Previous Article | Next Article 
Molecular and Cellular Biology, January 1999, p. 321-329, Vol. 19, No. 1
CRC Centre for Cell and Molecular Biology,
Chester Beatty Laboratory, Institute of Cancer Research, London,
SW3 6JB, United Kingdom
Received 13 May 1998/Returned for modification 29 June
1998/Accepted 15 September 1998
To investigate the contribution that ERK/mitogen-activated protein
kinase signalling makes to cell cycle progression and gene expression,
we have constructed cell lines to express an inducible version of
activated MEK1. Using these cells, we show that activation of MEK leads
to the expression of Fra-1 and Fra-2 but not c-Fos. Treatment of
Ras-transformed cells with the MEK inhibitor PD098059 blocks expression
of Fra-1 and Fra-2, showing that in Ras transformation ERK signalling
is responsible for Fra-1 and Fra-2 expression. Activation of MEK1
in growth-arrested cells leads to DNA synthesis; however, ERK
activation alone is insufficient because the induction of DNA synthesis
is blocked by inhibition of phosphatidylinositol 3-kinase (PI3-kinase).
Activation of PI3-kinase is indirect, perhaps through autocrine growth
factors, and is required for the induction of cyclin D1.
The ERK/mitogen-activated protein
kinase (MAP kinase) pathway has been identified as a major signalling
pathway activated through p21Ras (12). This signalling
pathway has been shown to be required for growth factors to stimulate
cell proliferation via Ras (10, 15, 47). However much
evidence has accumulated demonstrating that activation of ERKs is only
one of the effector pathways of Ras signalling. At least 10 proteins
have been identified which are candidates for effectors of Ras
signalling because they interact with Ras only in the active GTP-bound
form. These candidate effector proteins include Ras GTPase-activating
proteins (1, 38), Raf family protein kinases (60, 64,
70), phosphatidylinositol 3-kinases (PI3-kinases)
(51), guanine nucleotide exchange factors for Ral family
GTPases (24, 29, 37, 59, 67), protein kinase C-zeta
(14), MEKK-1 (53), and proteins of unknown
function (61). The use of effector mutants of Ras which
selectively interrupt interactions with some of these proteins has
strongly suggested roles for Raf-1 (65), PI3-kinase
(52), and the Ral GTPase activator Ral-GDS (66)
in cell transformation. Similarly, effector mutants have been used to
show that activation of multiple signalling pathways by Ras is required
to stimulate DNA synthesis (28, 65). In contrast to these
studies with Ras effector mutants, the observation that activated forms
of Raf and MEK act as oncoproteins suggests that activation of the ERK
pathway may be sufficient to stimulate DNA synthesis (10,
35).
Signalling through the ERK/MAP kinase pathway is thought to depend at
least in part on the activation of gene expression. ERK/MAP kinases
have been shown to phosphorylate transcription factors of the ELK/SAP
family as well as the Ets family and STAT family (23).
Phosphorylation of Elk-1 by ERK/MAP kinases has been shown to be
required for transcriptional activation of the immediate-early gene
product c-Fos (36). Ras-transformed cells have been shown to
have elevated levels of Fra-1, Fra-2, c-Jun, and JunB, which contribute
to elevated AP-1 activity (41). However, c-Fos expression is
not elevated in Ras-transformed cells even though ERK/MAP kinase
activity is raised (41). Blocking of AP-1 activity in
Ras-transformed cells has been shown to suppress transformation (34). The signalling pathways that result in elevated Fra-1, Fra-2, c-Jun, and JunB expression have not been elucidated, so the
mechanism by which these Fos and Jun family members are upregulated in
Ras-transformed cells is not clear.
To address these issues, we have constructed inducible forms of
activated MEK1 to see whether induction of MEK activity is sufficient
to permit entry into DNA synthesis and induction of Fra-1, Fra-2,
c-Jun, and JunB. Ras-mediated activation of Raf activates MEK (13,
25, 31), which is the immediate upstream activator of ERK1 and
ERK2 (45). To date, no substrates of MEK other than ERKs
have been identified. Inducible versions of activated signalling
components have an advantage over constitutive expression in that
long-term effects of expression of an activated signalling molecule can
be avoided and the kinetics of responses can be monitored. Using cells
with an inducible activated MEK, we show that MEK activation induces
DNA synthesis in quiescent NIH 3T3 cells. However, induction of DNA
synthesis in this system also requires participation of the PI3-kinase
pathway. Activation of MEK appears to act as a switch which not only
directly activates the ERK intracellular signalling pathway but also
may indirectly activate other signalling pathways.
Construction of the MEK/ Cell culture and transfection.
NIH 3T3 cells were grown in
Dulbecco's modified Eagle's medium (DMEM) containing 10% donor calf
serum (DCS) (Gibco-Life Sciences), and v-Ras-transformed NIH 3T3 cells
were grown in DMEM containing 5% DCS. For transfection with
Lipofectamine (Gibco-Life Sciences), 1.5 × 105 cells
were plated into 30-mm-diameter tissue culture dishes. On the following
day the cells were washed with serum-free medium, and DNA-Lipofectamine
complexes (0.4 µg of DNA and 5 µl of Lipofectamine) were added to
the cells in 1 ml of serum-free medium. The cells were incubated for
6 h in the presence of the DNA-Lipofectamine complexes, washed,
and incubated in 10% DCS-DMEM. After 24 h, the cells were
trypsinized and plated at low densities in the presence of 2.5 µg of
puromycin (Sigma) per ml. Cell clones were picked with sterile cotton
buds and characterized for MEK/
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Activated MEK Stimulates Expression of AP-1 Components
Independently of Phosphatidylinositol 3-Kinase (PI3-Kinase) but
Requires a PI3-Kinase Signal To Stimulate DNA Synthesis
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
NEE-ER fusion gene expression
vector.
Rabbit MEK1 cDNA, which contains the mutations that lead
to replacement of serine by glutamic acid at positions 218 and 222 (2), was digested with StuI and subsequently
religated to generate the activating deletion in the amino terminus
(amino acids 32 to 51) (35). The construct was then
linearized with BamHI and partially digested with
EagI. The BamHI-EagI MEK1 cDNA
fragment and an EagI-SalI fragment encoding the
mutant hormone binding domain of the mouse estrogen receptor (ER) were
ligated into BamHI- and SalI-digested pBABEpuro
(43).
NEE-ER expression by Western blot
analysis and immunofluorescence.
20°C.
PD098059 was provided by A. Saltiel (Parke-Davis), made up as a 1 mM
stock solution in dimethyl sulfoxide, and stored at 20°C. LY294002
was purchased from Biomol Research Laboratories, made up as a 50 mM
stock solution in ethanol, and stored at 20°C.
Antibodies. ERK2 polyclonal rabbit antiserum no. 122 was generated against a C-terminal ERK2 peptide (32), MEK1 polyclonal rabbit antiserum no. 179 was raised against recombinant glutathione S-transferase-MEK1 (2). Mouse monoclonal antibodies against MEK1 and p27Kip1 were from Transduction Laboratories. Anti-RSK1 was a gift from P. Cohen, Dundee, United Kingdom. Anti-cFos, anti-FosB, anti-Fra-1, anti-Fra-2, anti-cJun, and anti-JunB were purchased from Santa Cruz Biotechnology. Phospho-specific anti-protein kinase B (PKB)/Akt rabbit polyclonal antiserum (against serine 473) was kindly provided by J. Downward, Imperial Cancer Research Fund, London, United Kingdom. Anti-cyclin D1 antibody was from G. Peters, ICRF. Anti-CDK2 and anti-CDK4 were from Santa Cruz Biotechnology or from C. Sherr, Memphis, Tenn.
Preparation of cell extracts and analysis by Western blotting. Cells were washed twice in ice-cold phosphate-buffered-saline (PBS) and lysed in 20 mM Tris (pH 8.0)-40 mM sodium pyrophosphate-50 mM sodium fluoride-5 mM MgCl2-100 mM sodium vanadate-10 mM EGTA-1% Triton X-100-0.5% sodium deoxycholic acid-20 mg of leupeptin per ml-20 mg of aprotinin per ml-1 mM phenylmethylsulfonyl fluoride. Cell debris was removed by centrifugation at 12,000 rpm in an Eppendorf microcentrifuge and protein concentrations were determined by the Bradford protein assay (Bio-Rad). Cell extracts (50 µg) were electrophoresed through sodium dodecyl sulfate (SDS)-polyacrylamide gels and Western blotted onto nitrocellulose. Western blots were probed with the appropriate dilutions of primary antibodies for at least 1 h at room temperature.
DNA synthesis assay. DNA synthesis was determined by uptake and incorporation of bromodeoxyuridine (BrdU) (Amersham Life Sciences) from the culture medium. Cells were made quiescent either by incubation for 48 h in serum-free DMEM or by contact inhibition in the presence of 5% DCS. After 20 to 24 h of exposure to 10 µM BrdU, cells were fixed and permeabilized for immunofluorescence as described below. BrdU incorporation into DNA was detected by incubating the fixed cells with a mouse monoclonal antibody against BrdU (Boehringer Mannheim) at 5 µg/ml in the presence of 1 mg of DNase I per ml for 1 h at 37°C.
Immunofluorescence. Cells were fixed in 4% formaldehyde in PBS for 15 min, washed in several changes of PBS for 30 min, permeabilized in 0.2% Triton X-100 in PBS for 15 min, and blocked by incubation in 10% fetal bovine serum in PBS for 30 min. Primary antibody incubations were performed for 1 h at room temperature; after a 15-min wash in PBS, the cells were incubated with appropriate fluorescent second antibodies (Jackson Immunoresearch Laboratories) for 1 h at room temperature. Stained preparations were mounted under glass coverslips and examined with a Bio-Rad MRC 1000 or 1024 confocal imaging system in conjunction with a Nikon Diaphot epifluorescence microscope.
ERK2 assay.
Fifty micrograms of cell lysate was diluted to a
volume of 200 µl with lysis buffer and immunoprecipitated for 90 min
with 5 µl of ERK2 antiserum (no. 122) applied to 20 µl of protein
A-agarose (Bio-Rad) on a revolving wheel at 4°C. The reaction mixture
was washed twice with lysis buffer, once with 30 mM Tris (pH 8.0), and
once with kinase buffer (30 mM Tris [pH 8.0], 20 mM
MgCl2, 2 mM MnCl). The washed immunoprecipitates were
drained of excess liquid and incubated with 30 µl of kinase buffer
containing 10 µM ATP (Sigma), 0.25 mg of myelin basic protein
(Sigma), and 66 µCi of [
-32P]ATP (Amersham) per ml
at 30°C for 30 min. The reaction was stopped by the addition of SDS
sample buffer (80 mM Tris [pH 6.8], 2% SDS, 10% glycerol, 100 µM
dithiothreitol, 0.02% bromophenol blue), boiled for 5 min, and
analyzed on an SDS-15% polyacrylamide gel. The gel was blotted onto
nitrocellulose which was subsequently autoradiographed and analyzed on
a Molecular Dynamics PhosphorImager, using ImageQuant software.
RSK assay.
Two hundred micrograms of cell lysate was
immunoprecipitated for 90 min with 2.7 µg of RSK1 antiserum applied
to 20 µl of protein G-Sepharose (Sigma) on a revolving wheel at
4°C. The reaction mixture was washed twice with lysis buffer, once
with 50 mM MOPS (morpholinepropanesulfonic acid) (pH 7.0), and once
with kinase buffer (50 mM MOPS [pH 7.0], 10 mM MgCl2, 0.1 mM EGTA). The washed immunoprecipitates were drained of excess liquid
and incubated with 50 µl of kinase buffer containing 50 µM ATP
(Sigma), 30 µM peptide KKKNRTLSVA, and 50 µCi of
[-32P]ATP (Amersham) per ml at 30°C for 15 min. The
reactions were terminated by spotting 40 µl of the reaction mixture
onto P81 paper (Whatman), which was subsequently washed five times with 75 mM orthophosphoric acid. RSK1 activity was quantified by counting the radioactivity on the P81 paper in a Cerenkov counter.
Assays for CDK activity.
Serum-starved cells were lysed in
100 mM HEPES (pH 7.0)-500 mM NaCl-10 mM EDTA-20 mM
-glycerophosphate-20 mM sodium fluoride-2 mM sodium vanadate-0.5
mM dithiothreitol-0.2% Triton X-100-20 µg of aprotinin per ml-20
µg of leupeptin per ml-1 mM phenylmethylsulfonyl fluoride. Lysates
were centrifuged at 12,000 rpm to remove insoluble material, and
protein concentrations were determined with the Bradford assay
(Bio-Rad). Five hundred micrograms of cleared extract was
immunoprecipitated with antibodies coupled to protein G-Sepharose (Sigma) for 90 min at 4°C. Immune complexes were washed three times
with lysis buffer, once with 50 mM HEPES (pH 7.4), and then once with
kinase buffer (50 mM HEPES, 10 mM MgCl2, 10 mM
MnCl2, 10 mM -glycerophosphate, 1 mM dithiothreitol, 20 µg of aprotinin per ml, 20 µg of leupeptin per ml, and 1 mM
phenylmethylsulfonyl fluoride). CDK4-associated kinase activity was
measured by using as a substrate 0.5 µg of a glutathione
S-transferase fusion protein containing amino acids 763 to
928 of human pRb (a generous gift of S. Mittnacht) in 20 µl of kinase
buffer with 50 µM ATP and 5 µCi of [-32P]ATP. For
CDK2 assays the substrate was 2 µg of histone H1 (Boehringer Mannheim). After 15 min of incubation at 30°C, reactions were stopped
by addition of SDS sample buffer, and the mixtures were boiled and
resolved by SDS-polyacrylamide gel electrophoresis. After
electrophoresis, proteins were transferred to polyvinylidene difluoride
membranes (Immobilon; Millipore), and radioactivity was detected with a
PhosphorImager (Molecular Dynamics). Radioactivity was quantitated by
the ImageQuant program.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Morphological transformation induced by regulatable
MEK1.
In order to analyze the role of MEK1 in cell
transformation and cell cycle regulation, we established a
conditionally activated MEK1 by fusion to the hormone binding domain of
a mutant mouse ER which responds to 4-OHT but not to estradiol
(33). A variety of constructs were generated, in which
different activating mutations were combined with the ER either amino
terminal or carboxy terminal to MEK1. Placement of the hormone binding
domain of the ER at the C terminus of an activated MEK1 generated by
mutation of the amino acids serine 218 and 222 to glutamic acid in the
catalytic domain, which mimics the activating phosphorylations (2,
10), and deletion of a region in the amino terminus (amino acids
32 to 51) which has been described to enhance further the kinase activity of the mutant MEK (16, 35) created the most
effective version (Fig. 1A).
Deletion of amino acids 32 to 51 also removes the nuclear export signal
of MEK1 (17); however, the MEK/NEE-ER fusion protein was
present only in the cytoplasm (Fig. 1C), presumably as a
consequence of fusion to the ER.
|
NEE-ER cells).
Western blotting analysis and immunofluorescence staining
showed that there was a significant increase in the level of the fusion
protein detectable after 4-OHT induction over time (Fig. 1B and C).
Similar observations showing an increase in the levels of an ER fusion protein with time were described by Samuels et al. with a Raf-ER fusion
protein (55).
Individual NIH:MEK/NEE-ER cell clones were analyzed for their
morphology. In the absence of 4-OHT, the cells are flat and untransformed, but the induction of the MEK1-ER fusion protein by
addition of 4-OHT results in a refractile phenotype (Fig. 1C) and
disruption of actin stress fibers (data not shown). The first morphological changes are detectable about 16 h after addition of
4-OHT; after 24 to 30 h, the transformed phenotype is
complete. MEK1-induced transformation occurs in the presence and
absence of serum with similar kinetics. The morphological
transformation induced by MEK/NEE-ER was blocked with the
MEK-specific inhibitor PD098059 (15) in a
concentration-dependent manner. Treatment of the cells with 30 µM
PD098059 completely prevented MEK-induced transformation (data not shown).
Activated MEK1 induces low levels of ERK and RSK activities.
In order to characterize the downstream events after induction of
activated MEK, we analyzed the activity of the MEK substrate ERK2 after
stimulation of MEK/NEE-ER with 4-OHT (Fig.
2A). Between 1 and 5 h after 4-OHT
addition, endogenous ERK2 activity was induced twofold; at 16 to
24 h after induction, ERK2 activity was increased to three- to
fourfold. After 48 h, the level of ERK2 activity was further
increased to approximately sixfold. In comparison, the induction of
ERK2 activity after stimulation with 10% serum for 1 h was about
30-fold. Thus, activation of inducible MEK1 with 4-OHT leads to a
sustained but very weak activation of ERK2. Similar results were
obtained for ERK1 activation (data not shown).
|
NEE-ER fusion protein was low compared to that after induction
with 10% serum for 1 h (sixfold). However, these results show
that the low level of ERK2 activity after induction of the
MEK/NEE-ER fusion protein with 4-OHT is sufficient to activate the
downstream target RSK1. Analysis of several other cell clones
expressing the MEK/NEE-ER fusion protein gave similar results (data
not shown).
Inducible activated MEK1 stimulates DNA synthesis.
Given that
induction of MEK activates endogenous ERK only two- to sixfold, it was
of interest to determine if this was sufficient to induce DNA
synthesis. When passaged in the absence of 4-OHT, MEK/NEE-ER-expressing cells, like wild-type NIH 3T3 cells, enter proliferation arrest when they are grown to confluency in 5% serum or
incubated in serum-free medium. In order to analyze whether the
induction of activated MEK1 leads to cell cycle progression, quiescent
MEK/NEE-ER cells were induced with 1 µM 4-OHT. DNA synthesis was
measured by the incorporation of BrdU after different labelling
periods following 4-OHT induction (Fig.
3A). Activation of
MEK/NEE-ER with 4-OHT was mitogenic, leading to
approximately 40% of the cells incorporating BrdU over the time
course of stimulation under serum-free conditions. When cells were
arrested by contact inhibition in the presence of serum, activation of
MEK/NEE-ER cells led to 60% of the cells beginning DNA synthesis,
which is comparable to serum stimulation of uninduced cells (data not
shown). In the presence of the solvent control, ethanol, approximately 5 to 15% of the NIH:MEK/NEE-ER cells incorporated BrdU. Consistent with the start of DNA synthesis, Fig. 3B shows that induction of
MEK/NEE-ER led to a decrease in the level of the cyclin-dependent kinase inhibitor p27Kip1, degradation of which is
associated with reentry into the cell cycle (48).
|
NEE-ER fusion protein was a delayed event, increases in CDK4 and
CDK2 kinase activities were apparent only at later time points, whereas
serum treatment of uninduced cells activated CDK4 and CDK2 within
16 h (Fig. 3C and D). While the level of CDK4 and CDK2 activity
following activation of inducible MEK was much less than that
produced by serum stimulation, it was of a magnitude similar to that
associated with cell cycle reentry resulting from activation of an
inducible B-Raf-ER construct (68).
Previous work by McCarthy et al. has shown that induction of Raf
activity can lead to the indirect stimulation of other signalling pathways through the induction of autocrine growth factors
(40); therefore, it was possible that activation of MEK in
our system was leading to the activation of other signalling pathways.
Since stimulation of DNA synthesis by some growth factors has been
shown to be blocked by inhibition of PI3-kinase (50), we
investigated whether induction of DNA synthesis by inducible MEK
was blocked by treatment with the PI3-kinase inhibitor LY294002
(63). Figure 4A shows that
treatment with LY294002 blocked induction of DNA synthesis,
demonstrating that signalling pathways in addition to ERK
activation are required for activated MEK to induce DNA synthesis.
Figure 4B shows that PI3-kinase activity was required for the earliest
step in cell cycle progression: the induction of cyclin D1
expression following activation of MEK. To investigate the kinetics of
induction of PI3-kinase, we monitored the phosphorylation of PKB, a
downstream signalling event involving PI3-kinase (11). While
platelet-derived growth factor (PDGF) stimulation of PKB in a
PI3-kinase-dependent manner occurs within 5 min of treatment, phosphorylation of PKB following induction of MEK becomes
apparent only at 16 h following induction. This phosphorylation of
PKB was dependent on PI3-kinase, as shown by its sensitivity to
LY294002 (Fig. 4C), and parallels the induction of cyclin D1
expression. The effect of LY294002 on cyclin D1 expression and DNA
synthesis was not due to downregulation of expression of MEK/NEE-ER
(Fig. 1B) or suppression of ERK2 activation by the inducible MEK (Fig. 4D). The slow kinetics of PKB phosphorylation following the 4-OHT induction of MEK suggest that this is indirect. Evidence for indirect activation of PI3-kinase via autocrine production following ERK activation is provided by the observation that MEK/NEE-ER-induced PKB phosphorylation is partially blocked by treatment with suramin (4), an inhibitor of autocrine pathways (data not shown).
Consistent with the delay before PI3-kinase activation, entry into DNA
synthesis following activation of MEK was delayed compared to that for
cells stimulated with serum.
|
Induction of AP-1 components by inducible MEK1.
The
transcription factor AP-1 has been described to be involved in cell
proliferation. Dysregulation of the AP-1 component c-Fos or c-Jun
leads to cell transformation, suggesting that AP-1 plays an important
role in the process of transformation. The ERK/MAP kinase pathway has
been described as being involved in the regulation of the expression of
c-Fos (23). In order to analyze whether the activation of
MEK1 is sufficient to induce members of the Fos and Jun families, we
induced serum-starved NIH:MEK/NEE-ER cells with 4-OHT for
different lengths of time and analyzed the expression of endogenous
AP-1 components by Western blotting (Fig.
5). The c-Fos gene product was
undetectable at all time points we analyzed. However, using the same
cell clone, we were able to detect c-Fos expression after
induction with 10% serum for 1 h. Furthermore, induction of
activated MEK1 did not suppress the ability of serum to induce c-Fos.
We then examined the expression of the other Fos family members: FosB,
Fra-1, and Fra-2. Previous studies demonstrated that serum stimulation
leads to a distinct pattern of expression of the individual Fos
proteins. c-Fos and FosB are expressed transiently with similar
kinetics, whereas the expression of Fra-1 and Fra-2 is delayed but the
proteins are expressed for a prolonged time period (9, 30,
46). Activation of MEK1 did not lead to the induction of FosB;
however, the Fos-related gene products Fra-1 and Fra-2 were induced
between 4 and 8 h after induction of the MEK/NEE-ER fusion
protein with 4-OHT, and their expression was sustained (Fig. 5A). Thus,
activation of the ERK/MAP kinase pathway induces expression of the
Fos-related gene products Fra-1 and Fra-2 but not that of c-Fos and
FosB. The elevated expression of Fra-1 and Fra-2 following activation of the inducible MEK did not require PI3-kinase activity, because it
was unaffected by treatment with LY294002 (Fig. 5B). These results
demonstrated that activation of MEK1 and thereby ERK1 and ERK2 leads to
expression of Fra-1 and Fra-2. Consistent with the lack of a
requirement for PI3-kinase activity for Fra-1 and Fra-2 expression,
elevated levels of Fra-1 and Fra-2 could be detected at 4 to 8 h,
before PKB activation or cyclin D1 expression could be detected (16 h).
To determine whether MEK activation is required for growth
factor-mediated activation of Fra-1 and Fra-2, we used the MEK
inhibitor PD098059. Serum-induced expression of Fra-1 and Fra-2 was
blocked by treatment with PD098059 (Fig. 5C). We could also detect a
drastic decrease in c-Fos expression when MEK1 was blocked with the
inhibitor. We therefore conclude that the induction of Fra-1 and Fra-2
and also c-Fos by serum requires ERK activation but that c-Fos
induction requires additional signals.
|
NEE-ER fusion protein with
4-OHT. Even in quiescent cells c-Jun was apparent, but after 16 h
of 4-OHT treatment the level of c-Jun increased (Fig. 5D). Although
this delayed elevation of c-Jun expression may reflect an indirect
effect, unlike the expression of cyclin D1 it is not via PI3-kinase, as
LY294002 had no effect (data not shown). By the specific activation of
the ERK/MAP kinase pathway with an inducible MEK1 protein, we can show
that activation of the MAP kinase pathway leads to the induction of the
Fos family members Fra-1 and Fra-2 and the Jun family members c-Jun and JunB.
It has been shown that Ras-transformed cells contain elevated AP-1
activity but that only certain AP-1 components are upregulated by Ras
transformation. Mechta et al. (41) have demonstrated that
c-Jun, JunB, Fra-1, and Fra-2, but not c-Fos, are upregulated in
Ras-transformed cells. Since we could show that the activation of MEK
induces the expression of Fos and Jun family members, we analyzed
whether the upregulation of these AP-1 components in Ras-transformed
cells is also mediated by the ERK/MAP kinase pathway. Figure
6 shows that the expression of
Fra-1, Fra-2, and c-Jun is elevated in v-Ras-transformed cells.
Treatment of these cells with the MEK inhibitor PD098059 for
different lengths of time led to the downregulation of these AP-1
components to levels comparable to those in serum-starved wild-type NIH
3T3 cells. JunB is not upregulated in these cells; therefore, treatment
with the MEK inhibitor has no effect. At 24 h after addition of
PD098059, the Ras- transformed cells were almost completely
morphologically reverted (data not shown).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
We have established NIH 3T3 cell clones expressing a conditionally
regulatable version of activated MEK1. Induction of the MEK/NEE-ER
fusion protein by 4-OHT leads to morphological transformation and
stimulates cell cycle entry. These results show that activation of MEK1 results in cell cycle progression in quiescent NIH 3T3 cells.
Interestingly, induction of MEK/NEE-ER with 4-OHT activates ERK at
only low levels (two- to sixfold) (Fig. 2), indicating that this low
level of ERK activation is sufficient to permit cell cycle entry. This
finding is consistent with the observation that cell cycle progression
by a conditionally active form of Raf (Raf-ER) is dependent on the
level of Raf activity. High levels of Raf activity induce cell
cycle arrest by the induction of the cyclin-dependent kinase inhibitor
p21Waf1/Cip1 (57, 68).
While we show that activation of inducible MEK in quiescent cells
results in DNA synthesis, direct MEK signalling is itself insufficient
for cell cycle entry. Inhibition of PI3-kinase blocks DNA synthesis
following induction of MEK activity, because PI3-kinase activity is
required for expression of cyclin D1. This activation of PI3-kinase
appears to be indirect, because phosphorylation of PKB as a measure of
PI3-kinase signalling was detectable only at 16 h, whereas ERK
activity was elevated at 1 to 2 h postactivation of inducible MEK.
A possible explanation of this delayed activation of PI3-kinase is that
it is a consequence of signalling through autocrine growth factors
induced by ERK signalling. Previous work by McCarthy et al. has shown
that inducible Raf indirectly activates Jnks as a consequence of Raf
signalling leading to the expression of autocrine heparin-binding
epidermal growth factor (40). Treatment of the
inducible MEK/NEE-ER-expressing cells with suramin, which blocks autocrine signalling (4), partially blocked
MEK-induced PKB phosphorylation (data not shown), indicating that
activation of PI3-kinase is at least in part indirect and mediated by
autocrine growth factors.
In related studies using a different inducible MEK system, Cheng et al.
(7) also concluded that MEK signalling alone was insufficient for DNA synthesis. However, in their system activation of
MEK did not lead to DNA synthesis unless cyclin D1 and CDK4 were
overexpressed to titrate out p27Kip1 (7). In
contrast to the studies of Cheng et al. (7), we detect
downregulation of p27Kip1 (Fig. 3B). Furthermore, while
Cheng et al. (7) monitored induction of DNA synthesis up to
20 h, we have found that DNA synthesis is induced only at later
time points when the PI3-kinase pathway has been activated. Therefore,
the differences between the studies reported here and those of Cheng et
al. (7) reflect in part the time period in which DNA
synthesis was examined but may also reflect differences in ERK
activation or culture conditions between the two inducible systems. In
our system ERK activation was low (a maximum of sixfold), whereas
persistent high levels of ERK activity are known to elevate levels of
p21Waf1/Cip1 such that DNA synthesis is inhibited (57,
68). In the system described here, p21Waf1/Cip1 was
induced to similar levels by MEK/NEE-ER and serum (data not shown).
In order to delineate transcription factor induction mediated by MEK
activation, we investigated whether activation of the ERK/MAP kinase
pathway is sufficient to induce AP-1 components. Although the ERK/MAP
kinase pathway signalling cascade has been described as being involved
in the induction of the immediate-early gene product c-Fos via
phosphorylation and activation of the ternary complex factor ELK1
(36), we could not detect any induction of endogenous c-Fos
protein after activation of MEK1. FosB, another Fos family member,
which is induced with kinetics similar to those of c-Fos, was also not
induced by the ERK/MAP kinase pathway. However, the expression of the
immediate-early gene products c-Fos and FosB was induced by stimulation
of the same NIH:MEK/NEE-ER cell clone with serum (Fig. 5A),
demonstrating that the lack of c-Fos and FosB induction by MEK1 was not
due to a complete loss of inducibility of these genes as described for
Ras-transformed cells (41). In contrast to c-Fos and FosB,
the Fos-related gene products Fra-1 and Fra-2 were highly upregulated
after induction of activated MEK1. Fra-1 and Fra-2 induction was rapid
following MEK activation and was not blocked by inhibition of
PI3-kinase, arguing that it is a direct signalling response to ERK
activation, although we cannot rule out an effect of other signalling
pathways induced by activated MEK.
The observation that the inducible MEK-expressing cells begin DNA
synthesis without induction of c-Fos is consistent with the finding
that knockout of the mouse c-Fos gene shows that c-Fos is not
essential for the viability, profileration, and differentiation of
most cell types (27). Correspondingly,
FosB
/ mice are viable (5, 20). Furthermore,
c-Fos/ fibroblasts grow with kinetics similar to those
of Fos+/+ fibroblasts (20) and can be
transformed with oncogenic Ras (26), indicating again that
c-Fos is not essential for cell proliferation and also not necessary
for cellular transformation induced by oncogenic Ras or MEK1.
Furthermore, the expression of c-Fos leads to morphological
transformation independent of the cell cycle (42).
It has been suggested that the induction of Fra-1 and Fra-2 by serum
growth factors may be an indirect effect via induction of c-Fos-c-Jun
heterodimers acting on the TRE (3, 56, 58). However,
activation of MEK1 in NIH:MEK/NEE-ER cells leads to Fra-1 and Fra-2
induction in the absence of c-Fos protein. This is consistent with the
observation that Fra-1 is inducible in Fos/
fibroblasts, although to a lesser extent than in wild-type cells (6). Here we show that induction of the ERK/MAP kinase
pathway leads to phosphorylation of Fra proteins, which may increase
the transcriptional activity of Fra-Jun heterodimers (44)
and result in the positive autoregulation of Fra-1 and Fra-2 via AP-1
sites in their promoters. Although it has been shown in some studies that Fra-1 and Fra-2 repress AP-1 activity (58, 69), these results are contradictory to the observation that Fra-1 and Fra-2 are
overexpressed in Ras- and v-Src-transformed cells and lead to an
increased AP-1 activity (41, 44).
All four Fos family members are able to heterodimerize with members of
the Jun family to form AP-1 complexes. We show that the induction of
the MEK/NEE-ER fusion protein in NIH 3T3 cells induces not only the
Fos family members Fra-1 and Fra-2 but also c-Jun and JunB expression.
Similar to that of Fra-1 and Fra-2, c-Jun gene expression is mediated
by positive autoregulation via an AP-1 binding site present in its
promoter, which is recognized by c-Jun-ATF2 heterodimers
(62). An AP-1-independent mechanism of regulation via ERK
phosphorylation of Ets-2 may be responsible for Ras-induced JunB
expression (8, 18, 39).
The transcription factor AP-1 seems to be involved in the regulation of many different processes, e.g., proliferation and differentiation. It is likely that specificity is achieved by the differential induction of individual AP-1 components and their phosphorylation status. Previous studies showed that different dimer combinations have different binding affinities which are also dependent on the specific TRE sequence and the promoter context (21, 22, 49, 54). AP-1 activity is increased in Ras-transformed cells, and dominant negative Jun has been shown to inhibit Ras transformation. Mechta et al. (41) have shown that the elevated AP-1 activity in Ras-transformed cells is a consequence of elevated expression of Fra-1 and Fra-2 together with c-Jun and JunB; however, the identity of the Ras-dependent signalling pathway leading to elevated AP-1 activity has not been clear. Our data obtained by using inducible MEK and treatment of Ras-transformed cells with the MEK inhibitor PD098059 show that ERK activation is the major Ras-dependent signalling pathway leading to elevated AP-1 activity.
| |
ACKNOWLEDGMENTS |
|---|
I.T. was supported by a European Union Human Capital and Mobility Fellowship, and C.J.M. is a Gibb Life Fellow of the Cancer Research Campaign.
We thank S. Mittnacht for helpful discussions and P. Cohen, J. Downward, G. Peters, and C. Sherr for antibody reagents.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratory, Institute of Cancer Research, 237 Fulham Rd., London, SW3 6JB, United Kingdom. Phone: 44 (0)171-352-9772. Fax: 44 (0)171-352-5630. E-mail: chrism{at}icr.ac.uk.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
| 1. |
Adari, H.,
D. R. Lowy,
B. M. Willumsen,
C. J. Der, and F. McCormick.
1988.
Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain.
Science
240:518-521 |
| 2. | Alessi, D. R., Y. Saito, D. G. Campbell, P. Cohen, G. Sithanandam, U. Rapp, A. Ashworth, C. J. Marshall, and S. Cowley. 1994. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J 13:1610-1619[Medline]. |
| 3. | Bergers, G., P. Graninger, S. Braselmann, C. Wrighton, and M. Busslinger. 1995. Transcriptional activation of the fra-1 gene by AP-1 is mediated by regulatory sequences in the first intron. Mol. Cell. Biol. 15:3748-3758[Abstract]. |
| 4. |
Betsholtz, C.,
A. Johnsson,
C. H. Heldin, and B. Westermark.
1986.
Efficient reversion of simian sarcoma virus-transformation and inhibition of growth factor-induced mitogenesis by suramin.
Proc. Natl. Acad. Sci. USA
83:6440-6444 |
| 5. | Brown, J. R., H. Ye, R. T. Bronson, P. Dikkes, and M. E. Greenberg. 1996. A defect in nurturing in mice lacking the immediate early gene fosB. Cell 86:297-309[Medline]. |
| 6. | Brusselbach, S., U. Mohle-Steinlein, Z.-Q. Wang, M. Schreiber, F. C. Lucibello, R. Muller, and E. F. Wagner. 1995. Cell proliferation and cell cycle progression are not impaired in fibroblasts and ES cells lacking c-Fos. Oncogene 10:79-86[Medline]. |
| 7. |
Cheng, M.,
V. Sexl,
C. J. Sherr, and M. F. Roussel.
1998.
Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1).
Proc. Natl. Acad. Sci. USA
95:1091-1096 |
| 8. | Coffer, P., M. de Jonge, A. Mettouchi, B. Binetruy, J. Ghysdael, and W. Kruijer. 1994. junB promoter regulation: Ras mediated transactivation by c-Ets-1 and c-Ets-2. Oncogene 9:911-921[Medline]. |
| 9. |
Cohen, D. R., and T. Curran.
1988.
fra-1: a serum-inducible, cellular immediate-early gene that encodes a Fos-related antigen.
Mol. Cell. Biol.
8:2063-2069 |
| 10. | Cowley, S., H. Paterson, P. Kemp, and C. J. Marshall. 1994. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77:841-852[Medline]. |
| 11. | Cross, D. A., D. R. Alessi, P. Cohen, M. Andjelkovich, and B. A. Hemmings. 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785-789[Medline]. |
| 12. | Denhardt, D. T. 1996. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem. J. 318:729-747. |
| 13. |
Dent, P.,
W. Haser,
T. A. J. Haystead,
L. A. Vincent,
T. M. Roberts, and T. W. Sturgill.
1992.
Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro.
Science
257:1404-1406 |
| 14. |
Diaz-Meco, M. T.,
J. Lozano,
M. M. Municio,
E. Berra,
S. Frutos,
L. Sanz, and J. Moscat.
1994.
Evidence for the in vitro and in vivo interaction of Ras with protein kinase C zeta.
J. Biol. Chem.
269:31706-31710 |
| 15. |
Dudley, D. T.,
L. Pang,
S. J. Decker,
A. J. Bridges, and A. R. Saltiel.
1995.
A synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc. Natl. Acad. Sci. USA
92:7686-7689 |
| 16. |
Fukuda, M.,
I. Gotoh,
M. Adachi,
Y. Gotoh, and E. Nishida.
1997.
A novel regulatory mechanism in the mitogen-activated protein (MAP) kinase cascade.
J. Biol. Chem.
272:32642-32648 |
| 17. |
Fukuda, M.,
I. Gotoh,
Y. Gotoh, and E. Nishida.
1996.
Cytoplasmic localization of mitogen-activated protein kinase kinase directed by its NH2 terminal, leucine-rich short amino acid sequence, which acts as a nuclear export signal.
J. Biol. Chem.
271:20024-20028 |
| 18. | Galang, C. K., C. J. Der, and C. A. Hauser. 1994. Oncogenic Ras can induce transcriptional activation through a variety of promoter elements including tandem c-Ets-2 binding sites. Oncogene 9:2913-2921[Medline]. |
| 19. | Gruda, M. C., K. Kovary, R. Metz, and R. Bravo. 1994. Regulation of Fra-1 and Fra-2 phosphorylation differs during the cell cycle of fibroblasts and phosphorylation in vitro by MAP kinase affects DNA binding activity. Oncogene 9:2537-2547[Medline]. |
| 20. |
Gruda, M. C.,
J. Vanamsterdam,
C. A. Rizzo,
S. K. Durham,
A. Lira, and R. Bravo.
1996.
Expression of FosB during mouse development normal development of FosB knockout mice.
Oncogene
12:2177-2185[Medline].
|
| 21. |
Hai, T., and T. Curran.
1991.
Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity.
Proc. Natl. Acad. Sci. USA
88:3720-3724 |
| 22. | Halazonetis, T. D., K. Georgopoulos, M. E. Greenberg, and P. Leder. 1988. c-jun dimerizes with itself and with c-fos forming complexes with different binding affinities. Cell 55:917-924[Medline]. |
| 23. | Hill, C. S., and R. Treisman. 1995. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80:199-211[Medline]. |
| 24. |
Hofer, F.,
S. Fields,
C. Schneider, and G. S. Martin.
1994.
Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.
Proc. Natl. Acad. Sci. USA
91:11089-11093 |
| 25. | Howe, L. R., S. J. Leevers, N. Gómez, S. Nakielny, P. Cohen, and C. J. Marshall. 1992. Activation of the MAP kinase pathway by the protein kinase raf. Cell 71:335-342[Medline]. |
| 26. | Hu, E., E. Mueller, S. Oliviero, V. E. Papaioannou, R. Johnson, and B. M. Spiegelmann. 1994. Targeted disruption of the c-fos gene demonstrates c-fos-dependent and -independent pathways for gene expression stimulated by growth factors or oncogenes. EMBO J. 13:3094-3103[Medline]. |
| 27. | Johnson, R. S., B. M. Spiegelman, and V. Papaioannou. 1992. Pleiotropic effects of a null mutation in the c-fos proto-oncogene. Cell 71:577-586[Medline]. |
| 28. | Joneson, T., M. A. White, M. H. Wigler, and D. Bar-Sagi. 1996. Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of Ras. Science 271:810-812[Abstract]. |
| 29. |
Kikuchi, A.,
S. D. Demo,
Z. H. Ye,
Y. W. Chen, and L. T. Williams.
1994.
ralGDS family members interact with the effector loop of Ras p21.
Mol. Cell. Biol.
14:7483-7491 |
| 30. |
Kovary, K., and R. Bravo.
1991.
Expression of different Jun and Fos proteins during the G0-to-G1 transition in mouse fibroblasts: in vitro and in vivo associations.
Mol. Cell. Biol.
11:2451-2459 |
| 31. | Kyriakis, J. M., H. App, X. F. Zhang, P. Banerjee, D. L. Brautigan, U. R. Rapp, and J. Avruch. 1992. Raf-1 activates MAP kinase-kinase. Nature 358:417-421[Medline]. |
| 32. | Leevers, S. J., and C. J. Marshall. 1992. Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 11:569-574[Medline]. |
| 33. |
Littlewood, T. D.,
D. C. Hancock,
P. S. Danielian,
M. G. Parker, and G. I. Evan.
1995.
A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins.
Nucleic Acids Res.
23:1686-1690 |
| 34. | Lloyd, A. C., N. Yancheva, and B. Wasylyk. 1991. Transformation suppressor activity of a Jun transcription factor lacking its activation domain. Nature 352:635-638[Medline]. |
| 35. |
Mansour, S. J.,
W. T. Matten,
A. S. Hermann,
J. M. Candia,
S. Rong,
K. Fukasawa,
G. F. Vande Woude, and N. G. Ahn.
1994.
Transformation of mammalian cells by constitutively active MAP kinase kinase.
Science
265:966-970 |
| 36. | Marais, R., J. Wynne, and R. Treismann. 1993. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 73:381-393[Medline]. |
| 37. |
Martelli, P.,
E. G. Lapetina,
C. J. Der, and G. C. White.
1996.
Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1.
J. Biol. Chem.
271:29903-29908 |
| 38. | Martin, G. A., D. Viskochil, G. Bollag, P. C. McCabe, W. Crosier, H. Haubruck, L. Conroy, R. Clark, P. O'Connel, R. M. Cawthorn, M. A. Innis, and F. McCormick. 1990. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell 63:843-849[Medline]. |
| 39. | McCarthy, S. A., D. Chen, B. S. Yang, R. J. Garcia, H. Cherwinski, X. R. Chen, M. Klagsbrun, C. A. Hauser, M. C. Ostrowski, and M. McMahon. 1997. Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1. Mol. Cell. Biol. 17:2401-2412[Abstract]. |
| 40. |
McCarthy, S. A.,
M. L. Samuels,
C. A. Pritchard,
J. A. Abraham, and M. McMahon.
1995.
Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes.
Genes Dev
9:1953-1964 |
| 41. | Mechta, F., D. Lallemand, C. M. Pfarr, and M. Yaniv. 1997. Transformation by ras modifies AP1 composition and activity. Oncogene 14:837-847[Medline]. |
| 42. |
Miao, G. G., and T. Curran.
1994.
Cell transformation by c-Fos requires an extended period of expression and is independent of the cell cycle.
Mol. Cell. Biol.
14:4295-4310 |
| 43. |
Morgenstern, J. P., and H. Land.
1990.
Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.
Nucleic Acids Res.
18:3587-3596 |
| 44. | Murakami, M., M. H. Sonobe, M. Ui, Y. Kabuyama, H. Watanabe, T. Wada, H. Handa, and H. Iba. 1997. Phosphorylation and high level expression of Fra-2 in v-src transformed cells: a pathway of activation of endogenous AP-1. Oncogene 14:2435-2444[Medline]. |
| 45. | Nakielny, S., P. Cohen, J. Wu, and T. W. Sturgill. 1992. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase. EMBO J. 11:2123-2129[Medline]. |
| 46. |
Nishina, H.,
S. Hironori,
S. Takeshi,
S. Moriyuki, and H. Iba.
1990.
Isolation and characterization of fra-2, an additional member of the fos gene family.
Proc. Natl. Acad. Sci. USA
87:3619-3623 |
| 47. |
Pagès, G.,
P. Lenormand,
G. L'Allemain,
J. C. Chambard,
S. Meloche, and J. Pouysségur.
1993.
Mitogen-activated protein kinase p42mapk and p44mapk are required for fibroblast proliferation.
Proc. Natl. Acad. Sci. USA
90:8319-8323 |
| 48. | Polyak, K., J. Y. Kato, M. J. Solomon, C. J. Sherr, J. Massague, J. M. Roberts, and A. Koff. 1994. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev 8:8-22. |
| 49. |
Rauscher, F. J., III,
P. J. Voulalas,
B. R. Franza, Jr., and T. Curran.
1988.
Fos and Jun bind cooperatively to the AP-1 site: reconstitution in vitro.
Genes Dev
2:1687-1699 |
| 50. |
Roche, S.,
M. Koegl, and S. A. Courtneidge.
1994.
The phosphatidylinositol 3-kinase alpha is required for DNA synthesis induced by some, but not all, growth factors.
Proc. Natl. Acad. Sci. USA
91:9185-9189 |
| 51. | Rodriguez, V. P., P. H. Warne, R. Dhand, B. Vanhaesebroeck, I. Gout, M. J. Fry, M. D. Waterfield, and J. Downward. 1994. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370:527-532[Medline]. |
| 52. | Rodriguez, V. P., P. H. Warne, A. Khwaja, B. M. Marte, D. Pappin, P. Das, M. D. Waterfield, A. Ridley, and J. Downward. 1997. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89:457-467[Medline]. |
| 53. |
Russell, M.,
C. C. Lange, and G. L. Johnson.
1995.
Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1).
J. Biol. Chem.
270:11757-11760 |
| 54. | Ryseck, R. P., and R. Bravo. 1991. c-Jun, JunB and JunD differ in their binding affinities to AT-1 and CRE consensus sequences: effect of Fos proteins. Oncogene 6:533-542[Medline]. |
| 55. |
Samuels, M. L.,
M. J. Weber,
M. Bishop, and M. McMahon.
1993.
Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human Raf-1 protein kinase.
Mol. Cell. Biol.
13:6241-6252 |
| 56. | Schreiber, M., C. Poirier, A. Franchi, R. Kurzbauer, J.-L. Guenet, G. F. Carle, and E. Wagner. 1997. Structure and chromosomal assignment of the mouse fra-1 gene, and its exclusion as a candidate gene for oc (osteosclerosis). Oncogene 15:1171-1178[Medline]. |
| 57. | Sewing, A., B. Wiseman, A. C. Lloyd, and H. Land. 1997. High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1. Mol. Cell. Biol. 17:5588-5597[Abstract]. |
| 58. | Sonobe, M. H., T. Yoshida, M. Murakami, T. Kameda, and H. Iba. 1995. fra-2 promoter can respond to serum-stimulation through AP-1 complexes. Oncogene 10:689-696[Medline]. |
| 59. |
Spaargaren, M., and J. R. Bischoff.
1994.
Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap.
Proc. Natl. Acad. Sci. USA
91:12609-12613 |
| 60. |
van Aelst, L.,
M. Barr,
S. Marcus,
A. Polverino, and M. Wigler.
1993.
Complex formation between Ras and Raf and other protein kinases.
Proc. Natl. Acad. Sci. USA
90:6213-6217 |
| 61. |
van Aelst, L.,
M. White, and M. H. Wigler.
1994.
Ras partners.
Cold Spring Harbor Symp. Quant. Biol.
59:181-186 |
| 62. | Van Dam, H., M. Duyndam, P. Pottier, A. Bosch, L. De Vries-Smits, P. Herrlich, A. Zantema, P. Angel, and A. van der Eb. 1993. Heterodimer formation of c-Jun and ATF-2 is responsible for induction of c-jun by the 243 amino acid adenovirus E1A protein. EMBO J. 12:479-487[Medline]. |
| 63. |
Vlahos, C. J.,
W. F. Matter,
K. Y. Hui, and R. F. Brown.
1994.
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J. Biol. Chem.
269:5241-5248 |
| 64. | Vojtek, A. B., S. M. Hollenberg, and J. A. Cooper. 1993. Mammalian Ras interacts directly with the serine threonine kinase Raf. Cell 74:205-214[Medline]. |
| 65. | White, M. A., C. Nicolette, A. Minden, A. Polverino, A. L. Van, M. Karin, and M. H. Wigler. 1995. Multiple Ras functions can contribute to mammalian cell transformation. Cell 80:533-541[Medline]. |
| 66. |
White, M. A.,
T. Vale,
J. H. Camonis,
E. Schaefer, and M. H. Wigler.
1996.
A role for the Ral guanine nucleotide dissociation stimulator in mediating Ras- induced transformation.
J. Biol. Chem.
271:16439-16442 |
| 67. | Wolthuis, R. M., B. Bauer, L. J. van't Veer, A. M. de Vries-Smits, R. H. Cool, M. Spaargaren, A. Wittinghofer, B. M. Burgering, and J. L. Bos. 1996. RalGDS-like factor (Rlf) is a novel Ras and Rap 1A-associating protein. Oncogene 13:353-362[Medline]. |
| 68. | Woods, D., D. Parry, H. Cherwinski, E. Bosch, E. Lees, and M. McMahon. 1997. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol. Cell. Biol. 17:5598-5611[Abstract]. |
| 69. |
Yoshioka, K.,
T. Deng,
M. Cavigelli, and M. Karin.
1995.
Antitumor promotion by phenolic antioxidants: inhibition of AP-1 activity through induction of Fra expression.
Proc. Natl. Acad. Sci. USA
92:4972-4976 |
| 70. | Zhang, X. F., J. Settleman, J. M. Kyriakis, E. Takeuchi-Suzuki, S. J. Elledge, M. S. Marshall, J. T. Bruder, U. R. Rapp, and J. Avruch. 1993. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature 364:308-313[Medline]. |
Molecular and Cellular Biology, January 1999, p. 321-329, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Shi, H., Scheffler, J. M., Pleitner, J. M., Zeng, C., Park, S., Hannon, K. M., Grant, A. L., Gerrard, D. E.
(2008). Modulation of skeletal muscle fiber type by mitogen-activated protein kinase signaling. FASEB J.
22: 2990-3000
[Abstract] [Full Text] -
Basbous, J., Chalbos, D., Hipskind, R., Jariel-Encontre, I., Piechaczyk, M.
(2007). Ubiquitin-Independent Proteasomal Degradation of Fra-1 Is Antagonized by Erk1/2 Pathway-Mediated Phosphorylation of a Unique C-Terminal Destabilizer. Mol. Cell. Biol.
27: 3936-3950
[Abstract] [Full Text] -
Villanueva, J., Yung, Y., Walker, J. L., Assoian, R. K.
(2007). ERK Activity and G1 Phase Progression: Identifying Dispensable Versus Essential Activities and Primary Versus Secondary Targets. Mol. Biol. Cell
18: 1457-1463
[Abstract] [Full Text] -
Kogkopoulou, O., Tzakos, E., Mavrothalassitis, G., Baldari, C. T., Paliogianni, F., Young, H. A., Thyphronitis, G.
(2006). Conditional up-regulation of IL-2 production by p38 MAPK inactivation is mediated by increased Erk1/2 activity. J. Leukoc. Biol.
79: 1052-1060
[Abstract] [Full Text] -
Kakumoto, K., Sasai, K., Sukezane, T., Oneyama, C., Ishimaru, S., Shibutani, K., Mizushima, H., Mekada, E., Hanafusa, H., Akagi, T.
(2006). FRA1 is a determinant for the difference in RAS-induced transformation between human and rat fibroblasts. Proc. Natl. Acad. Sci. USA
103: 5490-5495
[Abstract] [Full Text] -
Ahmed, S., Wang, N., Hafeez, B. B., Cheruvu, V. K., Haqqi, T. M.
(2005). Punica granatum L. Extract Inhibits IL-1{beta}-Induced Expression of Matrix Metalloproteinases by Inhibiting the Activation of MAP Kinases and NF-{kappa}B in Human Chondrocytes In Vitro. J. Nutr.
135: 2096-2102
[Abstract] [Full Text] -
Korrapati, M. C., Lock, E. A., Mehendale, H. M.
(2005). Molecular mechanisms of enhanced renal cell division in protection against S-1,2-dichlorovinyl-L-cysteine-induced acute renal failure and death. Am. J. Physiol. Renal Physiol.
289: F175-F185
[Abstract] [Full Text] -
Hoffmann, E., Thiefes, A., Buhrow, D., Dittrich-Breiholz, O., Schneider, H., Resch, K., Kracht, M.
(2005). MEK1-dependent Delayed Expression of Fos-related Antigen-1 Counteracts c-Fos and p65 NF-{kappa}B-mediated Interleukin-8 Transcription in Response to Cytokines or Growth Factors. J. Biol. Chem.
280: 9706-9718
[Abstract] [Full Text] -
Wang, X., Sonenshein, G. E.
(2005). Induction of the RelB NF-{kappa}B Subunit by the Cytomegalovirus IE1 Protein Is Mediated via Jun Kinase and c-Jun/Fra-2 AP-1 Complexes. J. Virol.
79: 95-105
[Abstract] [Full Text] -
Mirza, A. M., Gysin, S., Malek, N., Nakayama, K.-i., Roberts, J. M., McMahon, M.
(2004). Cooperative Regulation of the Cell Division Cycle by the Protein Kinases RAF and AKT. Mol. Cell. Biol.
24: 10868-10881
[Abstract] [Full Text] -
Narayanan, R., Sepulveda, V. A. T., Falzon, M., Weigel, N. L.
(2004). The Functional Consequences of Cross-talk between the Vitamin D Receptor and ERK Signaling Pathways Are Cell-specific. J. Biol. Chem.
279: 47298-47310
[Abstract] [Full Text] -
Holloway, J. N., Murthy, S., El-Ashry, D.
(2004). A Cytoplasmic Substrate of Mitogen-Activated Protein Kinase Is Responsible for Estrogen Receptor-{alpha} Down-Regulation in Breast Cancer Cells: The Role of Nuclear Factor-{kappa}B. Mol. Endocrinol.
18: 1396-1410
[Abstract] [Full Text] -
Li, J., Chen, H., Tang, M.-S., Shi, X., Amin, S., Desai, D., Costa, M., Huang, C.
(2004). PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells. JCB
165: 77-86
[Abstract] [Full Text] -
Scott, L. A., Vass, J. K., Parkinson, E. K., Gillespie, D. A. F., Winnie, J. N., Ozanne, B. W.
(2004). Invasion of Normal Human Fibroblasts Induced by v-Fos Is Independent of Proliferation, Immortalization, and the Tumor Suppressors p16INK4a and p53. Mol. Cell. Biol.
24: 1540-1559
[Abstract] [Full Text] -
Aarbiou, J., Verhoosel, R. M., van Wetering, S., de Boer, W. I., van Krieken, J. H. J. M., Litvinov, S. V., Rabe, K. F., Hiemstra, P. S.
(2004). Neutrophil Defensins Enhance Lung Epithelial Wound Closure and Mucin Gene Expression In Vitro. Am. J. Respir. Cell Mol. Bio.
30: 193-201
[Abstract] [Full Text] -
Deng, Q., Liao, R., Wu, B.-L., Sun, P.
(2004). High Intensity ras Signaling Induces Premature Senescence by Activating p38 Pathway in Primary Human Fibroblasts. J. Biol. Chem.
279: 1050-1059
[Abstract] [Full Text] -
Balmanno, K., Millar, T., McMahon, M., Cook, S. J.
(2003). {Delta}Raf-1:ER* Bypasses the Cyclic AMP Block of Extracellular Signal-Regulated Kinase 1 and 2 Activation but Not CDK2 Activation or Cell Cycle Reentry. Mol. Cell. Biol.
23: 9303-9317
[Abstract] [Full Text] -
Vial, E., Marshall, C. J.
(2003). Elevated ERK-MAP kinase activity protects the FOS family member FRA-1 against proteasomal degradation in colon carcinoma cells. J. Cell Sci.
116: 4957-4963
[Abstract] [Full Text] -
Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., Mascarelli, F.
(2003). Mutation of B-Raf in Human Choroidal Melanoma Cells Mediates Cell Proliferation and Transformation through the MEK/ERK Pathway. J. Biol. Chem.
278: 42409-42418
[Abstract] [Full Text] -
Vaidya, V. S., Shankar, K., Lock, E. A., Dixon, D., Mehendale, H. M.
(2003). Molecular Mechanisms of Renal Tissue Repair in Survival from Acute Renal Tubule Necrosis: Role of ERK1/2 Pathway. Toxicol Pathol
31: 604-618
[Abstract] -
Casalino, L., De Cesare, D., Verde, P.
(2003). Accumulation of Fra-1 in ras-Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization. Mol. Cell. Biol.
23: 4401-4415
[Abstract] [Full Text] -
Ahmed, S., Rahman, A., Hasnain, A., Goldberg, V. M., Haqqi, T. M.
(2003). Phenyl N-tert-Butylnitrone Down-Regulates Interleukin-1{beta}-Stimulated Matrix Metalloproteinase-13 Gene Expression in Human Chondrocytes: Suppression of c-Jun NH2-Terminal Kinase, p38-Mitogen-Activated Protein Kinase and Activating Protein-1. J. Pharmacol. Exp. Ther.
305: 981-988
[Abstract] [Full Text] -
Vandeput, F., Perpete, S., Coulonval, K., Lamy, F., Dumont, J. E.
(2003). Role of the Different Mitogen-Activated Protein Kinase Subfamilies in the Stimulation of Dog and Human Thyroid Epithelial Cell Proliferation by Cyclic Adenosine 5'-Monophosphate and Growth Factors. Endocrinology
144: 1341-1349
[Abstract] [Full Text] -
Delmas, C., Aragou, N., Poussard, S., Cottin, P., Darbon, J.-M., Manenti, S.
(2003). MAP Kinase-dependent Degradation of p27Kip1 by Calpains in Choroidal Melanoma Cells. REQUIREMENT OF p27Kip1 NUCLEAR EXPORT. J. Biol. Chem.
278: 12443-12451
[Abstract] [Full Text] -
Dhillon, A. S., Meikle, S., Peyssonnaux, C., Grindlay, J., Kaiser, C., Steen, H., Shaw, P. E., Mischak, H., Eychene, A., Kolch, W.
(2003). A Raf-1 Mutant That Dissociates MEK/Extracellular Signal-Regulated Kinase Activation from Malignant Transformation and Differentiation but Not Proliferation. Mol. Cell. Biol.
23: 1983-1993
[Abstract] [Full Text] -
Diehl, J. A., Yang, W., Rimerman, R. A., Xiao, H., Emili, A.
(2003). Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase. Mol. Cell. Biol.
23: 1764-1774
[Abstract] [Full Text] -
Gotoh, J., Obata, M., Yoshie, M., Kasai, S., Ogawa, K.
(2003). Cyclin D1 over-expression correlates with {beta}-catenin activation, but not with H-ras mutations, and phosphorylation of Akt, GSK3{beta} and ERK1/2 in mouse hepatic carcinogenesis. Carcinogenesis
24: 435-442
[Abstract] [Full Text] -
Jorritsma, P. J., Brogdon, J. L., Bottomly, K.
(2003). Role of TCR-Induced Extracellular Signal-Regulated Kinase Activation in the Regulation of Early IL-4 Expression in Naive CD4+ T Cells. J. Immunol.
170: 2427-2434
[Abstract] [Full Text] -
Habib, A. A., Chun, S. J., Neel, B. G., Vartanian, T.
(2003). Increased Expression of Epidermal Growth Factor Receptor Induces Sequestration of Extracellular Signal-Related Kinases and Selective Attenuation of Specific Epidermal Growth Factor-Mediated Signal Transduction Pathways. Mol Cancer Res
1: 219-233
[Abstract] [Full Text] -
Jiang, B., Xu, S., Brecher, P., Cohen, R. A.
(2002). Growth Factors Enhance Interleukin-1{beta}-Induced Persistent Activation of Nuclear Factor-{kappa}B in Rat Vascular Smooth Muscle Cells. Arterioscler. Thromb. Vasc. Bio.
22: 1811-1816
[Abstract] [Full Text] -
Pursiheimo, J.-P., Saari, J., Jalkanen, M., Salmivirta, M.
(2002). Cooperation of Protein Kinase A and Ras/ERK Signaling Pathways Is Required for AP-1-mediated Activation of Fibroblast Growth Factor-inducible Response Element (FiRE). J. Biol. Chem.
277: 25344-25355
[Abstract] [Full Text] -
Wang, W., Chen, J. X., Liao, R., Deng, Q., Zhou, J. J., Huang, S., Sun, P.
(2002). Sequential Activation of the MEK-Extracellular Signal-Regulated Kinase and MKK3/6-p38 Mitogen-Activated Protein Kinase Pathways Mediates Oncogenic ras-Induced Premature Senescence. Mol. Cell. Biol.
22: 3389-3403
[Abstract] [Full Text] -
Eriksson, M., Leppa, S.
(2002). Mitogen-activated Protein Kinases and Activator Protein 1 Are Required for Proliferation and Cardiomyocyte Differentiation of P19 Embryonal Carcinoma Cells. J. Biol. Chem.
277: 15992-16001
[Abstract] [Full Text] -
Brockman, J. L., Schroeder, M. D., Schuler, L. A.
(2002). PRL Activates the Cyclin D1 Promoter Via the Jak2/Stat Pathway. Mol. Endocrinol.
16: 774-784
[Abstract] [Full Text] -
Xu, L., Fukumura, D., Jain, R. K.
(2002). Acidic Extracellular pH Induces Vascular Endothelial Growth Factor (VEGF) in Human Glioblastoma Cells via ERK1/2 MAPK Signaling Pathway. MECHANISM OF LOW pH-INDUCED VEGF. J. Biol. Chem.
277: 11368-11374
[Abstract] [Full Text] -
Appleman, L. J., van Puijenbroek, A. A. F. L., Shu, K. M., Nadler, L. M., Boussiotis, V. A.
(2002). CD28 Costimulation Mediates Down-Regulation of p27kip1 and Cell Cycle Progression by Activation of the PI3K/PKB Signaling Pathway in Primary Human T Cells. J. Immunol.
168: 2729-2736
[Abstract] [Full Text] -
Alt, J. R., Gladden, A. B., Diehl, J. A.
(2002). p21Cip1 Promotes Cyclin D1 Nuclear Accumulation via Direct Inhibition of Nuclear Export. J. Biol. Chem.
277: 8517-8523
[Abstract] [Full Text] -
Wolter, S., Mushinski, J. F., Saboori, A. M., Resch, K., Kracht, M.
(2002). Inducible Expression of a Constitutively Active Mutant of Mitogen-activated Protein Kinase Kinase 7 Specifically Activates c-JUN NH2-terminal Protein Kinase, Alters Expression of at Least Nine Genes, and Inhibits Cell Proliferation. J. Biol. Chem.
277: 3576-3584
[Abstract] [Full Text] -
Young, M. R., Nair, R., Bucheimer, N., Tulsian, P., Brown, N., Chapp, C., Hsu, T.-C., Colburn, N. H.
(2002). Transactivation of Fra-1 and Consequent Activation of AP-1 Occur Extracellular Signal-Regulated Kinase Dependently. Mol. Cell. Biol.
22: 587-598
[Abstract] [Full Text] -
Tognon, C., Garnett, M., Kenward, E., Kay, R., Morrison, K., Sorensen, P. H. B.
(2001). The Chimeric Protein Tyrosine Kinase ETV6-NTRK3 Requires both Ras-Erk1/2 and PI3-Kinase-Akt Signaling for Fibroblast Transformation. Cancer Res.
61: 8909-8916
[Abstract] [Full Text] -
Schmitt, J. M., Stork, P. J. S.
(2001). Cyclic AMP-Mediated Inhibition of Cell Growth Requires the Small G Protein Rap1. Mol. Cell. Biol.
21: 3671-3683
[Abstract] [Full Text] -
von Gise, A., Lorenz, P., Wellbrock, C., Hemmings, B., Berberich-Siebelt, F., Rapp, U. R., Troppmair, J.
(2001). Apoptosis Suppression by Raf-1 and MEK1 Requires MEK- and Phosphatidylinositol 3-Kinase-Dependent Signals. Mol. Cell. Biol.
21: 2324-2336
[Abstract] [Full Text] -
Boss, V., Roback, J. D., Young, A. N., Roback, L. J., Weisenhorn, D. M. V., Medina-Flores, R., Wainer, B. H.
(2001). Nerve Growth Factor, But Not Epidermal Growth Factor, Increases Fra-2 Expression and Alters Fra-2/JunD Binding to AP-1 and CREB Binding Elements in Pheochromocytoma (PC12) Cells. J. Neurosci.
21: 18-26
[Abstract] [Full Text] -
Hansen, S. H., Zegers, M. M. P., Woodrow, M., Rodriguez-Viciana, P., Chardin, P., Mostov, K. E., McMahon, M.
(2000). Induced Expression of Rnd3 Is Associated with Transformation of Polarized Epithelial Cells by the Raf-MEK-Extracellular Signal-Regulated Kinase Pathway. Mol. Cell. Biol.
20: 9364-9375
[Abstract] [Full Text] -
Kang, K. W., Ryu, J. H., Kim, S. G.
(2000). The Essential Role of Phosphatidylinositol 3-Kinase and of p38 Mitogen-Activated Protein Kinase Activation in the Antioxidant Response Element-Mediated rGSTA2 Induction by Decreased Glutathione in H4IIE Hepatoma Cells. Mol. Pharmacol.
58: 1017-1025
[Abstract] [Full Text] -
Peyssonnaux, C., Provot, S., Felder-Schmittbuhl, M. P., Calothy, G., Eychène, A.
(2000). Induction of Postmitotic Neuroretina Cell Proliferation by Distinct Ras Downstream Signaling Pathways. Mol. Cell. Biol.
20: 7068-7079
[Abstract] [Full Text] -
Mirza, A. M., Kohn, A. D., Roth, R. A., McMahon, M.
(2000). Oncogenic Transformation of Cells by a Conditionally Active Form of the Protein Kinase Akt/PKB. Cell Growth Differ.
11: 279-292
[Abstract] [Full Text] -
Lu, R., Serrero, G.
(2000). Inhibition of PC cell-derived growth factor (PCDGF, epithelin/granulin precursor) expression by antisense PCDGF cDNA transfection inhibits tumorigenicity of the human breast carcinoma cell line MDA-MB-468. Proc. Natl. Acad. Sci. USA
97: 3993-3998
[Abstract] [Full Text] -
Lipton, P.
(1999). Ischemic Cell Death in Brain Neurons. Physiol. Rev.
79: 1431-1568
[Abstract] [Full Text] -
Montesano, R., Soriano, J. V., Hosseini, G., Pepper, M. S., Schramek, H.
(1999). Constitutively Active Mitogen-activated Protein Kinase Kinase MEK1 Disrupts Morphogenesis and Induces an Invasive Phenotype in Madin-Darby Canine Kidney Epithelial Cells. Cell Growth Differ.
10: 317-332
[Abstract] [Full Text] -
Cook, S. J., Aziz, N., McMahon, M.
(1999). 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. Mol. Cell. Biol.
19: 330-341
[Abstract] [Full Text] -
Craddock, B. L., Hobbs, J., Edmead, C. E., Welham, M. J.
(2001). Phosphoinositide 3-Kinase-dependent Regulation of Interleukin-3-induced Proliferation. INVOLVEMENT OF MITOGEN-ACTIVATED PROTEIN KINASES, SHP2 AND Gab2. J. Biol. Chem.
276: 24274-24283
[Abstract] [Full Text] -
Kristof, A. S., Marks-Konczalik, J., Moss, J.
(2001). Mitogen-activated Protein Kinases Mediate Activator Protein-1-dependent Human Inducible Nitric-oxide Synthase Promoter Activation. J. Biol. Chem.
276: 8445-8452
[Abstract] [Full Text] -
Delmas, C., Manenti, S., Boudjelal, A., Peyssonnaux, C., Eychene, A., Darbon, J.-M.
(2001). The p42/p44 Mitogen-activated Protein Kinase Activation Triggers p27Kip1 Degradation Independently of CDK2/Cyclin E in NIH 3T3 Cells. J. Biol. Chem.
276: 34958-34965
[Abstract] [Full Text] -
Phillips-Mason, P. J., Raben, D. M., Baldassare, J. J.
(2000). Phosphatidylinositol 3-Kinase Activity Regulates alpha -Thrombin-stimulated G1 Progression by Its Effect on Cyclin D1 Expression and Cyclin-dependent Kinase 4 Activity. J. Biol. Chem.
275: 18046-18053
[Abstract] [Full Text] -
Rimerman, R. A., Gellert-Randleman, A., Diehl, J. A.
(2000). Wnt1 and MEK1 Cooperate to Promote Cyclin D1 Accumulation and Cellular Transformation. J. Biol. Chem.
275: 14736-14742
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»