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Molecular and Cellular Biology, January 1999, p. 330-341, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Repertoire of Fos and Jun Proteins Expressed
during the G1 Phase of the Cell Cycle Is Determined by the
Duration of Mitogen-Activated Protein Kinase Activation
Simon J.
Cook,1,2,*
Natasha
Aziz,3 and
Martin
McMahon3
ONYX Pharmaceuticals, Richmond, California
948061;
Signalling Programme, The
Babraham Institute, Cambridge CB2 4AT, England2;
and
Department of Cell Signalling, DNAX Research Institute,
Palo Alto, California 943043
Received 13 May 1998/Returned for modification 29 June
1998/Accepted 15 September 1998
 |
ABSTRACT |
In Rat-1 fibroblasts nonmitogenic doses of lysophosphatidic acid
(LPA) stimulate a transient activation of mitogen-activated protein
kinase (MAPK), whereas mitogenic doses elicit a sustained response.
This sustained phase of MAPK activation regulates cell fate decisions
such as proliferation or differentiation, presumably by inducing a
program of gene expression which is not observed in response to
transient MAPK activation. We have examined the expression of members
of the AP-1 transcription factor complex in response to stimulation
with different doses of LPA. c-Fos, c-Jun, and JunB are induced rapidly
in response to LPA stimulation, whereas Fra-1 and Fra-2 are induced
after a significant lag. The expression of c-Fos is transient, whereas
the expression of c-Jun, JunB, Fra-1, and Fra-2 is sustained. The early
expression of c-Fos can be reconstituted with nonmitogenic doses of
LPA, but the response is transient compared to that observed with
mitogenic doses. In contrast, expression of Fra-1, Fra-2, and JunB and
optimal expression of c-Jun are observed only with doses of LPA which
induce sustained MAPK activation and DNA synthesis. LPA-stimulated
expression of c-Fos, Fra-1, Fra-2, c-Jun, and JunB is inhibited by the
MEK1 inhibitor PD098059, indicating that the Raf-MEK-MAPK cascade is required for their expression. In cells expressing a conditionally active form of Raf-1 (
Raf-1:ER), we observed that selective, sustained activation of Raf-MEK-MAPK was sufficient to induce expression of Fra-1, Fra-2, and JunB but, interestingly, induced little
or no c-Fos or c-Jun. The induction of c-Fos observed in response to
LPA was strongly inhibited by buffering the intracellular [Ca2+]. Moreover, although Raf activation or calcium
ionophores induced little c-Fos expression, we observed a synergistic
induction in response to the combination of
Raf-1:ER and ionomycin.
These results suggest that kinetically distinct phases of MAPK
activation serve to regulate the expression of distinct AP-1 components
such that sustained MAPK activation is required for the induced
expression of Fra-1, Fra-2, c-Jun, and JunB. However, in contrast to
the case for Fra-1, Fra-2, and JunB, activation of the MAPK cascade alone is not sufficient to induce c-Fos expression, which rather requires cooperation with other signals such as Ca2+
mobilization. Finally, the identification of the Fra-1, Fra-2, c-Jun,
and JunB genes as genes which are selectively regulated by sustained
MAPK activation or in response to activated Raf suggests that they are
candidates to mediate certain of the effects of Ras proteins in
oncogenic transformation.
 |
INTRODUCTION |
Growth factors and oncogenes exert
their effects on cells by activating intracellular signal pathways
which elicit changes in gene expression leading to cell cycle
progression or cellular transformation. The dimeric transcription
factor AP-1 is a major target of cell growth, differentiation, and
stress signalling pathways (7, 50, 101). AP-1 consists of
various combinations of Fos and Jun family members that dimerize via a
leucine zipper domain and bind to DNA via an adjacent basic region
(32, 51, 58). The Fos family consists of four gene products
(c-Fos, FosB, Fra-1, and Fra-2), while the Jun family is made up of
three gene products (c-Jun, JunB, and JunD). Fos proteins form
heterodimers with Jun and ATF family proteins (37), whereas
Jun proteins can form heterodimers with ATF2 (98) and can
form functional homodimers, albeit with reduced stability (38,
79).
AP-1 binds to a specific target DNA sequence, TGAC/GTCA,
named the TRE (for tetradecanoyl phorbol acetate-responsive
element) (6), which is found in the promoters of many genes,
including those for cell cycle regulators such as cyclin D1 (3,
41) and autocrine growth factors such as heparin-binding
epidermal growth factor (HB-EGF) (70) and vascular
endothelial growth factor (45). The binding affinity for a
given TRE is determined by the different AP-1 dimer combinations and
the context of the surrounding sequences (27, 38, 39, 81).
In addition, some Fos and Jun proteins possess transcriptional
activation domains which are regulated by phosphorylation, with the
result that different dimer combinations may exhibit different
transactivation properties (50, 101). Finally, the
expression of Fos and Jun proteins is temporally coordinated in
response to various stimuli, with the result that the composition of
AP-1 may change in the cell as a function of time and stimulus
(18, 26, 52, 53, 57, 74, 85). All of these factors
contribute to make AP-1 a versatile and dynamic transcriptional complex
able to respond to differing environmental cues.
Cell transformation by Ras oncoproteins is intimately linked to
increases in AP-1-mediated gene expression. Microinjection of Ras
proteins induces c-Fos expression (87), and chronic Ras transformation leads to an increase in AP-1 binding activity and up-regulation of at least four AP-1 components (Fra-1, Fra-2, c-Jun,
and JunB) (71, 96). In addition, dominant interfering mutants of c-Jun and c-Fos can revert the phenotype of Ras-transformed cells (63, 89), c-Jun is required for Ras-induced malignancy (48), and c-Fos is required for Ras-driven malignant
progression in a multistep skin carcinogenesis model in mice
(82).
The Ras-dependent Raf-MEK-mitogen-activated protein kinase
(Raf-MEK-MAPK) cascade is one of the key signalling pathways
responsible for transmitting signals from growth factor receptors to
the nucleus (15, 42, 47, 50, 66, 95, 101). Signals from
growth factor receptors lead to activation of the membrane-tethered Ras proteins, which recruit the serine/threonine kinase Raf to the plasma
membrane where it in turn is activated (61, 65, 88, 90, 97,
100). Raf phosphorylates and activates the dual-specificity MAPK
kinase, MEK (28, 44, 55), which in turn phosphorylates MAPK
at adjacent Thr and Tyr residues, thereby reactivating it (24). Activated MAPKs accumulate in the nucleus (13,
62, 91), where they can phosphorylate and activate the
transcription factors Elk-1 and Sap1a, leading to the enhanced
expression of genes such as that for c-Fos (34, 42, 43, 64, 94,
101). In this way the Raf-MEK-MAPK cascade serves to amplify
low-level extracellular signals into intracellular messages which are
transmitted into the nucleus, thereby connecting growth factor
receptors to changes in gene expression and cell fate. Expression of
c-Jun is also stimulated by Ras proteins, but the mechanism for this is
less clear. c-Jun expression can be stimulated by activation of
preexisting ATF2-c-Jun dimers at the c-Jun TRE/AP-1 site (30, 98). The transcriptional activity of ATF2 and c-Jun is stimulated by phosphorylation of sites in their transactivation domains by the p38
and Jun N-terminal kinases (JNK/stress-activated protein kinase)
(50, 101), but these kinases are only weakly activated by
Ras proteins (29) and growth factors (29, 56). In
addition, there is evidence to suggest that additional sites outside
the transactivation domain may also be important (1).
While the role of MAPK in regulating the c-Fos serum response element
is well recognized (42, 95, 101), this is an early and
transient event and yet serum and growth factors are required to be
present for 8 to 10 h to ensure cell cycle reentry, during which
they stimulate a sustained activation of MAPK (19, 20, 49,
99). Activation of MEK and MAPK is sufficient to cause differentiation and transformation (23), but it is the
duration of MAPK activation which appears to be a key determinant of
cell fate signalling decisions. In PC12 cells, nerve growth factor stimulates a sustained activation of MAPK which is required for cell
cycle arrest and terminal differentiation, whereas factors which elicit
transient activation of MAPK do not promote differentiation (40,
66, 91). In CCL39 fibroblasts, thrombin requires sustained MAPK
activation to stimulate DNA synthesis (49, 99), while in
Rat-1 cells, sustained MAPK activity is observed only in response to
doses of lysophosphatidic acid (LPA) which stimulate DNA synthesis (20). Finally, low-level activation of the Raf-MEK-MAPK
pathway promotes cell proliferation, whereas persistent high-level
activation promotes cell cycle arrest and a quasidifferentiated state
in NIH 3T3 cells (84, 103). Since sustained MAPK activation
is associated with nuclear accumulation of MAPKs (13, 60,
91), it has been proposed that the quantitative differences in
the duration of MAPK activation may be reflected in qualitative changes in gene expression, thereby determining cellular responses
(66). The logical conclusion from this model is that some
genes will be expressed only under conditions of sustained MAPK
activation. One example of such a gene is that for the cyclin-dependent
kinase inhibitor p21Cip1 (84, 103); however, the
links between the MAPK pathway and the cell cycle apparatus are not yet
fully understood.
Here we have examined the role of sustained MAPK activation in the
expression of different components of the AP-1 transcription factor
complex in response to LPA stimulation. We demonstrate that the
duration of MAPK activation determines the repertoire of Fos and Jun
proteins expressed during cell cycle reentry. Specifically, we
demonstrate that Fra-1, Fra-2, c-Jun, and JunB are targets for
sustained MAPK activation and therefore are likely to mediate the
longer-term effects of Ras and Raf in transformed cells.
 |
MATERIALS AND METHODS |
Materials.
LPA was purchased from Avanti Polar Lipids or
Sigma, 4-hydroxytamoxifen (4-HT) and
-estradiol (
-E2) were from
Sigma, and ionomycin and BAPTA-AM
[1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester] were from Calbiochem. Fetal bovine serum (FBS) and all other cell culture reagents were from Gibco Life
Technologies. Rabbit antipeptide antisera to c-Fos, Fra-1, Fra-2, and
JunB were from Santa Cruz Biotechnology. Antipeptide antiserum to c-Jun
was kindly provided by David Gillespie, CRC Beatson Laboratories,
Glasgow, United Kingdom. Horseradish peroxidase-conjugated secondary
antibodies were from Bio-Rad, and detection was with the enhanced
chemiluminescence (ECL) system (Amersham). Protease inhibitors were
from Boehringer Mannheim. All other reagents were of the highest grade
commercially available.
Cells and cell culture.
Rat-1 and NIH 3T3 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
glutamine, penicillin-streptomycin, and 7% (vol/vol) FBS.
R1
Raf-1:ER-4 cells (21) and NIH 3T3 c4 or c9 cells
(70, 83) expressing
Raf-1:ER were grown in the same
medium without phenol red and maintained in the presence of 400 µg of
G418 ml
1.
Stimulations and preparation of cell lysates.
For Rat-1
cells and their transfected derivatives, cells were grown to confluence
and prepared for stimulation by replacing the medium with serum-free
Dulbecco's modified Eagle's medium for 24 h to induce a
quiescent state (G0 arrest). In the case of NIH 3T3 cells,
this medium was modified to 0.5% (vol/vol) FBS with 500 mg of fatty
acid-free bovine serum albumin and 10 ml of
insulin-transferrin-selenium supplement (ITS-X; Gibco-BRL) per liter,
as we observed loss of attachment to the substratum if NIH 3T3 cells
were incubated overnight under serum-free conditions. Cells were
stimulated with the appropriate agonists by adding them as 10×
solutions to the culture dish, thereby causing minimal disturbance.
Incubations proceeded at 37°C with 5% CO2 for the indicated times and were terminated by aspiration of the medium and
addition of ice-cold TG lysis buffer (20, 21). Cell extracts were harvested and clarified by centrifugation at 14,000 × g at 4°C for 10 min, and the supernatants were boiled in
Laemmli sample buffer and stored at
20°C.
Western blot analysis.
Equal amounts of cell lysates,
typically 50 µg, were fractionated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% gels
(Hoefer Mighty Small system) and transferred to methanol-soaked
polyvinylidene difluoride (PVDF) membranes at 300 mA for 1 h in a
Bio-Rad mini-Transblot apparatus. After staining to confirm equal
loading, filters were incubated in phosphate-buffered saline plus 0.1%
(vol/vol) Tween 20 (TPBS) supplemented with 5% (wt/vol) powdered milk
(TPBS-milk) for at least 1 h before probing with antisera. First-
and second-antibody incubations were performed in TPBS-milk at room
temperature for 1 h with six intervening washes during 20 min.
Antibody-antigen complexes were detected by using the ECL system
according to the manufacturer's instructions. Exposures for c-Fos,
Fra-1, and Fra-2 were typically 5 to 15 s, and those for c-Jun and
JunB were 30 s to 5 min. Results were recorded on a charge-coupled
device video camera, and integrated optical densities were derived by
using the Solitaire Plus 512 Seescan Imaging System. Quantification was
performed with multiple exposures for each blot, and results were
independently confirmed by scanning densitometry.
RNase protection.
The Rat c-fos riboprobe was
prepared by using a Bluescript plasmid containing bases 303 to 453 of
rat c-fos cDNA and protected a fragment of 150 bp. A
riboprobe prepared from a plasmid containing a 120-bp fragment of the
human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was
included in all hybridizations and used as a loading control.
Riboprobes were generated from linearized cDNAs by reverse
transcription in the presence of [32P]CTP, and the
labelled riboprobes were purified from unincorporated nucleotides by
using G50 Sephadex columns (Boehringer Mannheim) or by gel
purification. Ten micrograms of total RNA (RNeasy kit; Qiagen) or tRNA
(as a negative control) was hybridized overnight at 45°C with
purified c-fos-specific and GAPDH riboprobes. Unhybridized RNA was digested with RNase A and RNase T1, extracted with
phenol, ethanol precipitated, and resolved by electrophoresis on 6%
acrylamide sequencing gels. Results were quantitated with a Molecular
Dynamics PhosphorImager.
 |
RESULTS |
Kinetics of LPA-stimulated expression of Fos and Jun proteins in
Rat-1 cells.
Initially we defined the kinetics of induction of
AP-1 proteins in quiescent, serum-starved Rat-1 cells stimulated to
reenter the cell cycle by LPA. In these studies we examined the
expression of c-Fos and c-Jun, the classical immediate-early gene
products, and the Fos-related proteins Fra-1 and Fra-2, which are
reported to exhibit delayed kinetics of induction in response to serum. In addition, we also examined the expression of JunB, which, together with Fra-1, Fra-2, and c-Jun, is reported to be expressed in
Ras-transformed cells (71, 96). Quiescent Rat-1 cells were
either untreated or stimulated with LPA for various times from 30 min
to 10 h. Under these conditions, Rat-1 cells start to enter S
phase 12 h after stimulation and pass the restriction point
approximately 10 h after LPA addition (20, 22). The
kinetics of expression of Fos and Jun proteins was analyzed by Western
immunoblotting of whole-cell lysates (Fig.
1A and B) as described in Materials and
Methods.

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FIG. 1.
Kinetics of expression of Fos and Jun proteins in Rat-1
cells stimulated with LPA. (A and B) Confluent, serum-starved Rat-1
cells were stimulated with 100 µM LPA for the times indicated.
Whole-cell detergent lysates were fractionated by SDS-PAGE, transferred
to PVDF membranes, and subjected to Western immunoblotting with
antibodies specific for c-Fos, Fra-1, Fra-2 (A) or c-Jun, JunB, or
phospho-MAPK (P-MAPK) (B). Fold increases in protein expression or MAPK
activation are shown above each lane for each blot. Note that the basal
level of JunB was not detectable; the basal level of 1 is set at the
lowest detectable value, making the fold increases an underestimate.
(C) Quiescent Rat-1 cells were stimulated for the indicated times with
50 µM LPA. Whole-cell detergent lysates were prepared, divided into
two equal portions, and incubated in the absence (Control) or presence
(AP) of 20 U of alkaline phosphatase at 37°C for 2 h. Both
samples were then fractionated by SDS-PAGE, transferred to PVDF
membranes, and subjected to Western immunoblotting with antibodies
specific for c-Fos, Fra-1, Fra-2, or JunB. Note that the apparent
molecular weights of all four proteins are reduced to a greater or
lesser extent by AP treatment. In the case of JunB, this is most
apparent at the 1-h time point, when the protein exists as a triplet
with the uppermost band being the major protein in control samples; AP
treatment causes dephosphorylation of the upper band so that JunB now
appears as three bands of approximately equal intensity. The results
shown are from a single experiment typical of at least three others
giving identical results.
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|
c-Fos expression was rapidly and strongly induced within 40 min after
LPA addition (Fig.
1A). Relative to that of the other
AP-1 proteins
studied, c-Fos expression in response to LPA was
transient, peaking
between 40 min and 1 h and returning to basal
levels 6 to 8 h
after LPA addition; this result is consistent
with the rapid and
transient induction of c-Fos mRNA (
35,
43,
54,
75,
93). The
c-Fos protein was resolved as a broad band
of 55 to 65 kDa, and
previous studies indicate that this is due
to differential
phosphorylation of the c-Fos protein (
25,
57).
Indeed, the
pattern of c-Fos-immunoreactive bands observed is
virtually identical
to that observed in Sf9 cells when c-Fos is
coexpressed with v-Raf and
MAPK (
2), and the hyperphosphorylated
forms of c-Fos can be
reduced to a lower-molecular-mass band by
treatment of cell lysates
with alkaline phosphatase (Fig.
1C)
(
57). The c-Fos protein
is reported to be phosphorylated on
at least two C-terminal regulatory
sites (Ser362 and Ser374 in
the rat sequence) by MAPK and the
MAPK-activated p90
RSK in vitro and in response to serum
stimulation in vivo (
14).
Phosphorylation of these two sites
is implicated in increasing
the half-life of the c-Fos protein, and we
note that the most
hyperphosphorylated forms of c-Fos persisted for the
longest time
in response to growth factor stimulation (Fig.
1A).
Indeed, cell
transformation by c-Fos correlates with persistent
expression
of a partially phosphorylated form of the c-Fos protein
(
60).
Relative to c-Fos, Fra-1 was induced with delayed kinetics but in a
sustained manner in response to LPA stimulation. Fra-1
expression was
unchanged until 2 h after LPA stimulation, at which
time Fra-1 was
strongly induced, and it was maintained at elevated
levels for at least
a further 8 h (Fig.
1A). Compared to Fra-1,
Fra-2 was more rapidly
induced but, unlike c-Fos, was maintained
at high levels for up to
10 h after addition of LPA. In the case
of Fra-2, we observed a
reduction in electrophoretic mobility
of the preexisting Fra-2 protein
which was most pronounced as
early as 10 min after addition of LPA (see
also Fig.
2B). We also
observed some heterogeneity in the molecular
weight of Fra-1 which
was apparent only after prolonged stimulation,
when the Fra-1
protein was detectable as a ladder with a major band
uppermost
and two or three fainter bands beneath. In both cases this
reduction
in mobility is most likely to be due to phosphorylation of
the
proteins, since it can be reversed by treating the lysates with
alkaline phosphatase (Fig.
1C) (
57).
c-Jun protein expression was induced after 30 to 40 min of LPA
stimulation, but the response differed from that for c-Fos
in three
ways. First, there was a higher basal level of c-Jun
expression
detectable in virtually all experiments (except with
NIH 3T3 cells
[see Fig.
6]), whereas c-Fos expression was rarely
detected in
serum-starved cells. Second, in all experiments the
magnitude of the
increase in c-Jun in response to LPA was modest
compared to the robust
accumulation observed for c-Fos. Finally,
the increase in c-Jun
expression was generally sustained for a
longer period of time than
that for c-Fos, such that elevated
levels of c-Jun were still observed
late in G
1 (see also reference
57). In
these experiments we observed some heterogeneity in
the electrophoretic
mobility of c-Jun in response to LPA, but
this differed in degree
between experiments (see, for example,
Fig.
1 versus Fig.
2 and
4). In
most cases c-Jun was detected
as a minor protein of approximately 39 kDa and a major protein
of approximately 41 kDa. The phosphorylation of
c-Jun at Ser63,
Ser73, Thr91, and Thr95 is reported to induce a similar
reduction
in mobility of c-Jun (
68,
78). However, the
SAPK/JNKs, which
are responsible for phosphorylation of Ser63 and
Ser73, are activated
only weakly in response to LPA stimulation
(
12). Under conditions
in which alkaline phosphatase reduced
the apparent mobilities
of c-Fos, Fra-1, Fra-2, and JunB, it had no
effect on that of
c-Jun (
22), consistent with previous
reports (
57). There may
be several reasons for this. It is
possible that candidate phosphorylation
sites are not accessible to the
phosphatase under these conditions.
In addition, it is known that c-Jun
undergoes a complex pattern
of phosphorylation and dephosphorylation
upon cell stimulation
which can adequately be monitored only by
2-dimensional phosphopeptide
mapping (
68,
78). Finally,
c-Jun is known to be targeted for
ubiquitination (
77), and
this pattern may represent such
modification.
The kinetics of expression of the JunB protein were similar to those
for Fra-2 except that the basal level of JunB was so
low as to be
undetectable in some experiments (Fig.
1B; see Fig.
6A). Increased
expression of JunB was detected 1 h after LPA stimulation
and
appeared as a doublet and in some cases a triplet of approximately
40 to 42 kDa. JunB expression was maximal between 1 and 2 h after
LPA
stimulation, at which time the upper band of the doublet was
the
predominant form. Strong expression of JunB persisted for
up to 10 h after addition of LPA. The form of JunB with the lowest
electrophoretic mobility most likely reflects a phosphorylation
event,
since the apparent molecular mass of this protein was reduced
upon
treatment with alkaline phosphatase (Fig.
1C) (
57).
We have previously observed that the activity of p44 MAPK persists for
up to 8 h in Rat-1 cells stimulated with a maximal
mitogenic dose
of LPA. Activation of both p42 and p44 MAPKs in
these experiments was
confirmed by Western immunoblot analysis
of cell extracts with an
antibody which specifically recognizes
the activated forms of MAPK. LPA
stimulated a pronounced peak
of MAPK activation at 10 min, which
persisted above basal levels
in a sustained second phase for at least
8 h (Fig.
1B). These
data confirm those from our previous
experiments in which sustained
activation of p44 MAPK was assessed by
immunocomplex kinase assay
(
20).
In a parallel series of experiments we observed that EGF stimulated the
expression of c-Fos, Fra-1, Fra-2, c-Jun, and JunB
in quiescent Rat-1
cells with kinetics similar to those with LPA,
although in general EGF
induced c-Fos expression to a level lower
than that observed in
response to LPA (
22).
Although we did not address the expression of FosB in this study, it
has recently been reported that FosB exhibits a kinetic
profile of
induced expression similar to that observed for c-Fos
in
serum-stimulated NIH 3T3 cells (
57). In addition, our
preliminary
experiments indicated that JunD was readily detectable in
unstimulated
Rat-1 cells and that its expression was not elevated in
response
to LPA (
22,
57).
The expression of Fra-1 and Fra-2 requires mitogenic doses of LPA
and sustained MAPK activation.
Optimally mitogenic doses of LPA
(e.g., 100 µM) stimulate a biphasic activation of MAPK in Rat-1 cells
in which the sustained phase persists for several hours (Fig. 1). In
contrast, nonmitogenic doses of LPA (e.g., 1 µM) can fully
reconstitute the early peak of MAPK activation but fail to elicit the
sustained phase of MAPK activation (Fig.
2). Accordingly, the 50% effective
concentration (EC50) for activation of the peak of MAPK
activity at 10 min is approximately 100 nM, whereas the
EC50 for the sustained phase of the response is
approximately 10 µM, which is similar to that for induction of DNA
synthesis (20). Given the distinct temporal pattern of
expression of AP-1 components described above, we examined the
expression of Fos and Jun proteins in response to mitogenic (100 µM)
and nonmitogenic (1 µM) doses of LPA.

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FIG. 2.
Expression of Fos and Jun proteins in response to
mitogenic and nonmitogenic doses of LPA. Confluent, serum-starved Rat-1
cells were stimulated with 100 or 1 µM LPA for the times indicated.
Whole-cell detergent lysates were fractionated by SDS-PAGE, transferred
to PVDF membranes, and subjected to Western immunoblotting with
antibodies specific for phospho-MAPK (P-MAPK), c-Fos, or c-Jun (A) or
Fra-1, Fra-2, or JunB (B). Fold increases in protein expression or MAPK
activation are shown above each lane for each blot. The results shown
are from a single experiment typical of at least three giving identical
results.
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|
Consistent with our previous observations, 100 µM LPA elicited a
robust activation of MAPK followed by a sustained phase,
whereas 1 µM
LPA fully reconstituted the early peak of MAPK activation
at 10 min but
did not cause sustained activation of MAPK (Fig.
2A). Stimulation of
quiescent Rat-1 cells with 100 µM LPA resulted
in the normal profile
of c-Fos and c-Jun expression that is shown
in Fig.
1. c-Fos and c-Jun
expression peaked at 1 h (Fig.
2A),
whereas Fra-1 and Fra-2 were
induced after a short lag period
but were strongly expressed after
4 h of LPA stimulation (Fig.
2B). Stimulation of cells with 1 µM
LPA elicited the same early
peak of c-Fos, but the response was more
transient, while expression
of c-Jun was greatly reduced (Fig.
2A).
Treatment of Rat-1 cells
with 1 µM LPA failed to induce the
expression of Fra-1 and Fra-2.
Furthermore, in these experiments we
observed that JunB was significantly
induced only at the mitogenic
doses of LPA (Fig.
2B). These results
indicate that LPA-induced
expression of Fra-1, Fra-2, c-Jun, and
JunB correlates with sustained
MAPK activation in Rat-1
cells.
The correlation between the MAPK signal duration and the expression of
c-Fos, Fra-1, Fra-2, and c-Jun was strengthened by
analyzing the
dose-response curves for the expression of these
proteins in response
to LPA (Fig.
3). LPA-stimulated c-Fos
expression,
measured after 1 h, was saturable with an
EC
50 of approximately
100 to 200 nM, similar to that
required for the early peak of
MAPK activation (
20). In
contrast, stimulated expression of
Fra-1, Fra-2, or c-Jun, assayed
4 h after LPA stimulation, was
observed only at micromolar doses
of LPA, with EC
50 values of
approximately 10 µM (Fig.
3).
These are the same doses of LPA
required to elicit a sustained phase of
MAPK activation and to
stimulate DNA synthesis.

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FIG. 3.
Dose-response curves for expression of Fos and Jun
proteins in response to LPA. Confluent, serum-starved Rat-1 cells were
stimulated with increasing doses of LPA for 1 h (c-Fos) or 4 h (Fra-1, Fra-2, or c-Jun) as indicated. Whole-cell detergent lysates
were fractionated by SDS-PAGE, transferred to PVDF membranes, and
subjected to Western immunoblotting with antibodies specific for c-Fos,
c-Jun, Fra-1, or Fra-2. Fold increases in protein expression are shown
above each lane for each blot. The results shown are from a single
experiment typical of at least three giving identical results.
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PD098059 inhibits LPA-stimulated expression of all AP-1
components.
Although the kinetic and pharmacological correlations
between sustained MAPK activation and the induced expression of Fra-1, Fra-2, c-Jun, and JunB were strong, we sought direct evidence of a role
for the Raf-MEK-MAPK pathway in the LPA-stimulated expression of these
genes. To address this, we utilized PD098059, a highly selective
inhibitor of MEK1 (5), to prevent activation of MEK1 and
MAPK in response to LPA stimulation. We have previously demonstrated that PD098059 completely inhibits both LPA- and EGF-stimulated MAPK
activation in Rat-1 cells with a 50% inhibitory concentration of
approximately 5 to 10 µM, with complete inhibition observed at 40 µM (21).
Pretreatment of Rat-1 cells with 40 µM PD098059 resulted in a
pronounced, though not complete, inhibition of LPA-stimulated
c-Fos
expression which was apparent at all times tested (Fig.
4A). These data suggest that induction of
c-Fos exhibits a strong
dependency on the activity of the MEK-MAPK
cascade. In addition,
the remaining c-Fos protein was predominantly in
the hypophosphorylated
state, suggesting that the Raf-MEK-MAPK cascade
plays a role in
the observed hyperphosphorylation of c-Fos in
LPA-stimulated cells.
These results support the proposal that
phosphorylation of c-Fos
is catalyzed by MAPK or the MAPK-dependent
p90
RSK pathway in whole cells (
14). Similarly,
pretreatment of Rat-1
cells with PD098059 also inhibited the
LPA-induced increased expression
of c-Jun (Fig.
4).

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FIG. 4.
Effect of PD098059 on LPA-stimulated expression of Fos
and Jun proteins. Confluent, serum-starved Rat-1 cells were pretreated
for 30 min with 40 µM PD098059 or a vehicle control as indicated
before being stimulated with 50 µM LPA for the times indicated.
Whole-cell detergent lysates were fractionated by SDS-PAGE, transferred
to PVDF membranes, and subjected to Western immunoblotting with
antibodies specific for c-Fos or c-Jun (A) or Fra-1, Fra-2, or JunB
(B). Fold increases in protein expression are shown above each lane for
each blot. The results shown are from a single experiment typical of at
least three giving identical results.
|
|
In the same series of experiments, pretreatment of Rat-1 cells with
PD098059 resulted in complete inhibition of the LPA-stimulated
expression of Fra-1, Fra-2, and JunB, strongly suggesting that
the
expression of these proteins requires the activity of MEK
and MAPK
(Fig.
4B). In addition, we also noted that the reduced
mobility of
Fra-2 which was observed after 10 or 15 min of LPA
stimulation was
prevented by treatment with PD098059. Indeed,
the time of maximal MAPK
activation (10 min) (Fig.
1) correlates
well with the kinetics of
appearance of the reduced-mobility forms
of Fra-2, suggesting that MAPK
may be responsible for phosphorylation
of preexisting Fra-2. It was not
possible to reliably confirm
if Fra-1 hyperphosphorylation was blocked
by PD098059, since PD098059
strongly inhibited the stimulated
expression of Fra-1, making
it difficult to detect any form of Fra-1.
However, it has been
reported that Fra-1 and Fra-2 are phosphorylated
in vitro by MAPK
(
36,
76).
Taken together, these results suggest that activation of the MAPK
cascade is required for the expression of the AP-1 proteins
studied
here but that expression of Fra-1, Fra-2, c-Jun, and JunB
in particular
requires sustained activation of this pathway. This
proposal is not
based solely on studies with LPA in Rat-1 cells,
as we have recently
made similar observations by studying a variety
of growth factors in
the CCL39 cell line (
9).
Activation of
Raf-1:ER is sufficient to induce expression of
Fra-1, Fra-2, and JunB but not c-Fos or c-Jun.
The preceding
experiments demonstrated a strong requirement for sustained MAPK
activation in regulating the expression of Fra-1, Fra-2, c-Jun, and
JunB, whereas the early phase of MAPK activation is associated with
expression of c-Fos and a modest increase in c-Jun. We wished to
determine if activation of the MAPK cascade alone would be sufficient
to induce expression of these genes. It has been shown that Fra-1,
Fra-2, c-Jun, and JunB are expressed at elevated levels in
Ras-transformed cells (71); however, Ras regulates at least
three different effector pathways (Raf, phosphatidylinositol 3'-kinase,
and RalGDS/Rlf) (67), and it is unclear what roles the
different pathways play in the regulation of gene expression
(102). For these studies we used Rat-1 (R1
RafER-4) and
3T3 (NIH 3T3 c4 or c9) cells expressing a conditionally active form of
oncogenic human Raf-1 (
Raf-1:ER). Activation of
Raf-1:ER in these
cells, by
-E2 or 4-HT, leads to rapid, protein synthesis-independent
activation of MEK and MAPK without the activation of other parallel
growth factor-stimulated signal pathways (83) until 15 to
20 h later (69, 73).
Treatment of quiescent R1

RafER-4 cells with serum resulted in
increased expression of c-Fos, c-Jun, Fra-1, Fra-2, and JunB
(Fig.
5A), indicating that the signalling
pathways regulated by
serum were not compromised by expression of

Raf-1:ER. Activation
of

Raf-1:ER for up to 4 h had no effect
on the expression of
c-Fos and c-Jun but significantly induced the
expression of Fra-1,
Fra-2, and JunB in R1

RafER-4 cells (Fig.
5A).
The expression
of Fra-1, Fra-2, and JunB observed in response to
activation of

Raf-1:ER was slightly less than that observed in
response to
serum but was still much stronger than that seen for c-Fos
or
c-Jun. Analysis of MAPK activation under these conditions, using
the
phosphospecific MAPK antibody, revealed that serum stimulation
and
activation of

Raf-1:ER induced similar levels of MAPK activation.

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FIG. 5.
Expression of Fos and Jun proteins in Rat-1 cells in
response to activation of Raf-1:ER. Confluent, serum-starved
R1 Raf-1:ER-4 cells were stimulated with either 20% FBS or 1 µM
-E2 for 1 to 4 h. Whole-cell detergent lysates were
fractionated by SDS-PAGE, transferred to PVDF membranes, and subjected
to Western immunoblotting with antibodies specific for c-Fos, Fra-1, or
Fra-2 (A) or c-Jun, JunB, or phospho-MAPK (P-MAPK) (B). Fold increases
in protein expression or MAPK activation are shown above each lane for
each blot. The results shown are from a single experiment typical of
three giving identical results.
|
|
A similar analysis was performed with NIH 3T3 c4 and c9 cells
expressing

Raf-1:ER. Again,

Raf-1:ER activation caused only
a
weak activation of c-Fos expression in these cells, although
the
response to LPA proceeded normally (Fig.
6A). The basal level
of c-Jun in NIH 3T3
cells was considerably lower than that in
Rat-1 cells and was not
detectable, making the fold increase in
c-Jun an underestimate.
Increased expression of c-Jun was observed
in response to LPA and in
response to activation of

Raf-1:ER
at early times (1 to 5 h),
but a much more pronounced induction
was observed 20 h after
activation of

Raf-1:ER, which has also
been observed by others
(
92). Under these conditions, it has
previously been
demonstrated that activation of

Raf-1:ER leads
to the expression and
release of growth factors such as HB-EGF,
which may act in an autocrine
manner to promote c-Jun expression
(
69,
73). In the same
experiments we observed strong expression
of Fra-1 and JunB in response
to both LPA and

Raf-1:ER activation.
Even after prolonged serum
withdrawal, NIH 3T3 c4 cells exhibited
high levels of Fra-2, so both
LPA and

Raf-1:ER activation elicited
only modest increases in Fra-2
expression. However, we again observed
that both LPA and

Raf-1:ER
caused a similar reduction in electrophoretic
mobility of the Fra-2
protein (Fig.
6). Indeed, the electrophoretic
mobility of Fra-2 was
reduced to its greatest extent at the time
of maximal MAPK activation
by LPA (10 min) or

Raf-1:ER (1 h).
Since the mobility shift of Fra-2
is abolished by treatment of
cells with PD098059 or treatment of
lysates with alkaline phosphatase,
these results suggest that the
Raf-MEK-MAPK cascade is both necessary
and sufficient for
hyperphosphorylation of Fra-2.

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FIG. 6.
Expression of Fos and Jun proteins in NIH 3T3 cells in
response to activation of Raf-1:ER. (A) Confluent, serum-starved c4
cells (NIH 3T3 cells expressing Raf-1:ER) were stimulated with
either 50 µM LPA or 1 µM 4-HT for the times indicated. Whole-cell
detergent lysates were fractionated by SDS-PAGE, transferred to PVDF
membranes, and subjected to Western immunoblotting with antibodies
specific for phospho-MAPK (P-MAPK), c-Fos, c-Jun, Fra-1, Fra-2, or
JunB. Fold increases in protein expression or MAPK activation are shown
above each lane for each blot. Note that the basal levels of c-Jun and
JunB were not detectable; the basal level of 1 is set at the lowest
detectable value, making the fold increases an underestimate. The
results shown are from a single experiment typical of three giving
identical results. (B) c4 cells were stimulated with 1 µM 4-HT, 50 µM LPA, or 20% FBS for the times indicated. Cell extracts were
prepared, and c-Fos transcripts were analyzed by RNase protection as
described in Materials and Methods. The graph represents a quantitative
analysis of the data shown in the autoradiograph. Con, control; PI,
phosphorimager units.
|
|
Since many studies have suggested a role for the MAPK cascade in
regulating c-Fos expression (
34,
42,
43,
50,
64,
95,
101),
we were intrigued by the fact that

Raf-1:ER activation
caused such a
small increase in c-Fos protein levels. We investigated
this further by
RNase protection analysis of c-Fos mRNA induction
following

Raf-1:ER
activation. Under these conditions, we observed
a very modest and
transient expression of c-Fos transcripts in
response to activation of

Raf-1:ER, whereas serum stimulation
elicited a robust response (Fig.
6B). For example, in a side-by-side
comparison,

Raf-1:ER stimulated
the expression of c-Fos protected
transcripts by 7-fold, LPA stimulated
the accumulation of c-Fos
transcripts by 25-fold, and serum did so by
73-fold. In these
experiments the maximal activations of MAPK seen in
response to
serum, LPA, and

Raf-1:ER were similar, although the
kinetics
of the response to

Raf-1:ER activation was slightly slower
than
that for serum or LPA (Fig.
5 and
6A). It is therefore unlikely
that the lack of inducibility of c-Fos by

Raf-1:ER is due to
insufficient activation of MAPK. Thus, while activation of MAPK
is
required for c-Fos expression, activation of that pathway alone
is not
sufficient to induce c-Fos.
In summary, selective and sustained activation of the MAPK cascade
alone is sufficient to induce expression of Fra-1, Fra-2,
and JunB,
whereas induced expression of c-Fos is very weak. c-Jun
expression is
induced weakly as a result of acute activation of
the MAPK pathway but
strongly after a prolonged activation (15
to 20 h), which may
reflect the action of an autocrine
factor.
Synergistic expression of c-Fos by
Raf-1:ER activation and
Ca2+.
In addition to activating the Ras-Raf-MAPK
cascade, LPA stimulates the release of Ca2+ from
intracellular stores, resulting in a transient increase in the
intracellular [Ca2+] ([Ca2+]i).
We have recently shown that an immediately-early gene, that for MAPK
phosphatase-1 (MKP-1), is unresponsive to the activation of
Raf-1:ER
in Rat-1 cells (21). In this case the expression of MKP-1 is
dependent upon both the MAPK cascade and Ca2+ mobilization
and can be reconstituted by a combination of ionomycin and
Raf-1:ER
activation. These results prompted us to examine a possible role for
Ca2+ in the regulation of c-Fos expression. Pretreatment of
Rat-1 cells with BAPTA-AM to buffer increases in
[Ca2+]i prior to stimulation with LPA
resulted in a strong, but not complete, inhibition of subsequent c-Fos
expression (Fig. 7A). These data suggest
that LPA-induced c-Fos expression requires an increase in
[Ca2+]i. In these experiments we noted that
the small amount of c-Fos protein which was still induced in response
to LPA plus BAPTA-AM was predominantly in the hyperphosphorylated form
(Fig. 7A). This may be due to enhanced MAPK-dependent phosphorylation
(14), since under these conditions, LPA-stimulated MKP-1
expression is inhibited and MAPK activation is greatly enhanced
(21).

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FIG. 7.
Effect of Ca2+ on c-Fos expression in
response to LPA and Raf-1:ER. (A) R1 Raf-1:ER-4 cells were
pretreated with 30 µM BAPTA-AM (BAP) or a vehicle control before
being stimulated with 50 µM LPA for the times indicated. Whole-cell
detergent lysates were fractionated by SDS-PAGE, transferred to PVDF
membranes, and subjected to Western immunoblotting with antibodies
specific for c-Fos. (B) R1 Raf-1:ER-4 cells were stimulated with
increasing doses of ionomycin in the absence or presence of 1 µM 4-HT
for 60 min. Whole-cell detergent lysates were fractionated by SDS-PAGE,
transferred to PVDF membranes, and subjected to Western immunoblotting
with antibodies specific for c-Fos or phospho-MAPK (P-MAPK). Fold
increases in protein expression or MAPK activation are shown above each
lane for each blot. Similar results were obtained in additional
experiments with R1 Raf-1:ER-4 and NIH 3T3 c4 cells.
|
|
To examine possible cooperation between Ca
2+ and the MAPK
pathway, we stimulated serum-starved R1

Raf-1:ER-4 cells with
increasing
concentrations of ionomycin in the absence or presence of
4-HT
to activate

Raf-1:ER and analyzed c-Fos expression by
immunoblotting.
In these experiments we observed that activation of

Raf-1:ER
in the presence of a low dose of ionomycin (125 or 250 nM)
resulted
in synergistic induction of c-Fos expression (Fig.
7B). Even
at
500 nM ionomycin, a dose sufficient to weakly induce c-Fos on
its
own, we observed synergy with

Raf-1:ER activation (Fig.
7B).
The
c-Fos protein induced by ionomycin alone (e.g., at 500 nM)
exhibited a
lower apparent molecular weight than that induced
in response to the
combination of

Raf-1:ER and ionomycin. Since
c-Fos is phosphorylated
by MAPK and MAPK-dependent kinases (
14),
this may be related
to the weak and very transient activation
of MAPK which is observed in
response to Ca
2+ ionophores in these cells (Fig.
7B)
(
20). In order to determine
if under these conditions

Raf-1:ER and Ca
2+ were not simply synergizing to
hyperactivate the MAPK cascade,
the same samples were immunoblotted
with the phosphospecific MAPK
antibody. In these experiments it was
evident that ionomycin alone
elicited little activation of MAPK and did
not amplify the activation
of MAPK observed in response to

Raf-1:ER
activation. Similar
results were obtained when we examined the synergy
between

Raf-1:ER
and ionomycin in NIH 3T3 c4 cells (data not shown).
These results
suggest that ionomycin and

Raf-1:ER activation
cooperate to induce
c-Fos expression by activating separate
pathways.
 |
DISCUSSION |
The duration of MAPK activation determines the expression of
distinct Fos and Jun proteins.
Considerable interest has been
focused on the regulation of gene expression following growth factor
stimulation or oncogenic transformation and how such patterns of gene
expression relate to the biological effects observed. It is well
documented that the strength and duration of signal pathway activation
can have profound effects on the biological responses of cells. For
example, Raf-activated signalling pathways can elicit either a
mitogenic response or cell cycle arrest in NIH 3T3 cells, depending on
the level of pathway activation (103). Other studies have
demonstrated that growth and/or differentiation factors can stimulate
the sustained activation of MAPK and that this correlates with cell
proliferation or differentiation, depending on the magnitude of the
response and the cell type (19, 20, 40, 49, 66, 91). Here we have demonstrated that the duration of MAPK activation determines the
pattern of expression of different Fos and Jun proteins, allowing the
MAPK cascade to impart significant variation to the repertoire of AP-1
complexes assembled during cell cycle reentry.
The LPA-stimulated expression of Fos and Jun proteins described here is
strongly dependent on the Raf-MEK-MAPK pathway (Fig.
4), but the
temporal pattern of expression is highly coordinated
(Fig.
1) and is
determined by the duration of MAPK activation
(Fig.
2 and
3). In
response to a maximal proliferative dose of
LPA, c-Fos expression peaks
within 1 h and then declines to basal
levels by 6 h, whereas
Fra-1, Fra-2, c-Jun, and JunB are expressed
in a sustained manner,
persisting throughout the G
1 phase of the
cell cycle. In
contrast, a nonmitogenic dose of LPA, which fails
to induce the
sustained phase of MAPK activation, still induces
c-Fos but fails to
induce the expression of Fra-1 and Fra-2 and
elicits a greatly reduced
expression of c-Jun and
JunB.
These results suggest that in Rat-1 cells one of the functions of the
sustained phase of MAPK activity is to determine the
pool of AP-1
proteins available to form dimers. For example, other
studies have
demonstrated that c-Fos makes only a transient contribution
to AP-1
complexes early in G
1, and the assembly of heterodimers
containing Fra-1, Fra-2, c-Jun, and JunB in the mid-to-late
G
1 phase has been documented (
52,
53). Based on
these observations,
it seems reasonable to speculate that dimers
containing Fra-1,
Fra-2, c-Jun, and JunB exhibit distinct properties
(such as recognition
of a subset of TREs and/or unique transcriptional
properties)
such that they activate or repress distinct AP-1-regulated
genes
(
38,
39,
81). In this way the changing pattern of Fos
and
Jun protein expression during cell cycle reentry could provide
the
link between sustained MAPK activation and the cell cycle
regulatory
apparatus.
Given their different kinetics of expression and dependency on
sustained MAPK activation, it seems pertinent to consider the
biological functions of c-Fos, Fra-1, Fra-2, c-Jun, and JunB in
relation to cell cycle reentry. In Rat-1 cells the maximal induction
of
c-Fos at 1 h is the same at both doses of LPA, but the response
is
clearly more transient in response to 1 µM LPA. These data
might
suggest a correlation between the more prolonged gradual
decline in
c-Fos expression, sustained MAPK activation, and DNA
synthesis.
However, in pharmacological terms there seems to be
a poor correlation
between LPA-stimulated c-Fos expression (EC
50 of 0.1 to 0.2 µM) and DNA synthesis (EC
50 of 10 to 20 µM)
(
20).
In this regard it is interesting that
c-Fos
/
mice are viable and that c-Fos
/
fibroblasts proliferate normally (
11,
45). Furthermore,
enforced
expression of c-Fos can cause morphological transformation
without
affecting cell cycle progression (
72), and

Raf-1:ER can induce
DNA synthesis (
103) without promoting
significant c-Fos expression
(Fig.
5 and
6). While our results do not
address the role of c-Fos
in cell cycle reentry directly, they are
consistent with many
studies in the literature which argue against a
role for c-Fos
in driving cell cycle reentry in most cell
types.
The kinetic and pharmacological correlation between sustained MAPK
activation and cell cycle reentry in response to LPA (
20)
certainly suggests that the expression of Fra-1, Fra-2, c-Jun,
and JunB
correlates well with proliferative efficacy. Overexpression
of Fra-1
leads to morphological transformation and anchorage-independent
growth
of Rat-1 cells (
10) and can cooperate with c-Jun in
transforming
NIH 3T3 cells (
71). Furthermore, Fra-1, Fra-2,
c-Jun, and JunB
are found to be overexpressed in Ras-transformed
thyroid cells
(
96) and NIH 3T3 cells (
71), and
antisense-mediated inhibition
of Fra-1 expression causes a partial
reversion of the Ras-transformed
phenotype (
96).
Microinjection of neutralizing antisera specific
for the individual Fos
or Jun proteins has revealed that Fra-1,
Fra-2, c-Jun, and JunB serve
an important function during the
G
1-to-S transition and in
normal asynchronous cell proliferation
(
52).
Our results have implications for the role of AP-1 in Ras signalling.
The major AP-1 components up-regulated in Ras-transformed
cells are
Fra-1, Fra-2, c-Jun, and JunB (NIH 3T3 cells) (
71)
and Fra-1
and JunB (thyroid cells) (
96). Since Ras regulates
at least
three different effector pathways (Raf, phosphatidylinositol
3'-kinase,
and RalGDS) (
67), it is conceivable that all three
pathways
may contribute to changes in gene expression. Our analysis
here shows
that reconstitution of just one of these pathways,
Raf-MEK-MAPK, is
sufficient to account for the effects of Ras
on Fra-1, Fra-2, c-Jun,
and JunB, and these results have been
independently confirmed
(
92).
Recent studies have demonstrated a link between the Ras-Raf-MEK-MAPK
pathway and the cell cycle apparatus. Inducible expression
of RasV12 or
conditional activation of Raf is sufficient to drive
the expression of
cyclin D1 (
84,
103), and MEK is required
for activation of
the cyclin D1 promoter (
59). The cyclin D1
promoter contains
an AP-1 site (
3,
41) which binds to c-Fos
and the three Jun
proteins (
3), but it seems unlikely that
c-Fos is required
for cyclin D1 expression, since activation of

Raf-1:ER induces
cyclin D1 (
103) without significant levels
of c-Fos
expression (Fig.
5 and
6). Based on these observations,
it will be
interesting to study the role of Fra-1, Fra-2, and
JunB in the
expression of cell cycle-regulatory genes such as
those for cyclin D1
and p21
Cip1, which are linked to sustained MAPK activation.
However, we emphasize
that AP-1 is unlikely to be the sole target of
sustained MAPK
activity. Indeed, cyclin D1 has also been proposed to be
a direct
transcriptional target of Ets-2 (
3); this may
provide a direct
link between the MAPK pathway and cyclin D1
expression, since
Ets-2 is phosphorylated and regulated by MAPK
(
70).
How does the duration of MAPK activation determine the expression of
these individual proteins? Given the temporal pattern
of Fos and Jun
protein expression, our results are consistent
with a model in which
the early peak of c-Fos expression directs
the subsequent expression of
Fra-1 and Fra-2 by forming functional
AP-1 complexes at AP-1 sites in
their promoter enhancer regions.
In this way the MAPK-dependent
expression of c-Fos would be reflected
in the expression of c-Fos
target genes such as that for Fra-1,
and such a model would be
consistent with the ability of c-Fos:ER
to induce Fra-1 expression
(
10). However,

Raf-1:ER induces
robust expression of
Fra-1 and JunB but little, if any, expression
of c-Fos (Fig.
5 and
6),
and serum-stimulated transcription of
Fra-1 is insensitive to
cycloheximide (
17). Indeed, in c-Fos
/
fibroblasts serum-induced expression of Fra-1 is reduced and
delayed,
but not abolished, and the expression of other AP-1 components
is
normal (
11,
45). This suggests that c-Fos is not required
for the expression of Fra-1, Fra-2, and JunB and may reflect redundancy
between c-Fos and other Fos family members or indeed other
transcription
factors. Fra-2 expression may be regulated directly by
the MAPK
cascade via an SRE site or indirectly via the AP-1 site
(
86).
JunB expression is regulated by Ras via tandem
inverted Ets binding
sites in the promoter (
16,
31). The
recent demonstration that

Raf-1:ER activation is sufficient to
promote Ets-2 phosphorylation
and the activation of both AP-1/Ets and
Ets/Ets
cis-acting elements
(
70,
104) suggests a
direct, AP-1-independent pathway between
the MAPK cascade and
expression of JunB. Our demonstration that
JunB expression is blocked
by PD098059 and can be reconstituted
by

Raf-1:ER alone suggests that
such a pathway may indeed operate
for JunB
expression.
c-Jun is reported to be up-regulated in Ras-transformed cells but
exhibits weak inducibility by

Raf-1:ER. This may suggest
that c-Jun
expression requires integration of two signals, both
of which can be
provided by Ras, whereas Raf can only provide
one. Alternatively, the
enhanced expression of c-Jun in constitutively
Ras-transformed cells
(
71) may reflect activation of an autocrine
loop. Indeed,
activation of the MAPK cascade leads to the expression
and release of
HB-EGF, which can act as an autocrine factor to
promote activation of
JNK (
69,
73); in this way expression
of c-Jun could result
from autoregulation of its own promoter
(
30,
98). The
delayed expression of c-Jun in response to activation
of

Raf-1:ER
(Fig.
6) or MEK (
92) may be due to such an autocrine
loop.
It is important to stress that the changes in steady-state protein
levels reported here may not simply reflect changes in
the
transcription rate. The duration of MAPK activation may regulate
mRNA
stability, and there is certainly precedent for MAPK or SAPK
regulating
the stability of Fos and Jun proteins. For example,
MAPK phosphorylates
c-Fos, thereby increasing its half-life and
transforming potential
(
14), while MAPK- or SAPK-catalyzed phosphorylation
of c-Jun
reduces its ubiquitination, thereby stabilizing the c-Jun
protein
(
77). Future studies will aim to address the mechanism
by
which the duration of MAPK activation determines the pattern
of protein
expression reported
here.
Activation of the MAPK cascade by
Raf-1:ER is sufficient for
expression of Fra-1, Fra-2, and JunB but not c-Fos.
One of the
central paradigms to have emerged in signal transduction is that
activation of the MAPK cascade regulates c-Fos expression (34, 42,
43, 50, 64, 95, 101). However, our results, together with those
of others, suggest that c-Fos regulation is more complex than this. In
R1
Raf:ER-4 cells we did not observe expression of c-Fos in response
to activation of
Raf-1:ER, while in NIH 3T3 cells RNase protection
assays confirmed that c-Fos transcripts were only weakly induced by
activation of
Raf-1:ER. In both cells serum- and LPA-stimulated
c-Fos expression was normal. In addition, it is clear that the
inability to induce c-Fos does not reflect a generalized failure of
Raf-1:ER to activate gene expression, since other genes, such as
those for Fra-1, Fra-2, and JunB (Fig. 5 and 6), cyclin D1
(103), and c-Myc (8), are induced strongly by
activation of
Raf-1:ER.
The results with PD098059 (Fig.
4) and

Raf-1:ER (Fig.
5) suggest
that the MAPK cascade is necessary but not sufficient for
c-Fos
expression. One possibility is that expression of c-Fos
requires
activation of multiple signals and it is the synergistic
integration of
these signals which leads to induced c-Fos expression.
This model is
supported by experiments in which

Raf-1:ER activation
and ionomycin
synergized to induce expression of c-Fos without
amplifying activation
of the MAPK cascade. Since buffering of
increased
[Ca
2+]
i inhibited LPA-induced c-Fos
expression (Fig.
7A) without blocking
MAPK activation (
21),
these results are most consistent with
induced c-Fos expression
requiring the integration of MAPK- and
Ca
2+-driven signals.
This could occur at the level of the c-Fos promoter
itself, which
contains both MAPK-responsive elements (ternary
complex factor/serum
response element) and Ca
2+-responsive elements (
33,
46). Robertson et al. have shown
that both the
Ca
2+-responsive element and SRE sites are required for
optimal expression
of c-Fos in response to growth factors
(
80). In addition to
the possible integration of signalling
pathways at different
cis-acting
promoter elements, a recent
study has demonstrated that dynamic
regulation of histone H4
hyperacetylation may also provide an
additional level of signal
cooperation to regulate c-Fos expression
(
4). Future studies
will aim to address the mechanism of the
observed cooperation between

Raf-1:ER and Ca
2+ in regulating c-Fos
expression.
Conclusion.
In summary, these results demonstrate that the
magnitude and duration of MAPK activation determine the repertoire of
Fos and Jun family members available to form AP-1 complexes at
different stages during the G1 phase of the cell cycle.
This is likely to lead to the formation of discrete AP-1 complexes with
distinct properties which could, in principle, allow for changes in
AP-1-regulated gene expression as the cell proceeds through
G1. These results provide strong support for the proposal
that quantitative differences in duration of MAPK activation will lead
to qualitative changes in gene expression (66) and suggest
that the temporal changes in AP-1 composition may represent a suitable
experimental paradigm to test this model further.
 |
ACKNOWLEDGMENTS |
We thank Kathryn Balmanno, Allan Balmain, Jerlyn Beltman, Gideon
Bollag, Michelle Garrett, Frank McCormick, John Pascall, and Doug Woods
for stimulating discussions and particularly Iris Treines, Hugh
Paterson, and Chris Marshall for discussion of their unpublished results.
The DNAX Research Institute is supported by Schering Plough
Corporation. This work was initiated at ONYX Pharmaceuticals as part of
a project supported by a collaborative research agreement with Bayer AG
and was continued at The Babraham Institute, where it was supported by
a competitive strategic grant from the BBSRC.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Signalling
Programme, The Babraham Institute, Babraham Hall, Cambridge, CB2 4AT,
England, United Kingdom. Phone: (44) 1223-496453. Fax: (44)
1223-496043. E-mail: simon.cook{at}bbsrc.ac.uk.
 |
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Molecular and Cellular Biology, January 1999, p. 330-341, Vol. 19, No. 1
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