate the late phase of ~44”~~~ activation by stimulating
protein kinase C. Indeed down-regulation
of protein
kinase C resulted in partial attenuation of cu-thrombininduced p44”=p” activation, providing evidence for the
requirement of additional signaling events (Kahan, C.,
K. Seuwen, S. Meloche, and J. Pouyssegur, submitted).
Similar results were obtained by Gotoh et al. (39) who
found that late phase activation of MAP kinase by NGF
was partly dependent on protein kinase C (39).
The demonstration
that a mitogen like cu-thrombin
induces a late and sustained activation of ~44”~~~ is
important in light of the observation that continuous
presence of the protease for at least 4 h is required for
DNA synthesis (31, 32). The following arguments indicate that there is a very close correlation between the
ability of a growth factor to induce the late phase of
MOL
852
ENDO.
1992
Vol6
p44”“Pk response and its mitogenic potential. cu-Thrombin, a very potent mitogen for CCL39 ceils, induces a
biphasic activation of ~44”=~~, while carbachol, which
has no mitogenic potential, stimulates the first but not
the second peak in the same cells expressing the Ml
muscarinic receptor (Kahan, C., K. Seuwen K, S. Melache, and J. Pouyssegur, submitted). Very similar data
were obtained for the stimulation of pp70 S6 kinase,
an enzyme thought to play a critical role in cell proliferation (40; Kahan, C., K. Seuwen, S. Meloche, and J.
Pouyssegur J, submitted). Early removal of a-thrombin
with hirudin, which prevents DNA synthesis, also eliminates the second phase of ~44”~~~ response. Serotonin, which is not a mitogen by its own, stimulates the
first peak but has no effect on the second peak of
p44”@ activation. However, the synergistic effect of
serotonin on basic FGF mitogenicity (35) is paralleled
by the synergistic activation of late kinase response.
Pretreatment of CCL39 ceils with pertussis toxin, which
inhibits thrombin- (30) and serotonin-induced
mitogenicity (35) completely abolishes the second phase while
partially reducing the first phase of ~44”“~~ activation
(data not shown for serotonin). These results provide
further support for a role of G,-activated pathways in
the mitogenic response (1,49, 50). Interestingly, Gotoh
et al. (39) reported that NGF stimulates late phase
activation of MAP kinase in PC12 cells, while EGF,
which has no differentiation potential, only stimulates a
transient kinase response. Therefore, we propose that
late and sustained ~44”“~~ activation is an obligatory
event for growth factor-induced
cell cycle progression.
MATERIALS
AND METHODS
No. 5
NaCI, 50 mM NaF, 5 mM EDTA, 40 mM P-glycerophosphate,
200 PM sodium orthovanadate,
10m4 M phenylmethylsulfonyl
fluoride, 1 pg/ml leupeptin,
1 PM pepstatin
A,-and 1% Triton
X-100) for 25 min at 4 C. Insoluble material was removed
bv
centriiugation
at 12,000 x g for 15 min at 4 C. Proteins from
cell lysates were resolved
on 10% acrylamide/6
M urea gels
and electrophoretically
transferred
to Hybond-C
Extra membranes (Amersham)
in 25 mrv Tris, 190 mM glycine. Membranes
were blocked in TBS (20 mM Tris-HCI,
pH 7.5, 137 mM NaCI)
containing
3% nonfat dry milk. The membranes
were then
incubated
with antibody
837 (1 :lOOO) in blocking
solution for
2-4 h at 25 C, washed
with TBS, and incubated
with horseradish peroxidase-conjugated
goat anti-rabbit
immunoglobulin
G (1:lOOO; Sigma) in blocking solution for 1 h at 25 C. The
blots were developed
by adding horseradish
peroxidase
substrate diaminobenzidine
in the presence
of NiCl*.
Immune
Complex
Kinase
Assay
of p44”@
Quiescent
CCL39
cells in 12-well
plates were incubated
in
HEPES-buffered
DMEM
before stimulation.
Cells were stimulated with growth factors for the indicated
times at 37 C. The
cells were then washed twice with cold PBS and lysed in 0.3
ml Triton X-l 00 lysis buffer for 25 min at 4 C. After clarification
by centrifugation
at 12,000 x g for 15 min at 4 C, the lysates
were precleared
for 1 h at 4 C with 1 ~1 normal rabbit serum
and protein A-Sepharose
(Pharmacia,
Piscataway,
NJ). The
lysates were then incubated
for 2 h or 24 h at 4 C with 2 ~1
antiserum
837 preadsorbed
to protein
A-Sepharose
beads.
Note that incubation
for 2 or 24 h with antiserum
gave the
same results. Immune complexes
were collected
by centrifugation, washed four times with Triton X-l 00 lysis buffer, and
one time with kinase buffer (20 mM HEPES, pH 7.4, 10 mM
MgCl*,
1 mM dithiothreitol,
and 10 mM p-nitrophenyl
phosphate). MBP kinase activity was assayed by resuspending
the
final pellet in a total vol of 40 ~1 kinase buffer containing
0.25
mg/ml MBP and 50 PM [y-3ZP]ATP
(SA, -5500
cpm/pmol).
Reactions
were initiated with ATP and incubated
at 30 C for
10 min. Assays were stopped
by the addition of 40 ml 2 x
Laemmli’s
sample buffer. After heating at 95 C for 5 min, the
samples were analyzed
by sodium dodecyl sulfate (SDS)-gel
electrophoresis
on 12% acrylamide
gels. The gels were stained
with Coomassie
blue, dried, and subjected to autoradiography.
Phosphate incorporation was measured by excising substrate
Highly purified human a-thrombin
and recombinant
basic FGF
were generous
gifts of J. W. Fenton
II (New York State
Department
of Health, Albany,
NY) and D. Gospodarowicz
(University
of California
Medical Center,
San Francisco,
CA).
Recombinant
hirudin was kindly provided
by Transgene
(Strasbourg, France).
Serotonin
and bovine
MBP were obtained
from Sigma (St. Louis, MO). Ketanserin
was obtained
from
Janssen. Pertussis toxin was from List Biological Laboratories.
32P, and [y3’P]ATP
were from Amersham
(Arlington
Heights,
IL). Triton X-100 was from Pierce (Rockford.
IL). Antibodv
837
is’a rabbit polyclonal
antibody
raised to a C-terminal
peptide
of ERKl and generously
provided
by M. Cobb (University
of
Texas) (29).
Cell Culture
The Chinese hamster lung fibroblast
line CCL39 (ATCC, Rockville, MD) was cultured in Dulbecco’s
modified Eagle’s medium
(DMEM;
GIBCO,
Grand Island, NY) supplemented
with 7.5%
fetal calf serum and antibiotics
(50 U/ml penicillin and 50 pg/
ml streptomycin)
at 37 C in a 95% sir/5% CO2 atmosphere.
Quiescent
cells were obtained by incubating
confluent
cultures
of CCL39 cells in serum-free
medium for 24 h.
Western
Blot Analysis
CCL39 cells were washed twice with cold
Triton X-100 lysis buffer (50 mM Tris-HCI,
PBS and lysed in
pH 7.5, 100 mM
bands from the gel and counting
the radioactivity
in a liquid
scintillation
counter.
Protein kinase activities are expressed
as
picomoles
of phosphate
incorporated
per min/mg protein.
For the experiments
with pertussis toxin, quiescent
CCL39
cells were incubated
4-6 h in HEPES-buffered
DMEM containing 100 rig/ml toxin before the addition of growth factors, and
the cells were stimulated
in the continuous
presence
of the
toxin.
32P Labeling
and lmmunoprecipitation
Quiescent
CCL39 cells in loo-mm
plates were labeled for a
total time of 6 h at 37 C in bicarbonateand phosphate-free
HEPES-buffered
DMEM containing
500 &i/ml
[32P]orthophosphate. The cells were stimulated
by addition of growth factors
to the labeling medium for the last 2 h or 10 min of incubation.
The cells were then washed and lysed in 0.8 ml Triton X-100
lysis buffer.
Lysates
were processed
as described
above,
precleared
with 2 /II normal rabbit serum, and incubated
overnight at 4 C with 5 ~1 antiserum
837 preadsorbed
to protein
A-Sepharose
beads. Immune
complexes
were washed
five
times with Triton X-100 lysis buffer, one time with 100 mM
Tris-HCI
(pH 7.5) 500 mM LiCI, 40 mM P-glycerophosphate,
and 200 PM sodium orthovanadate,
and one time with 10 mM
Tris-HCI
(pH 7.5). 40 mM B-glycerophosphate,
and 200 FM
sodium orthovanadate.
The final pellet was resuspended
in
Laemmli’s
sample buffer, heated at 95 C for 5 min, and the
proteins
were resolved
by SDS-gel
electrophoresis
on 10%
acrylamide
gels. The gels were stained, dried, and subjected
Sustained
Activation
of ~44”“~~
by Growth
Factors
to autoradiography
at -70 C for visualization
of the total 32P
content of proteins.
For detection
of phosphotyrosine-containing
proteins,
the
fixed gels were rinsed in water before incubation
in 1 M KOH
at 55 C for 2 h (51). The alkali-treated
gels were neutralized
in
10% acetic acid/l 0% methanol
and dried before autoradiography at -70 C. Experiments
with pertussis toxin were carried
out as described
above,
except
that the toxin was added
directly to the labeling medium.
Measurement
of DNA Synthesis
Quiescent
CCL39
cells in 24-well
plates were stimulated
in
serum-free
DMEM/Ham’s
F12 (1:l) medium
containing
0.5
&i/ml
[3H]thymidine
with the indicated
growth
factors.
After
24 h incubation,
the cells were fixed and washed four times
with cold 5% trichloroacetic
acid. The radioactivity
incorporated into trichloroacetic
acid-precipitable
material was measured after solubilization
in 0.1 M NaOH.
For experiments
with pertussis
toxin, the quiescent
cells
were treated for 4 h with 100 rig/ml toxin before addition of
growth
factors,
and the toxin was maintained
during
the
stimulation
period.
Other
Methods
Protein concentrations
were measured
using the BCA protein
assay kit (Pierce) with BSA as standard.
SDS-polyacrylamide
gel electrophoresis
was performed
using the discontinuous
buffer system of Laemmli (52).
Acknowledgments
We wish to thank Melanie Cobb for her generous
gift of ERKl
antibodies,
Fergus McKenzie
for his help with acrylamide
urea
gels and for valuable discussions,
Dominique
Grail for help in
tissue culture, and Mar-tine Valetti for secretarial
assistance.
Received
October
31, 1991. Revision
received
March 3,
1992. Accepted
March 9, 1992.
Address requests
for reprints to: Dr. Jacques
Pouyssegur,
Centre de Biochimie-CNRS.
Part Valrose. 06108 Nice C&fex
2, France.
This work was supported
by grants from the Centre National
de la Recherche
Scientifique
(UMR 134) the lnstitut National
de la Sante et de la Recherche
Medicale,
the Fondation
pour
la Recherche
Medicale,
and the Association
pour la Recherche
contre le Cancer.
Recipient
of a Centennial
Fellowship
from the Medical
Research
Council of Canada.
Present
address:
Centre
de
Recherche,
Hotel-Dieu
de Montreal,
3850 St.-Urbain,
Montreal, Quebec,
Canada H2W lT8.
l
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