A Dominant Role for the Raf-MEK Pathway in Forskolin, 12-O-Tetradecanoyl-phorbol Acetate, and Platelet-Derived Growth Factor-Induced CREB (cAMP-Responsive Element-Binding Protein) Activation, Uncoupled from Serine 133 Phosphorylation in NIH 3T3 Cells
Ole Morten Seternes,
Bjarne Johansen and
Ugo Moens1
1 Department of Gene Biology Institute of Medical Biology
University of Tromsø N-9037 Tromsø, Norway
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ABSTRACT
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In this study we describe that
platelet-derived growth factor (PDGF),
12-0-tetradecanoyl-phorbol-acetate (TPA), and forskolin
induced CREB (cAMP-responsive element-binding protein) Ser-133
phosphorylation with comparable magnitude and kinetics in NIH 3T3
cells. While forskolin was the most potent activator of CREB, TPA or
PDGF modestly increased CREB activity. The role of protein kinase C,
protein kinase A, and the Raf-MEK kinase pathway in the
activation and Ser-133 phosphorylation of CREB by these three stimuli
was investigated. We found that inhibition of the Raf-MEK kinase
pathway efficiently blocks transcriptional activation of CREB by all
three stimuli. This dominant involvement of Raf-MEK in CREB
transcriptional activation seems to be uncoupled from CREB Ser-133
phosphorylation. We further demonstrate that although inhibition of
Raf-MEK represses forskolin-induced CREB activation, forskolin by
itself failed to activate ERK1/2 and Elk-1 mediated transcription.
These results suggest that a basal level of Raf-MEK activity is
necessary for both PDGF- and forskolin-induced CREB activation,
independent of CREB Ser-133 phosphorylation.
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INTRODUCTION
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Regulation of gene expression in response to extracellular stimuli
occurs often through signal transduction pathways that involve a
cascade of biochemical events eventually leading to phosphorylation of
specific transcription factors (1). One of the best characterized
signaling pathways is the cAMP/protein kinase A (PKA) pathway. Genes
whose activity can be induced by increased intracellular cAMP levels
often contain so-called cAMP response elements (CRE) in their promoter
region to which the cAMP-responsive element binding protein (CREB), a
member of the large CREB/CREM/ATF family of transcription factors, can
bind (2, 3, 4). PKA has been shown to phosphorylate CREB at the serine
residue 133 (Ser-133) in vivo, indicating that CREB mediates
cAMP-induced gene expression (5). This residue seems to be crucial for
transcriptional activation of CREB since replacing Ser-133 by alanine
rendered CREB unresponsive to both cAMP and PKA, and this mutated form
of CREB failed to activate transcription of CRE-responsive promoters
(5). Several recent studies have shown that the cAMP/PKA pathway is not
the only signaling pathway able to induce Ser-133 phosphorylation and
transcriptional activation of CREB. CREB is phosphorylated and
activated upon nerve growth factor, epidermal growth factor, fibroblast
growth factor, hepatocyte growth factor/scatter factor/mast/stem cell
growth factor, endothelin-1, UV irradiation, cross-linking surface Ig,
and arginine vasopressin treatment (6, 7, 8, 9, 10, 11). Once phosphorylated at
Ser-133, CREB can make contact with a large transcriptional coactivator
termed CREB-binding protein (CBP). The importance of this interaction
for phospho-CREB-activated transcription has been shown by several
laboratories (12, 13, 14, 15, 16).
Although Ser-133 phosphorylation of CREB is necessary for CREB to
interact with CBP and activate transcription, Ser-133 phosphorylation
alone is not sufficient for transcriptional activation in
vivo. For example, Ca2+/Cam kinase II can
phosphorylate CREB at Ser-133 (8, 17, 18), but this kinase is unable to
activate transcription even though CREB phosphorylated by Cam kinase II
can physically interact with CBP (19). The authors showed that Cam
kinase II also phosphorylated CREB at Ser-142 and that this
phosphorylation inhibited the transcriptional activity of CREB.
Treatment of Jurkat T-cells with
12-O-tetradecanoyl-phorbol-acetate (TPA) resulted in
Ser-133-phosphorylated CREB protein that was transcriptionally inactive
as measured in a CREBGAL4 system. Simultaneous costimulation, however,
with suboptimal doses of the cAMP agonist forskolin resulted in a
transcriptionally active CREB (20). Finally, CREB became
phosphorylated, but not activated, upon stimulation of CD28 on T
lymphocytes. Cotreatment with TPA or anti-CD3 allowed CREB activation
(21).
We investigated whether platelet-derived growth factor (PDGF), TPA, and
forskolin could induce Ser-133 phosphorylation and transcriptional
activation of CREB in NIH 3T3 cells. Our results show that all agents
can induce CREB phosphorylation with comparable kinetics and magnitude,
but the transcriptional activity of CREB induced by TPA and PDGF was
less efficient than that measured after forskolin stimulation. The role
of PKC, PKA, and the Raf/mitogen-activated protein (MAP) kinase
pathways in the activation of CREB by each of these stimuli was
investigated. Our results indicate that the Raf/MAP kinase pathway is
the main mediator of PDGF-induced CREB activation. Forskolin-induced
CREB activation is mediated by the PKA but also by the Raf/MAP kinase
pathway. PDGF- or forskolin-induced Ser-133 phosphorylation of CREB,
however, was not affected by the MEK-specific inhibitor PD98059.
Inhibition of PKC, PKA, or the Raf-MAP kinase-signaling pathway all
abrogated the transcription activity of CREB induced by TPA. Together,
these results fail to establish a direct correlation between CREB
Ser-133 phosphorylation and transcriptional activation and demonstrate
that different pathways can activate CREB in vivo.
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RESULTS
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Growth Factors, TPA, and Forskolin Induce Phosphorylation of CREB
at Ser-133 in NIH 3T3 Cells with Comparable Levels and Kinetics as the
cAMP-Elevating Agent Forskolin
CREB was originally characterized as a cAMP-responsive
transcription factor whose transcriptional activation is mediated via
PKA phosphorylation at Ser-133 (2). Several laboratories have reported
that growth factor stimulation and cellular stress can induce Ser-133
phosphorylation of CREB as well as activate CREB (6, 9, 10, 20, 22). We
wanted to compare the ability of different growth stimuli and the
adenylate cyclase activator forskolin to induce CREB Ser-133
phosphorylation in murine fibroblasts NIH 3T3 cells. Forskolin has been
shown to stimulate both Ser-133 phosphorylation and CREB activation in
NIH 3T3 cells (23, 24, 25). Whole-cell extracts were prepared from NIH 3T3
cells serum-starved for 2024 h and from serum-starved cells
stimulated with either 10 µM forskolin, 10 ng/ml PDGF,
10% newborn calf serum (NCS) or 50 ng/ml TPA for 15, 30, 60, or 180
min, respectively. Western blot analysis with an antibody that
recognizes the Ser-133 phosphorylated epitope of CREB were performed.
Forskolin rapidly induced Ser-133 phosphorylation of CREB with the peak
between 15 and 30 min, followed by a gradual decline in phosphorylation
after 60 and 180 min (Fig. 1
).
Stimulation with NCS, PDGF, and TPA induced CREB phosphorylation with
comparable magnitude and time kinetics as forskolin. Due to the
structural similarities between CREB and ATF-1, this antibody also
reacts with the Ser-63-phosphorylated form of ATF-1, the
phosphorylation kinetics of which parallels that observed for
phospho-CREB.

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Figure 1. Forskolin, NCS, PDGF, and TPA Stimulate CREB
Ser-133 Phosphorylation
NIH 3T3 cells (35-mm wells) were serum starved for 2024 h and treated
with either 10 µM forskolin, 10% NCS, 10 ng/ml PDGF, or
50 ng/ml TPA for the times indicated before harvesting directly in 50
µl SDS-sample buffer. Extract (20 µl) was separated by 10%
SDS-PAGE, electroblotted onto polyvinylidene fluoride membranes,
and probed with an antibody specific for Ser-133-phosphorylated CREB
(P-CREB) and the Ser-63 phosphorylated form of ATF-1. Total CREB
expression was analyzed by reprobing the stripped blots with an
antibody recognizing CREB regardless of its phosphorylation status
(lower panel). The positions of P-CREB and
phospho-ATF-1 are indicated.
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PDGF and TPA, but Not Serum, Moderately Induce the Transcriptional
Activity of CREB
Ser-133 phosphorylation has been regarded as the rate-limiting
step in transcriptional activation by CREB. Since PDGF, NCS, or TPA
induced Ser-133 phosphorylation with levels comparable to
forskolin-treated cells, we wanted to investigate whether these
mitogens also could induce CREB-mediated transcription. Therefore, NIH
3T3 cells were cotransfected with a reporter plasmid containing the
luciferase gene under control of the minimal promoter of the adenovirus
E1b gene and five binding motifs for the yeast transcription
factor GAL4 and with a GAL4-CREB expression plasmid. This chimeric
protein consists of the DNA-binding domain of GAL4 fused to full-length
CREB. Treating such cotransfected cells with forskolin resulted in a
more than 20-fold increase in luciferase activity compared with
unstimulated cells (Fig. 2A
). Neither
increasing the concentration of forskolin (up to 100 µM)
nor cotreatment with the phospodiesterase inhibitor
isobutymethylxanthine (0.5 mM) elevated this
induction level (results not shown). TPA and PDGF were less potent
inducers of luciferase activity with only a 3- and 5-fold enhancement
of luciferase expression, respectively. NCS failed to induce the
transcriptional activity of GAL4-CREB but was able to induce
phosphorylation of CREB (Fig. 2A
). Costimulation with either TPA or
PDGF had no negative influence on forskolin-induced CREB activation
(our unpublished data), thus indicating that no concomitant inhibitory
signal could be the reason for the relative low induction of CREB
activity observed after PDGF and TPA stimulation. Cotransfection with a
plasmid expressing only the GAL4-DNA-binding domain did not induce any
transactivation with either stimuli (data not shown). Similar
experiments were performed with a mutant GAL4-CREB hybrid in which
serine-133 was replaced by an alanine. This single-point mutation
abolishes the PKA-induced transcriptional activity of CREB (5). None of
the stimuli tested here could induce transcription activity of the CREB
Ala-133 mutant (Fig. 2B
). Our results confirm that Ser-133 is crucial
for transcriptional activation of CREB induced by forskolin, PDGF, or
TPA in NIH 3T3 cells, but fail to illustrate a direct correlation
between the degree of CREB Ser-133 phosphorylation and transcriptional
activation.

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Figure 2. Activation of CREBGAL4-Mediated Transcription
A, Growth stimuli are inefficient activators of CREB-mediated
transcription. NIH 3T3 cells (35-mm wells) were transfected with 1 µg
p5GE1bLuc plus 0.5 µg pCMVCREBGAL4 together with 0.2 µg pCH110 to
correct for transfection efficiency. Cells were serum starved for
20 h posttransfection before stimulation with either 10
µM forskolin, 10% NCS, 10 ng/ml PDGF, or 50 ng/ml TPA
for 3 h. The mean luciferase/ß-gal ratio was calculated (±
SD, n = 6) The activity from unstimulated cells
transfected with CREBGAL4 was arbitrary set as 1. B, Transfections and
stimulation were done as in panel A except that ALA 133
CREBGAL4 was used as transactivator.
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PKA Is Involved in Forskolin- and TPA-, but Not PGDF-Induced,
Activation of CREB
Since PKA seems to be the major kinase responsible for Ser-133
phosphorylation and activation of CREB induced by a wide variety of
stimuli, we tested whether forskolin, TPA, or PGDF-induced CREB
activation in NIH 3T3 cells was PKA dependent. Previous studies have
shown that both Ca2+- and PKC-mediated CREB activation in
several cell lines is dependent on PKA (15). This experiment was also
justified by the recent report of a cAMP-independent route for
activation of PKA. The authors showed that the C
catalytic subunit
of PKA resides in an inactive complex with the transcription factor
NF-
B (26). This catalytic subunit could be released by agents that
induce phosphorylation/degradation of I
B, e.g. by TPA
which is a potential inducer of I
B phosphorylation/degradation. NIH
3T3 cells were transfected with a plasmid that expresses a heat-stable
inhibitor of PKA (PKI). PKI has been previously described as a specific
and potent inhibitor of PKA activation and PKA-induced gene expression
(27). To exclude squelching by the distinct promoters of the different
plasmids, transfections were also done with a plasmid that expressed a
nonfunctional PKI protein (PKImut). Overexpression of PKI strongly
repressed forskolin (80% inhibition)- and TPA (50% reduction)-induced
CREBGAL4 gene expression (Fig. 3A
). In
contrast, PKI expression had no effect on PDGF-induced CREBGAL4
activity. Expression of PKI did not reduce the levels of CREBGAL4
fusion protein as determined by Western blotting (results not shown).
Thus TPA- and forskolin-, but not PDGF-mediated, CREB activation seems
to be dependent on PKA.

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Figure 3. PKA Inhibitor Protein PKI Represses TPA- and
Forskolin-Induced CREB Activation
A, NIH 3T3 cells were transfected with 1 µg p5GE1bLuc plus 0.5
µg pCREBGAL4 and either 1 µg pPKImut or 1 µg pPKI. After 20
h maintenance in medium containing 0.5% serum, the transfected cells
were stimulated with 50 ng/ml TPA, 10 ng/ml PDGF, or 10
µM forskolin (F) for 3 h. The mean luciferase
activities (± SD, n = 36) are presented. B, NIH 3T3
cells transfected with 1 µg p5GE1bLuc plus 0.5 µg GAL4ElkC and
either 1 µg pPKImut or 1 µg pPKI. After transfection the cells were
kept for 20 h in 0.5% NCS and then treated with 50 ng/ml TPA for
3 h. Each bar represents the mean
luciferase/ß-gal ratio (± SD, n = 3). Activity from
untreated cells transfected with GAL4 ElkC was arbitrarily set as 1.
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Previous studies have indicated that TPA activates the Raf/MAP kinase
signaling pathway, a signaling pathway that has been implicated in CREB
activation by nerve growth factor (NGF) (22, 28, 29). It is
therefore theoretically possible that PKI exerts its negative effect on
TPA-induced CREB activation through disturbance of TPA-induced
activation of the MAP kinase pathway. To investigate whether the
inhibitory effect of PKI on TPA-induced CREB activation targets TPA
ability to activate ERK1/2, we cotransfected cells with a GAL4-Elk-1
expression plasmid together with the PKI expression vectors. The
transcription factor Elk-1 is a direct substrate for ERK1/2, and ERK
phosphorylation of the C-terminal part of Elk-1 leads to
transcriptional activation (30). Stimulation with TPA resulted in a
30-fold increase in Elk-1-mediated expression, while no influence on
either basal or TPA-induced GAL4-Elk-1 activity was observed in the
presence of PKI (Fig. 3B
). These observations strongly suggest that PKI
does not interfere with TPA-induced activation of the MAP kinase
pathway.
PDGF- or Forskolin-Mediated CREB Transactivation and Ser-133
Phosphorylation Are PKC Independent
Ser-133 of CREB also forms the target for a putative PKC
phosphorylation motif. Indeed, PKC phosphorylates CREB at Ser-133
in vitro with similar efficiency as PKA (20, 31). Phorbol
esters such as TPA are believed to activate certain forms of PKC
by mimicking the second messenger diacylglycerol (32). PDGF can
activate classic PKCs (cPKC) through activation of phospholipase
C
, which leads to generation of diacylglycerol and
subsequently cPKC activation (33). Cross-talk between the cAMP and PKC
pathways is well illustrated (34). This suggested that PKC could be a
kinase that was indirectly responsible for TPA-, forskolin-, and
PDGF-mediated transcriptional activation of CREB. To test this, NIH 3T3
cells were pretreated for 1 h with 0.5 µM of the
PKC-specific inhibitor GF109203X, able to block both classical PKC
subtypes and the novel PKC
and -
, but not -
(35, 36) before
stimulation with TPA, forskolin, or PDGF. Pretreatment with GF109203X
completely abolished TPA-induced CREBGAL4 activity but had no influence
on PDGF-induced CREB activity (Fig. 4
and
results not shown). GF109203X alone had no effect on basal luciferase
activity (Fig. 4
). We also examined whether the PKC inhibitor GF109203X
abrogated phosphorylation of CREB (Fig. 5
). GF109203X clearly reduced TPA-induced
CREB Ser-133 phosphorylation, e.g. compare lanes 2 and 6,
but interfered imperceptibly with PDGF-induced phosphorylation,
e.g. lanes 3 and 7, or forskolin-induced phosphorylation
(data not shown). The compound had no effect on either
forskolin-induced CREB activation or Ser-133 phosphorylation (data not
shown). This indicates that PDGF- and forskolin-induced CREB
activation/Ser-133 phosphorylation are independent of the phorbol
ester-induced PKCs. The lack of involvement of cPKCs in PDGF-mediated
CREB phosphorylation was also shown by depletion of PKC by exhausting
NIH 3T3 cells for 20 h with 400 ng/ml TPA before stimulation. This
pretreatment with TPA had little effect on subsequent PDGF-stimulated
Ser-133 phosphorylation, whereas it completely blocked TPA-induced CREB
phosphorylation (results not shown).

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Figure 4. PKC Mediates TPA-Induced CREB Activation
NIH 3T3 cells were transfected as described in the legend of Fig. 2B .
Where indicated, cells were pretreated with 500 nM PKC
inhibitor GF109203X for 30 min before stimulation with TPA or PDGF.
Cells were harvested 3 h later, and luciferase and
ß-galactosidase activities were determined as described in
Materials and Methods. Each bar
represents the mean luciferase/ß-galactosidase ratio (±
SD, n = 36). The activity in unstimulated
CREBGAL4-transfected cells was set as 1.
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Figure 5. Both PKC Inhibitor GF109203X and MEK Inhibitor
PD98059 Act Differently on TPA- and PDGF-Induced CREB Ser-133
Phosphorylation
Serum-starved (20 h) NIH 3T3 cells were pretreated with either PD98059
(50 µM) or GF109203X (0.5 µM) for 60 min
and then stimulated with 50 ng/ml TPA or 10 ng/ml PDGF for 30 min. The
cells were directly lysed in SDS-sample buffer, and the lysates were
boiled for 3 to 5 min. Lysate (20 µl) was separated on 10% SDS-PAGE.
The gel was blotted and Ser-133-phosphorylated CREB and
Ser-69-phosphorylated ATF-1 were visualized as described in the
Materials and Methods. Total CREB level in each sample
was determined by stripping and reprobing the membrane with an antibody
that recognizes CREB regardless of its phosphorylation status
(lower panel). A representative experiment is shown.
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The Raf-MEK Pathway Is Involved in Forskolin-, TPA-, and
PGDF-Induced CREB Activation
Growth factors, including PDGF, and phorbol esters are both potent
activators of the MAP kinases, ERK1 and ERK2, in NIH 3T3 cells (our
unpublished results and Ref. 28). Growth factors such as nerve growth
factor and epidermal growth factor have been shown to induce
CREB phosphorylation in a Ras/Raf-dependent manner, and MAPKAP-kinase
1b (also known as RSK2) and MAPKAP-K-2 have been identified as these
CREB kinases (22). MAPKAP-K2 is activated by the p38 MAP kinase, while
MAPKAP-K1b is directly activated by the MAP kinases ERK1/2 (37), and
the only known activators of ERK1/2 are the dual specificity kinases
MEK1 and MEK2. Activation of ERK1/2 by phorbol esters in NIH 3T3
cells is believed to occur through Raf, which in turn activates
MEK1/MEK2 (29). To test whether PKC-induced CREB activation occurs
through the Raf-MEK-MAP kinase pathway, we first cotransfected with an
expression plasmid for a dominant negative mutant of c-Raf (Raf 1130)
expressing only the N-terminal regulatory domain of c-Raf-1 (38).
Expression of Raf 1130 inhibited TPA-induced CREB activation by more
than 70%, while it repressed the forskolin-induced CREBGAL4 activity
by almost 70% (Fig. 6
). Coexpression of
a dominant negative mutant of c-Raf did not inhibit basal CREBGAL4
transcriptional activity or ß-galactosidase expression from the
cotransfected pCH110 plasmid (results not shown), which argues against
a general effect of the inhibitors on either transcription or
translation of the reporter gene.

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Figure 6. Raf Is Involved in Forskolin- and TPA-Induced CREB
Activation
NIH 3T3 cells were transfected as described in Fig. 2B , except that the
cells were either pretreated with 50 µM PD98059 for 90
min or cotransfected with 1 µg of dominant negative Raf expression
plasmid (pRaf 1130) before stimulation with either 10
µM forskolin or 50 ng/ml TPA for 3 h. Luciferase and
ß-gal activities were measured as in Fig. 2B . Each bar
represents the mean luciferase/ß-gal ratio (± SD, n
= 6). Luciferase activity corrected for ß-gal activity in
unstimulated cells was set as 1.
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To further explore the involvement of the Ras/Raf/MAP kinase pathway in
CREB activation by PGDF, TPA, and forskolin, we made use of the
recently developed flavone compound PD98059, an efficient and specific
inhibitor of MEK1/2 (39, 40). TPA-induced, as well as
forskolin-mediated, CREBGAL4 activation was reduced by 60% in the
presence of PD98059 (see Fig. 7
). The
real inhibitory effect of PD98059 on TPA- or forskolin-induced CREB
activation is probably more since PD98059 alone slightly stimulated
CREB-mediated luciferase activity. Pretreatment with this MEK inhibitor
strongly repressed PDGF-induced CREBGAL4 activation (Fig. 7
). PDGF has
also been reported to activate ERK1/2 independent of MEK via
phosphatidylinositol-3-kinases (PI3K) (41). Pretreatment of cells with
wortmannin, a specific inhibitor of PI3K did not influence PDGF-induced
CREB activation (data not shown), indicating that CREB activation by
PDGF involved MEK rather than PI3K.

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Figure 7. Forskolin-, TPA-, and PDGF-Stimulated CREBGAL4
Activity Is MEK Dependent
NIH 3T3 cells were transfected with pG5E1bLuc and pCREBGAL4 as in Fig. 2B , except that some of the cells were pretreated with 50
µM PD98059 for 60 min, before stimulation with either 10
µM forskolin, 50 ng/ml TPA, or 10 ng/ml PDGF for 3
h. Relative luciferase activity was calculated as explained in the
legend of Fig. 2B . Each bar represents the mean (±
SD, n = 3).
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The results above show for the first time that the Raf-MAP kinase
pathway is involved in forskolin-mediated CREB activation. Studies have
demonstrated that forskolin and PKA can stimulate ERK1/2 activation via
B-Raf in PC12 cells (42). To determine whether forskolin stimulation
resulted in ERK1/2 activation, we transfected NIH 3T3 cells with an
Elk-1 GAL4 fusion protein where the C-terminal part of Elk-1
(containing the ERK1/2 phosphorylation sites) was fused to the
DNA-binding domain of GAL 4. Forskolin was unable to stimulate
transcriptional activity from GAL4-Elk-1 (Fig. 8
), indicating that ERK1/2 was not
activated by forskolin. This is agreement with our earlier finding that
forskolin was unable to stimulate ERK1/2 activation in NIH 3T3 cells
(43). The only known downstream targets for MEK are the MAP kinases
ERK1/2. MEK activates ERK1/2 by dual phosphorylation of ERK1/2 on both
tyrosine and threonine. The MAP kinases can efficiently be
deactivated by a class of dual specificity phosphatases that are able
to specifically dephosphorylate the tyrosine and threonine residues
phosphorylated by MEK (reviewed in Ref. 44). CL 100 and Pyst-1 are two
members of this family of phosphatases. While CL 100 is able to
dephosphorylate/deactivate several MAP kinase types (p38, SAPK, and
ERKs), Pyst-1 seems to be more specific toward ERK1/2. To determine
whether blocking downstream effectors of MEK could also repress
forskolin-mediated CREBGAL-4 activity, we cotransfected expression
vectors for CL100 and Pyst-1 together with CREBGAL4 and p5GE1bLuc.
Coexpression of either phosphatases efficiently repressed both basal
and forskolin-induced CREBGAL4-mediated luciferase activity (Fig. 9
). Taken together, the Raf/MAP kinase
pathway is involved in forskolin-mediated CREB activation, but
forskolin by itself is not able to activate this pathway. The
inhibitory effect of the MEK inhibitor PD98059 on forskolin-induced
CREB activation could therefore be the result of a negative effect on
PKA activation by forskolin. We tested whether PD98059 interfered with
forskolin-induced PKA activation. The results are shown in Fig. 10A
. Exposure to 10 µM
forskolin resulted in a 3-fold increase in PKA activity compared with
unstimulated cells, but no effect on either basal (not shown) or
forskolin-induced PKA activity was measured. These results support the
findings that the Raf/MAP kinase is involved in forskolin-induced CREB
activation.

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Figure 8. Forskolin Fails to Stimulate Transcription from
GAL4ElkC
NIH 3T3 cells were transfected with 1.0 µg p5GE1bLuc and 0.5 GALElkC
together with 0.2 µg pCH110. After serum starvation for 20 h,
the cells were treated with 10 µM forskolin (F) or left
untreated (U) and 3 h later the cells were harvested. The results
represent the mean (± SD) of one experiment with three
independent parallels. Relative luciferase activities were calculated
as described in Fig. 2B .
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Figure 9. MAP-Kinase Phosphatases Inhibit Forskolin-Induced
CREB Activation
Cells were transfected and treated as in Fig. 2A except that either 1
µg pSG5 (Vector), 1 µg CL100, or 1 µg Pyst 1 expression plasmid
was added.
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Figure 10. MEK Inhibitor Does Not Interfere with
Forskolin-Induced PKA Activation or CREB Ser-133 Phosphorylation
A, PKA activity measured in whole-cell extracts from untreated,
forskolin-treated, or forskolin plus PD98059-treated cells. After
maintenance in medium containing 0.5% serum, the cells were incubated
with 50 µM PD98059 for 90 min before stimulation with 10
µM forskolin for 30 min. Preparation of cell extracts and
PKA assays were done as described in Materials and
Methods. The results are the mean of the PKA activity in three
to six separate extracts measured in duplicate. B, PD98059 does not
alter forskolin-induced CREB Ser-133 phosphorylation. Western blot
analyses with a CREB phospho Ser-133-specific antibody of cells
pretreated for 90 min with 50 µM PD98059 and stimulated
for 30 min with 10 µM forskolin were performed as
described in the legend to Fig. 1 . The blot was stripped and reprobed
with an antibody recognizing CREB regardless of its phosphorylation
status (lower panel). The positions of phospho-CREB
(P-CREB), phospho-ATF-1(P-ATF-1), and CREB are indicated.
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Next we examined whether PD98059 affected Ser-133 phosphorylation of
CREB. TPA-induced Ser-133 phosphorylation of CREB was reduced in
PD98059-treated cells (Fig. 5
, lanes 2 and 4). However, PD98059
treatment did not repress forskolin-induced Ser-133 phosphorylation
(Fig. 10B
). In contrast to the efficient repression of PDGF-mediated
CREB activity by the MEK inhibitor, PDGF-induced CREB Ser-133
phosphorylation was poorly affected by pretreatment with the compound
(Fig. 5
, lanes 3 and 5).
The Q2 Domain of CREB Is Activated by Forskolin, TPA, and PDGF
CREB is regarded as a bipartite transcription factor where both a
kinase- inducible domain (KID, amino acids 101160) and a
glutamine-rich domain termed Q2 (amino acids 160284) are necessary
for full CREB activation in response to external stimuli (45). Earlier
investigators have shown that the Q2 domain fused to a heterolog
DNA-binding domain (GAL4 from yeast) functions as a constitutive
transcriptional activator. As we found that the Raf-MEK pathway was
necessary for full CREB activation in response to forskolin, TPA, and
PDGF independently of the KID domain, we asked whether the Q2 domain
could be a possible target. To test this, the cells were transfected
with a GAL4 Q2 construct in which amino acids 160284 of CREB were
fused to the DNA-binding domain of GAL4 and stimulated with forskolin,
TPA, and PDGF. The GAL4Q2 protein was activated by all three stimuli
(Fig. 11A
). The magnitude of activation
by the different stimuli was similar to what was observed with the
full-length CREB (Fig. 2A
). Interestingly, GAL4Q2 was not activated by
overexpression of PKA at doses that transactivated the full-length CREB
20- to 30-fold (Fig. 11A
and results not shown). PKA activity actually
decreased GAL4Q2 activity. We found also that the MEK-specific
inhibitor PD98059 was able to repress basal GAL4Q2 activity (Fig. 11B
).

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Figure 11. The Glutamine-Rich Q2 Domain of CREB Can Mediate
Forskolin-, PDGF-, or TPA-Induced Transcription
A, TPA, PDGF, and forskolin, but not coexpression of PKA,
efficiently activates Q2. NIH 3T3 cells were transfected as in Fig. 2
except that a GAL4Q2 expression vector was used instead of CREBGAL4.
After serum starvation for 2024 h, the cells were treated for 3
h with 10 µM forskolin (F), 10 ng/ml PDGF or 50 ng/ml TPA
(T), or left untreated (U). Some cells were also cotransfected with 1.0
µg expression plasmid for the catalytic subunit of PKA (PKA). B,
PD98059 represses basal pGAL4 Q2 activity. NIH 3T3 cells were
transfected as above. After serum starvation for 2024 h, the cells
were treated for 4 h with either PD98059 or vehicle (U). The
results from one representative experiment are presented as ratio
luciferase/ß-galactosidase activity with unstimulated GAL4 Q2
activity as 1 (n = 3).
|
|
In conclusion, the Ras/Raf/MAP kinase pathway is involved in CREB
activation by forskolin, PGDF, and TPA, but seems to be dispensable for
forskolin- or PGDF-induced Ser-133 phosphorylation because the MEK
inhibitor PD98059 did not affect CREB phosphorylation in PGDF- or
forskolin-treated NIH 3T3 cells.
 |
DISCUSSION
|
---|
CREB was originally isolated as a cAMP-responsive transcription
factor whose transcriptional potential was activated through
PKA-mediated phosphorylation of Ser-133 in the kinase-inducible domain
of CREB (reviewed in Refs. 45, 46). Recent studies have shown that
mitogenic stimuli are also able to induce CREB Ser-133 phosphorylation.
The identities of these mitogen-regulated CREB kinases are still
controversial, but both the MAP kinases, ERK1 and ERK2, and
p38-activated kinases have been proposed (9, 22). In the present study
we observed that in NIH 3T3 cells, PDGF, serum, and TPA induced CREB
Ser-133 phosphorylation with comparable kinetics and quantitative
levels as the adenylate cyclase activator forskolin. Thus our results
and those of others indicate that CREB can function as an integrator of
multiple signaling pathways activated by both mitogenic stimuli and
cAMP agonists. However, serum could not increase the activity of CREB
while PDGF and TPA could only moderately increase CREB activity
as measured in the CREBGAL4 system. Other investigators have failed to
detect CREB-mediated transcription by phorbol ester or growth factors
despite the fact that these stimuli could induce Ser-133
phosphorylation of CREB (6, 20, 47). These reports, combined with our
observation that serum failed to induce CREB-mediated transcription,
suggest that Ser-133 phosphorylation per se may not be
sufficient for CREB activation and that additional events, involving
separate signaling pathways, may be necessary for activation of
CREB-mediated transcription.
Since the PKA-, PKC-, and Raf-MEK-dependent signaling pathways have
been implicated in the regulation of CREB transcriptional activation
(5, 7, 48) we wanted to investigate the role of each of these signaling
pathways in CREB-mediated transcription induced by either forskolin,
TPA, or PDGF. The fact that recent reports have demonstrated that cAMP-
and mitogen-stimulated signaling pathways mediate extensive cross-talks
also at levels upstream of CREB further justified such an investigation
(26, 42).
PKA has a central role in CREB-mediated transcription, not only in
response to cAMP, but also for PKC and calcium-induced CREB activation
(20, 49). Our results from overexpression of PKI indicated a positive
role for PKA in TPA- induced CREB activation in NIH 3T3 cells. This
role for PKA in mitogen-stimulated gene expression is in agreement with
the previous observation by Day and co-workers (27), who found that PKI
repressed TPA-induced gene expression from a PRL promoter. A positive
role for PKA in MAP kinase-mediated signaling has been demonstrated
involving a novel Ras-independent pathway via the Ras-related small G
protein Rap-1. Rap-1 is a selective activator of B-Raf and an inhibitor
of c-Raf-1 (42). We found, however, that expression of neither B-Raf
nor c-Raf-1 was induced by forskolin, and the levels of B-Raf and
c-Raf-1 proteins in our NIH 3T3 cells were comparable (results not
shown). Moreover, if forskolin could activate the MAP kinase pathway in
NIH 3T3 cells, one should expect that inhibition of PKA also would
repress TPA-induced Elk-1 activation since Elk-1 is a well known target
for the MAP kinase pathway (30). However, we failed to detect any
effect of PKI overexpression on TPA-induced Elk-1 activation. An
alternative explanation comes from the study by Zhong et al.
(26), which showed that a fraction of the C
subunit of PKA resides
in complex with NF-
B. This activity can be released from NF-
B
independent of cAMP. Phorbol esters as TPA are well known inducers of
NF-
B in different cell types (50, 51). Therefore, TPA-induced CREB
activation in NIH 3T3 cells may be assigned to the kinase activity of
the PKA C
subunits liberated from NF-
B. If this were true, TPA
and PDGF must activate NF-
B differently in NIH 3T3 cells, since
PDGF-induced CREB activation was independent of any PKA activity as
shown by the use of PKI. While we found that TPA alone could moderately
increase the transcriptional activity in NIH 3T3 cells, Brindle and
co-workers (20) showed that suboptimal concentrations were necessary to
activate CREBGAL4 in phorbol ester-stimulated Jurkat cells. A threshold
level of activated PKA seems to be required to activate CREB by phorbol
ester. We observed a basal level of PKA level in serum-starved NIH 3T3
cells (Fig. 10A
). This residual PKA activity may be sufficient to
obtain CREB activation by phorbol ester alone, while in Jurkat cells
suboptimal concentrations of forskolin are required to obtain a
critical PKA activity. Expression of PKA inhibitor protein will block
this basal PKA activity and, therefore, phorbol ester-induced CREB
activation.
The fact that PDGF activates CREB independently of PKA strongly
suggests that alternative signaling pathways are involved in
CREB-mediated transcription. Since PDGF can activate classical PKCs
(cPKC) through activation of phospholipase C
and PKC has been shown
to induce CREB-mediated transcription, there is a possibility that a
PKC-dependent signaling pathway may be involved. Our results with the
inhibitor GF109203X or PKC depletion by long-term exposure to TPA
indicate that neither the classical PKCs nor the novel PKC
and -
isoforms are necessary for PDGF-induced CREB activation. It does not,
however, exclude that other PDGF-activated PKC isoforms such as
or
, which are not inhibited by GF109203X or TPA down-regulation, could
be responsible (52).
By the use of a dominant negative mutant of Raf or the MEK inhibitor
PD98059 we found that PDGF-, TPA-, and forskolin-induced CREB
activation were dependent on the Raf-MEK signaling pathway. PDGF and
TPA are believed to transmit signals from the membrane to the nucleus
through MEK and the downstream MAP kinases ERK1 and -2. Therefore, it
was not surprising to find that inhibition of MEK blocked
transcriptional activity of CREB induced by PDGF. However, the fact
that PD98059 repressed both PDGF- and forskolin-induced CREB
activation, but not CREB Ser-133 phosphorylation, was more intriguing.
Recent studies have shown that cAMP and PKA can activate MAP kinase and
Elk-1 in PC 12 cells independently of Ras via the Ras-related G protein
RAP-1 (42). The observation that forskolin failed to activate Elk-1-
mediated transcription, combined with our previous results which showed
that forskolin was unable to activate the MAP kinases ERK1/2 in NIH 3T3
cells, means that a basal level of MEK activity rather than activation
of the MEK pathway is necessary for maximal CREB activation in response
to forskolin (43). Although activation of MEK without subsequent
activation of ERK has been reported, these studies suggested
that the ultimate function of MEK is dependent on the manner in which
MEK is activated (53). One of the upstream activators of MEK is Raf-1
(54). We found that overexpression of a dominant negative mutant of
Raf-1 repressed forskolin-induced CREB activation, which indicates
involvement of an upstream signal to MEK induced by forskolin. It is
also likely that the Ras-binding domain of Raf-1 can interact with
other members of the Ras family, such as Rap-1. Therefore, the dominant
negative Raf-1 mutant used in this study may interfere with Rap-1
effectors. Recent studies have pointed out that Ras has multiple
effectors in addition to Raf-1, and forskolin activation of MEK may
involve upstream activators other than Raf-1 (reviewed in Ref. 55).
Furthermore, we demonstrated, by using the two dual specificity
phosphatases, CL100 and Pyst1, a role for a downstream effector of MEK
in forskolin-induced CREB activation. The fact that the phosphatase
Pyst 1 deactivates ERK1/ERK2 100-fold better than other members of MAP
kinases, combined with the known specificity of MEK, indicates that
this effector operates at the level of the ERK1/2 or further downstream
of MAPK, represented by the MAPKAP kinases (56, 57). The use of a
dominant-negative mutant of MAPKAP-K1a and GF109203X (a potent
inhibitor of MAPKAP-K1b), however, indicates that these kinases
downstream of ERK1/2 are not involved (Fig. 3
and data not shown; Ref.
58).
In conclusion, we found that PDGF and forskolin induce at least two
signals that target CREB. One of these signals triggers CREB Ser-133
phosphorylation, while the other signal governs the transcriptional
activity state of CREB. This latter signal is MEK dependent and may
involve a novel signaling pathway from forskolin through MEK down to
CREB. Experiments from other cell lines or in vitro
transcriptional systems further support the model that more than one
signal is needed for full transcriptional activation of CREB (20, 59, 60). CREB is a bipartite transcription factor consisting of a KID and a
noninducible constitutive glutamine-rich Q2 domain (45). These two
domains synergize to induce transcription by CREB. Further examination
of the mechanism required for CREB-mediated transcription of target
genes after its phosphorylation at Ser-133 led to the proposal of a
two-signal model for target gene activation: a
phospho-Ser-133-dependent interaction of CREB with RNA polymerase II
via the coactivator CBP, and a glutamine-rich domain interaction with
TFIID via hTAFII130 (60). Recruitment of CBP-RNA pol II complex
per se was not sufficient for transcriptional activation,
but activator-mediated recruitment of TFIID was also required for
induction of signal-dependent genes (60). Our results indicate that a
signal through the MAP kinase pathway may effect the activator-mediated
recruitment of TFIID to the complex via Q2 domain of CREB and TAFII130.
The inhibition of transcriptional activity from a GAL4 Q2 chimeric
protein by the MEK inhibitor supports this assumption (Fig. 11B
). A
previous study has demonstrated that the Q2 domain of CREB functions as
a constitutive activator when fused to GAL4 in F9 cells (45). However,
it appears that the Q2 domain when fused to GAL4 is inducible to both
growth stimuli and cAMP in NIH 3T3 cells. Whether these signals
influence the Q2 domain directly or the proteins that interact with the
Q2 domain at the promoter is still open for investigation.
 |
MATERIALS AND METHODS
|
---|
Materials
Forskolin, TPA, and PDGF were purchased from Sigma Chemical Co. (St. Louis, MO). NCS was from BioWhittaker, Inc. (Verviers, Belgium). Cell culture medium was obtained from
Gibco BRL, Gaithersburg, MD). Phospho-CREB(133) and CREB
antibodies, biotinylated protein molecular weight standard, and
CDP-Star were all obtained from New England Biolabs, Inc.
Beverly, MA). Alkaline phosphatase-conjugated swine antirabbit antibody
was purchased from DAKO Corp. (Copenhagen, Denmark). Dual
assay kit was acquired from Perkin Elmer-Tropix. PD98059 was
from New England Biolabs, Inc. GF109203X was purchased
from Calbiochem (La Jolla, CA). Heat-stable PKA inhibitor
peptide PKI TTYADFIASGRTGRRNAIHD was from Sigma Chemical Co. CREBtide KRREILSRRPSYRK was synthesized by Ø. Rekdal
(University of Tromsø).
Cell Culture
NIH 3T3 cells (ATCC CRL 1658, American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented
with 10% (vol/vol) NCS, HEPES, NaHCO3, penicillin (100
U/ml), and 100 µg/ml streptomycin (Life Technologies,
Gaithersburg, MD).
Plasmids
The GAL4 fusion protein expression vectors, pCMVCREBGAL4 and
pCMVALA133 CREBGAL4, have been described previously; pCMVPKI and
pCMVPKImut are improved versions of the expression plasmids of the
heat-stable inhibitor of PKA and were all generously provided by
R. A. Maurer (17, 27). GAL Q2 expressing the Q2 domain of CREB
fused to GAL4 has been described previously and was provided by M.
Montminy (45). The catalytic subunit of PKA
was expressed from a
pSR vector provided by K. Tasken (43). The plasmid G5E1bLuc, which
contains five binding sites for the yeast transcription factor GAL4
upstream of an adenovirus E1b TATA-box and a luciferase gene, was
provided by R. J. Davis (61). Plasmid pCH110 was purchased from
Pharmacia Biotech (Uppsala, Sweden) and contains the
bacterial lacZ gene driven by a SV40 early promoter. The two
dual-specificity phosphatases, Pyst1 and CL100, were expressed as
myc-tagged proteins from the eukaryote expression vector pSG5 and were
generously provided by S. M. Keyse (56). The dominant-negative
mutant of c-Raf was expressed from the plasmid pRaf 1130 provided by
C. J. Der (38).
Transient Transfection and Luciferase Assay
For reporter gene assays, NIH 3T3 cells (35-mm wells) were
transfected with 0.5 µg of a GAL4 fusion protein expression plasmid
together with 1.0 µg of a GAL4 luciferase reporter plasmid pG5E1bLuc
using the Ca-phosphate coprecipitation method (62). In some
experiments, cotransfections were performed with various amounts of
expression plasmids as indicated in the figure legends. The total
amount of plasmid DNA in each transfection reaction was maintained
constant at 4 µg by adjusting with the prokaryotic plasmid pGEM3Zf.
The cells were serum starved 2024 h posttransfection and then
stimulated for 3 h with either 10 µM forskolin, 10%
NCS, 10 ng/ml PDGF, or 50 ng/ml TPA before harvesting in 100 µl
potassium phosphate, pH 7.8, 0.2% Triton X-100, and 0.5 mM
dithiothreitol. Cotransfection with 0.2 µg of a ß-galactosidase
reporter (pCH110, Pharmacia Biotech) was performed to
correct for variations in transfection efficiency and sample handling.
Luciferase and ß-galactosidase activities were determined in 10 µl
lysate using the Dual-Assay Kit (Perkin Elmer-Tropix) and a Luminoscan
RT (Labsystems OY, Helsinki, Finland) luminometer according to
the manufacturers instructions.
Protein Kinase Assay
Whole-cell extracts were made as described previously (63). NIH
3T3 cells were serum starved for 2024 h before they were stimulated
for 30 min (as described in figure legends), and the 100-mm culture
dish was placed on ice. The medium was removed and the monolayer washed
two times with PBS before the cells were scraped off in 500 µl lysis
buffer (10 mM Tris-HCl, pH 7.05, 50 mM NaCl, 50
mM NaF, 1% Triton X-100, 30 mM
Na4P2O7, 5 µM
ZnCl2, 100 µM Na3VO4,
1 mM dithiothreitol, 2.8 µg aprotinin per ml, 2.5 µg
each of leupeptin and pepstatin per ml, 0.5 mM benzamidine,
and 0.5 mM phenymethylsulfonyl fluoride (all from
Sigma Chemical Co.). After vigorous vortexing for 45 sec
at 4 C, the lysates were cleared by centrifugation at 10,000 x
g for 10 min at 4 C and frozen in aliquots at 70 C. PKA
activity was determined as described previously (43). Briefly, 10 µl
extract were incubated in a total volume of 40 µl containing 50
mM Tris, pH 7.5, 10 mM MgCl, 100
µM(
-32 P), 0.25 mg/ml BSA, and 50
µM of CREB-tide as a substrate. Reaction mixtures were
incubated for 10 min at 30 C and spotted onto nitrocellulose paper. The
filters were washed twice in 1% phosphoric acid and twice in water,
and radioactivity was determined by scintillation counting. Total PKA
activity was determined in the presence of 10 µM cAMP.
PKA activity was defined as that sensitive to the inhibitor peptide PKA
inhibitor peptide (1 µM). Each value represents the mean
of at least three parallels.
Immunoblotting
Cells were grown in 35-mm wells in medium containing 0.5% NCS
for 2024 h and treated with 10 µM forskolin, 10 ng/ml
PDGF, 10% NCS, or 50 ng/ml TPA for the times indicated. The cell
extracts were prepared as described previously (43). Twenty microliters
of lysate were separated by 10% SDS-PAGE, and proteins were detected
by immunoblotting using phospho-CREB or CREB control antibody as
described previously (43). The blots were developed using a rabbit
alkaline phosphatase-conjugated antibody and the chemiluminiscence
substrate CDP-Star (Perkin Elmer/Tropix). Molecular weights were
estimated using a biotinylated broad range molecular weight protein
standard and alkaline phosphatase-conjugated antibiotin antibody
(New England Biolabs, Inc.).
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank R. J. Davis, C. J. Der, T.
Johansen, S. M. Keyse, R. Maurer, M. Montminy, and K. Tasken for
providing the following plasmids: p5GE1bLuc, pRaf (1130), pGAL4-ElkC,
pSGCL100, pSGPyst1, pCMVCREBGAL4, pCMVALA 133 CREBGAL4, pCMVPKI,
pCMVPKImut, pGAL4-Q2, and pSRPKA-
respectively; Ø. Rekdal for
synthesizing the CREB-tide; and R. Robberrecht for critically reading
the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Ugo Moens, Institute of Medical Biology, Department of Gene Biology, University of Tromsø, Tromsø, Norway N-9037.
This work was supported by fundings from the Norwegian Cancer Society
(D.N.K.) and the Erna and Olav Aakre Foundation.
Received for publication May 21, 1998.
Revision received January 15, 1999.
Accepted for publication March 16, 1999.
 |
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