From the Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel, Switzerland
Received for publication, October 16, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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In this study, we have identified novel
regulatory steps involved in the cross-talk between protein kinase B
(PKB) and MAPK signaling pathways. We found that PKB down-regulates the
Ras-Raf-MEK-ERK pathway by reducing the activity of ERK, which leads to
inactivation of the transcription factor Elk1. In addition, PKB is able
to reduce protein levels of Elk1. Both events lead to suppression of
serum response element (SRE)-dependent transcription and a consequent decrease in the transcription of SRE-containing genes, such
as c-fos. Because activation of the Ras/MAPK cascade is
reported to increase c-fos transcription before apoptosis,
our results are consistent with a specific role for PKB in promoting
cell survival. Decrease in c-Fos protein levels in glioblastoma cells with constitutively active PKB provides further support for our observations. Therefore, our findings delineate a novel mechanism regulating immediate-early transcription, which may be involved in the
initial steps in PKB-induced oncogenic transformation.
Protein kinase B (PKB,1
also termed Akt) belongs to a family of serine/threonine kinases that
are down-stream of the phosphoinositide 3-kinase (PI3K). PKB mediates a
variety of biological responses to insulin/insulin-like growth factor 1 (IGF-1) and other growth factors and is, thus, an important regulator
of glycogen and protein synthesis, glucose and amino acid transport,
translational regulation, survival, and cardiovascular homeostasis (for
review, see Refs. 1 and 2).
Upon activation, PKB translocates from the membrane to the nucleus (3),
where it directly regulates transcription. The targets of PKB
regulation are either genes whose products are involved in insulin
metabolism or transcription factors regulating cell survival (for
review, see Ref. 4). Thus, PKB mediates transcription of several
hepatic genes by acting through the insulin response sequences within
the respective promoters. Insulin-induced genes include fatty acid
synthase and GLUT1, whereas insulin also represses transcription of the
IGF-binding protein-1 gene (for review, see Ref. 5).
Several transcription factors are also regulated by PKB.
Phosphorylation by PKB is reported to stimulate the activity of
cAMP-response element-binding protein (CREB) (6). In addition, PKB
participates in I Another key mediator of cellular response to external stimuli is the
mitogen-activated protein kinase pathway, which is highly conserved in
all eukaryotes (for review, see Refs. 11 and 12). It is activated by a
GTP-binding protein, p21 Ras, which leads to recruitment of Raf
(MAPKKK) to the plasma membrane. Activated Raf phosphorylates MEK
(MAPKK), a dual specificity kinase that phosphorylates the
Thr-X-Tyr motif in the activation loop of
extracellular-signal-regulated kinase (ERK). Upon activation, ERK
translocates to the nucleus and regulates the activity of many
transcription factors, which results in distinct biological responses
(for review, see Refs. 11 and 12).
The most-studied transcription factors regulated by MAPK are ternary
complex factors (TCFs). TCF proteins belong to the ETS family of
transcription factors, which have a characteristic DNA binding domain
(the ETS-DBD), forming a highly conserved helix-turn-helix structural
motif (for review, see Ref. 13). The C terminus of TCFs comprises a
transactivation domain with multiple serine and threonine residues that
can be phosphorylated by different groups of MAP kinases. Whereas
serum, growth factors and TPA stimulate phosphorylation of TCFs via the
Raf-MEK-ERK pathway, interleukin-1, tumor necrosis factor PKB and ERK signaling cascades are activated under similar conditions,
which provides the possibility for cross-regulation. Thus, under
specific conditions PKB can phosphorylate Raf1 (16, 17) or B-Raf (18),
which inactivates the MAPK pathway. On the other hand, PI3K can
stimulate the activity of the MAPK cascade via the Rac1-PAK pathway by
activating Raf (19). It has been reported recently that Rho-family
GTPases, Rac1 and Cdc42, inhibit MAPK signaling at the level of Raf
through PI3K/PKB activation (20). Inactivation of Rac1/Cdc42 signaling
mimics the loss of cell anchorage, which activates the Raf-MEK-ERK
pathway and induces apoptosis (20).
The aim of this study was to identify novel regulatory points in the
cross-talk between PKB and MAPK pathways that contribute to the
PKB-mediated changes in transcription. We found that PKB down-regulates
the Ras-Raf-MEK-ERK pathway by reducing the activity of ERK, which
leads to inactivation of transcription factor Elk1. Significantly,
active PKB also indirectly induces a reduction of Elk1 protein levels.
Both events lead to suppression of SRE-dependent transcription and consequently change the immediate-early gene response. Our results delineate a novel mechanism of regulating transcription from the SRE/c-fos promoters that may be
involved in oncogenic transformation by PKB.
Expression Vectors--
Mammalian expression pCMV5 constructs
encoding hemagglutinin (HA) epitope-tagged PKB and pSG5-FLAG-C-terminal
modulator protein (CTMP) have been described previously (3, 21).
HA-RasV12 cDNA was sub-cloned by PCR into the BamHI
site of pSG5 (Stratagene). pcDNA1.RafBXB, pEXV3.EEMEK,
and pcDNA-HA-ERK2 were obtained from Dr. Y. Nagamine (Friedrich
Miescher Institute), and pCMV5-Myc-ERK was from Dr. M. Cobb
(University of Texas, Dallas, TX). SRE and TCF constructs were obtained
from Drs. B. Wasylyk (CNRS, Illkirch, France), P. E. Shaw
(University of Nottingham), and A. D. Sharrocks (University of
Manchester), and pfos-luc was provided by Dr. J. L. Bos
(University Medical Center Utrecht).
(Gal)5-TK-luc was obtained by subcloning the promoter of pG4CAT
(Clontech) in pGL2-Basic vector (Promega) in front
of the TK promoter. pGL2-(Pal)8-TK-luc was obtained by subcloning the (Pal)8-TK fragment of the (Pal)8-TK-CAT reporter in pGL2-Basic. Secreted alkaline phosphatase (SEAP) reporters were from the Mercury Pathway Profiling System from Clontech.
Cell Culture, Transient Transfection, and Stimulation
Conditions--
HEK 293 and glioblastoma cell lines LN229, U87MG, and
U343MG were maintained as described previously (21). The cells were seeded at 60% confluency and transfected the following day by a
modified calcium phosphate method (22). The cells were incubated for
24 h with the transfection mix, washed twice with Dulbecco's modified Eagle's medium, and serum-starved for 24 h, in some
cases in the presence of 20 µM UO126 (Tocris).
After starvation, the cells were stimulated with 10% fetal calf serum
for 6 h, or 100 ng/ml TPA or 50 ng/ml IGF-1 as indicated.
Luciferase and SEAP Assays--
HEK 293 cells were lysed with
200 µl of buffer (cell culture lysis reagent; Promega)
supplemented with 1 µM microcystin-LR (Alexis), 1 mM phenylmethylsulfonyl fluoride, and 1 mM
benzamidine. The lysates were centrifuged at 15,000 × g for 15 min at 4 °C. Protein concentrations were
determined by the method of Bradford (23). The luciferase activity was
assayed according to the Promega protocol and corrected for
transfection efficiency. SEAP assays were done according to the
manufacturer's instructions using the Great EscAPe chemiluminescence
protocol (Clontech).
Immunoprecipitation and in Vitro Kinase Assays--
HEK 293 cells were lysed with lysis buffer containing 50 mM
Tris-HCl (pH 7.5), 1% Nonidet P-40, 2 µM microcystin-LR
(Alexis), 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM sodium pyrophosphate, 0.1 mM sodium orthopervanadate, and 10 mM NaF. The
lysates were centrifuged at 15,000 × g for 15 min at
4 °C, and protein concentrations were determined as described above. The HA-tagged PKB
For endogenous PKB and ERK kinase assays, proteins were
immunoprecipitated from 2 mg of cell extracts, and immune complexes were washed as above. In vitro kinase assays were performed
for 30 min at 30 °C with crosstide (PKB) or myelin base
protein (ERK) as described (24, 25).
Immunoblot Analysis--
HEK 293, LN229, U87MG, and U343MG cells
were lysed as described above, and 20-40 µg of the cell extracts was
resolved by SDS-10% PAGE and transferred to Immobilon P membranes. The
filters were blocked for 30 min in 1× Tris-buffered saline blocking
buffer containing 5% skimmed milk, 1% Triton X-100, 0.5% Tween 20 followed by a 2-h incubation with rabbit polyclonal antibodies specific for active MAPK (Promega), PKB (26), phospho-Akt Ser-473 (Cell Signaling Technology), and ERK1 (C-16; Santa Cruz Biotechnology) or
with the following mouse monoclonal antibodies: pan-actin (Ab5; NeoMarkers), c-Fos (4-10G; Santa Cruz Biotechnology), 12CA5 anti-HA, or 9E10 anti-Myc antibody. The secondary antibodies used were alkaline
phosphatase-conjugated anti-rabbit IgG (Sigma) or alkaline phosphatase-conjugated anti-mouse Ig (Southern Biotechnology
Associates). Detection was performed using alkaline phosphatase
color development reagents from Bio-Rad.
Protein Kinase B Is Able to Suppress Transcription from the
SRE--
To investigate possible cross-talk between PKB and other
signaling cascades, we performed a screen of signal transduction pathways by monitoring the transcriptional activity of several reporter
constructs. For this purpose, we used the SEAP reporter system, where a
specific response element is located upstream of a minimal thymidine
kinase promoter fused to secreted alkaline phosphatase. Secreted
phosphatase activity represents a direct readout for induction of
specific signaling pathways. Thus, AP1 mirrors mitogen-activated JNK
signaling, cAMP-response element (CRE) responds to polypeptide hormone-
and neurotransmitter-activated protein kinase A and the
stress-activated JNK/p38 pathway, NF
To monitor the influence of PKB on other signaling cascades,
constitutively active membrane targeted myristoylated/palmitoylated PKB
(m/pPKB) was co-transfected with different SEAP reporter vectors. As
shown in Fig. 1A, the basal
activity of the tested response elements was already high in HEK 293 cells, even in the absence of stimuli. Under these conditions,
co-expression of m/pPKB led to a 20 and 30% increase in AP1- and
NF
The most significant change in transcription was observed when m/pPKB
was co-transfected with the SRE-dependent construct, the
activity of which decreased 85% compared with the control. This
suggests that PKB plays an important role in the regulation of ERK/JNK
pathways and/or ternary complex factor Elk1/Sap1-mediated transcription.
To further confirm whether the SRE can be regulated by the PKB pathway,
a luciferase reporter gene was constructed that contained binding sites
for the TCFs and SRF as well as an AP1-binding site (Fig.
1B). As shown in Fig. 1B, the expression of
increasing amounts of wild-type PKB reduced the basal level of
transcription of the SRE in a dose-dependent manner in HEK
293 cells. In contrast, the same effect could not be observed with an
inactive form of the kinase lacking the C-terminal 14 amino acids
(PKB-KD), which indicates that the kinase activity of PKB is required
for the effect on the SRE. Importantly, wild-type PKB promoted 50%
inhibition at a concentration as low as 1 ng/ml transfected DNA,
whereas inactive PKB had no effect on transcription.
To further test whether this effect was due to PKB kinase activity, we
compared the activity of constitutively active PKB mutants with that of
wild-type PKB. For this purpose, we expressed in HEK 293 cells
constitutively active m/pPKB as well as the constitutively active
cytosolic PKB mutant in which both the regulatory sites Thr-308 and
Ser-473 were mutated to aspartate (PKB-DD). As shown in Fig.
1C, both constitutively active mutants suppressed the basal
level of SRE trans-activation, whereas the inactive PKB-KD had no
effect on transcription (Fig. 1C, open bars). The
effect of PKB mutants was in accordance with their previously described activity (3).
Because it is well known that mitogens induce transcription by acting
on SRE, we examined whether this activation is blocked by PKB. Serum
stimulation led to a significant increase in SRE transcription (Fig.
1C). However, all the active PKB mutants not only prevented
activation of the SRE but also lowered its activity to below basal
levels. As observed for basal conditions, PKB-KD was also unable to
repress induction of the promoter by serum (Fig. 1C,
black bars).
Taken together, these results demonstrate that PKB can suppress basal
as well as activated transcription from SRE. These data also suggest
that PKB interfered with one or more pathways involved in SRE regulation.
Protein Kinase B Down-regulates the Human c-fos
Promoter--
The SRE is the major cis-element activated in
response to growth factors and is, thus, responsible for rapid and
transient activation of immediate early genes, including
c-fos and egr-1 (27). To examine the effect of
PKB on SRE within a physiologically relevant promoter, we performed
experiments similar to those shown in Fig. 1, with the luciferase
reporter gene under the control of a 750-bp proximal human
c-fos promoter containing Sis-inducible enhancer (SIS) and
CRE elements in addition to SRE (Fig.
2A).
Consistent with our results with the SRE reporter (Fig. 1), wild-type
but not kinase-inactive PKB reduced basal transcription from the
c-fos promoter by 90% (Fig. 2B). The effect of
PKB was dose-dependent and caused a 50% inhibition of
c-fos transcription at concentrations as low as 4 ng/ml
transfected expression vector. Significantly, PKB and the
constitutively active mutants also abolished the activity of the
c-fos promoter after serum stimulation (Fig. 2C).
Moreover, reduction in c-fos transcription in HEK 293 cells
correlated with the level of activity of endogenous PKB induced by
upstream activators such as serum, TPA, or insulin (data not shown).
Similar results were obtained with NIH 3T3 and COS-1 cells (data
not shown), indicating that this regulation is not cell type-specific
but may be a general mechanism of transcriptional regulation.
PKB Inhibits the Ras-Raf-MEK Pathway--
We showed that PKB was
able to suppress transcription mediated by SRE, which results in
inhibition of its function to promote transcription of serum-responsive
genes, such as c-fos. Mitogens activate immediate-early gene
transcription through all three Ras-induced MAPK signaling pathways.
The SRE is mainly activated via the pathway involving Ras, Raf, MEK,
and ERK (for review, see Ref. 15). We first searched for a direct
target of PKB regulation in this pathway using the c-fos
promoter construct as a readout. For this purpose, HEK 293 cells were
transfected with constitutively active components of the pathway (Fig.
3, A-C). As expected,
constitutively active RasV12 stimulated c-fos transcription
20-fold (Fig. 3A). This was almost completely inhibited by a
MEK-specific inhibitor UO126 (Fig. 3A) and indicates that
the MEK-ERK signaling cascade was the main pathway involved in
c-fos transcription. Constitutively active PKB, when
coexpressed with Ras, displayed the same effect as the MEK inhibitor,
decreasing c-fos transcription below the basal level,
indicating that PKB affects the same pathway (Fig. 3A). To
investigate whether PKB acts downstream or upstream of Raf, PKB was
co-transfected with constitutively active Raf BXB. As shown in Fig.
3B, Raf BXB induced c-fos transcription 45-fold, which was completely abolished when UO126 was added to the cells. Similar to the experiments with RasV12, constitutively active PKB was
able to decrease Raf-induced transcription from the c-fos promoter (Fig. 3B), suggesting that PKB does not act
directly on Ras but, rather, downstream in the pathway.
By a similar strategy, constitutively active EE-MEK was co-transfected
with PKB. Transfection of EE-MEK alone stimulated transcription 10-fold
(Fig. 3C). PKB diminished the activity of the promoter to
its basal level, similar to that observed with UO126 (Fig. 3C). Taken together, these data indicate a PKB target
downstream of Ras, Raf, and MEK, which directed us to investigate
transcription factors regulated by ERK.
PKB Suppresses TCF-dependent Transcription--
The
c-fos SRE is constitutively occupied by an SRF dimer, which
recruits monomeric TCFs, Elk1, Sap1, or Sap2/ERP/Net, to form a ternary
complex with SRF (for review, see Refs. 13 and 14). All three MAPK
pathways converge on the TCFs, and ERK, JNK, and p38 are all able to
phosphorylate Elk1 (for review, see Refs. 13 and 14). Therefore, we
examined TCFs as possible targets of PKB downstream of MAP kinases. All
three ETS-domain-containing transcription factors that can form ternary
complexes on SREs were transfected with a minimal reporter containing
the ETS palindromic site joined to the luciferase reporter. Both Elk1
(35-fold) and Sap1 (4-fold) activated, whereas Net inhibited (70%)
transcription from the luciferase reporter (Fig.
4A and data not shown), as previously demonstrated (28). PKB repressed Elk- as well as Sap-induced
transcription but did not interfere with Net-mediated inhibition (Fig.
4A and data not shown). As a positive control, we used an
Ets1 expression vector, which specifically binds to the ETS palindromic
site and activates transcription. As shown in Fig. 4A,
Ets1-induced transcription was also efficiently suppressed by PKB,
which can be explained by the fact that Ets1 can be regulated by MAP
kinases (29). In addition, TCF activity was sufficient to drive
transcription from this reporter, indicating that the presence of SRF
was not required and, therefore, strongly suggesting that inhibition of
the c-fos transcription was not
SRF-dependent.
Because Elk was a more potent activator of transcription than Sap and
it can be regulated by all MAP kinases, we next examined whether Elk is
a target of PKB. For these experiments we used an expression vector
containing the C-terminal MAP kinase-inducible activation domain of
Elk1 fused to the heterologous DNA binding domain of the yeast protein
Gal4 (Gal.ElkC). As shown in Fig. 4B, Elk promoted
transcription from the Gal4 reporter, but this was completely inhibited
by PKB, whereas PKB-KD had no effect on transcription. Furthermore,
UO126 only partially reduced Elk-stimulated transcription (Fig.
4B). This confirmed previous data suggesting that several
pathways contribute to Elk activation (Fig. 4B). PKB,
however, was able to abolish transcription completely and, thus, acts
on an effector common to all these pathways (Fig.
4B).
PKB Regulates ERK Activity--
We examined next whether PKB
directly phosphorylates Elk1 in vitro. Under the conditions
employed we could not find any evidence for Elk as a PKB substrate
(data not shown), which prompted us to investigate the effect of PKB on
Elk phosphorylation indirectly. ERK2 and PKB constructs were
co-expressed in HEK 293 cells, and Ras-activated ERK2 was used as a
positive control. In vitro kinase assays of
immunoprecipitated ERK2 and PKB were performed using glutathione
S-transferase-Elk (ERK) or crosstide (PKB) as a substrate. Unstimulated ERK showed low activity toward glutathione
S-transferase-Elk, whereas activation by RasV12 increased
phosphorylation of Elk by ERK more than 10-fold (Fig.
5A, middle panel).
Constitutively active, but not inactive PKB suppressed Ras-induced ERK
activity, resulting in 80% lower phosphorylation of Elk (Fig.
5A, middle panel, bar graph). The
level of repression of ERK activity (Fig. 5A, middle
panel, bar graph) was proportional to PKB activity (Fig. 5A, upper panel). Levels of
immunoprecipitated ERK on the beads were controlled by blotting the
same autoradiograph with the Myc monoclonal antibody. This revealed
similar amounts of ERK in each lane and, thus, excludes the possibility
that lower ERK activity in the presence of PKB is due to the lower
amount of ERK (Fig. 5A, lower panel). ERK is
activated by phosphorylation on Tyr and Thr in the activation loop.
This TEY phosphorylation is regularly regarded as a measure of
ERK activity. Therefore, to examine the phosphorylation status of ERK2
in our experiments, the membranes were stripped and re-probed with TEY
phospho-specific antibody. Surprisingly, in contrast to the observed
decrease in ERK activity by PKB (Fig. 5A, middle
panel, bar graph), the level of Ras-induced
phosphorylation of ERK2 was not significantly changed in the presence
of PKB (Fig. 5A, lowest panel). This implies that the level of ERK activity does not always necessarily correlate with
TEY phosphorylation in the activation loop. These results indicate that
in some cases, despite TEY phosphorylation, ERK activity may be
suppressed by a mechanism other than dephosphorylation. Therefore,
direct kinase assays provide a more accurate measure of ERK
activity.
To confirm these results, HEK 293 cells were stimulated by TPA, IGF-1,
or both at various time points, and endogenous kinase assays were
performed. As shown in the Fig. 5B, TPA was able to induce
ERK activity 14-fold after 10 min and 25-fold after 30 min (lower
panel), whereas it did not significantly affect activity of
endogenous PKB. Conversely, IGF-1 predominantly induced PKB activity.
When the cells were exposed to TPA and IGF-1 to activate both pathways,
we observed strongly suppressed activity of ERK (66% after 10 min or
80% after 30 min) as compared with TPA stimulation (Fig.
5B, lower panel). At the same time points PKB
activity was high (Fig. 5B, upper panel). Taken
together, these data provide evidence for PKB-promoted inhibition of
ERK kinase activity by a novel mechanism (see "Discussion").
PKB Regulates Protein Levels of Elk1--
The stability of some
transcription factors can be regulated by phosphorylation (for review,
see Ref. 30). Thus, phosphorylation is needed not only to activate the
transcription factor but also to protect it from degradation. For
example, c-Jun is protected from ubiquitination when phosphorylated on
Ser-73 by JNK (31). To investigate the effect of reduced ERK activity
on Elk1 stability, we co-expressed Elk together with active or inactive
PKB. The results in Fig. 6 show that PKB
and m/pPKB decreased the amount of Elk1 protein in a
dose-dependent manner. However, PKB-KD had no effect on
Elk1 protein levels (Fig. 6). Co-transfection of green fluorescent
protein and PKB showed that neither active nor inactive PKB
significantly changed the expression levels of green fluorescent
protein (data not shown), which excludes the possibility that the
decreased level of Elk1 protein was an unspecific consequence of
transfection. The proteasome inhibitor MG132 did not block PKB-promoted
reduction in Elk levels (data not shown), suggesting that this was not
proteasome-mediated.
These results were confirmed when the cells were treated with
cycloheximide to inhibit protein synthesis. Under such conditions, the
amount of Elk1 protein declined more rapidly when the cells were
co-transfected with PKB or treated with insulin (data not shown). These
findings indicate that PKB can reduce ERK activity and decrease the
level of Elk1 phosphorylation, leading to reduced levels of Elk protein
by a proteasome-independent mechanism.
Active PKB Can Reduce the Levels of Fos Protein in Vivo--
It is
well established that PKB activity can be regulated by the tumor
suppressor PTEN, a phosphatase that dephosphorylates phosphatidylinositol 3,4,5-trisphosphate. By dephosphorylating phosphatidylinositol 3,4,5-trisphosphate, PTEN acts in opposition to PI3K (for review, see Ref. 32). Recently, we discovered a PKB-interacting protein, termed CTMP, that inhibits phosphorylation of
PKB
Finally, we examined cells in which both the PKB and MAPK pathways were
constitutively active in vivo. In U87MG and U343MG glioblastoma multiforme cell lines with compromised PTEN function, CTMP
is expressed at low levels, which results in constitutive PKB activity.
However, in LN229 glioblastoma cells with normal PTEN status, CTMP is
also expressed at higher levels, thus inhibiting PKB activity (21).
Moreover, because PTEN is able to down-regulate the MAPK pathway, PTEN
Cross-talk between the MAPK and PI3K/PKB pathways apparently
operates at different levels and depends on specific conditions and the
cell types studied. The "classical" activation of ERK involves
signaling from Ras through Raf and MEK (for review, see Ref. 11),
although an alternative mechanism also exists involving activation of
the Raf-MEK-ERK cascade through signaling via PI3K (19, 34, 35). On the
other hand, under specific conditions, PKB can inhibit activity of the
Ras-MAPK pathway. It is reported that PKB negatively regulates Raf1
through phosphorylation of Ser-259 (16, 17) and B-Raf by
phosphorylating several residues in the N-terminal regulatory domain
(18). Loss of cell anchorage induces anoikis in fibroblasts. It
can be mimicked by inhibition of Rac1/cdc42 signaling through PI3K and
PKB, which activates Raf-MEK-ERK (20). This suggests under normal
conditions Rac1/cdc42 activates PKB, which then inhibits Raf signaling
(20).
In this study we identify ERK/Elk-mediated regulation of
c-fos transcription as a novel target for MAPK/PKB
cross-talk. The negative effect of PKB on c-fos promoter
transcription that we observed does not, however, appear to involve
previously described mechanisms. This is based on several lines of
evidence as follows. (i) The induction of the c-fos promoter
by constitutively active Raf lacking Ser-259 in the CR2 domain was
efficiently down-regulated by PKB (Fig. 3B); (ii)
c-fos promoter induction by constitutively active MEK was
also efficiently repressed by PKB (Fig. 3C); (iii) expression of PKB was sufficient to inhibit c-fos induction
by co-expressing either ERK or Jun kinase (data not shown). In our study, we identified two regulatory steps involved in the cross-talk between the Raf/MAPK and PI3K/PKB pathways. We demonstrated that PKB
can reduce activity of ERK toward its substrates, one of which is the
transcription factor Elk1. In addition, we showed that PKB reduces the
amount of Elk1 protein. Both of these events apparently result in
efficient down-regulation of SRE-dependent transcription. Our current model for PKB-mediated regulation of c-fos
induction is summarized in Fig. 8.
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ABSTRACT
INTRODUCTION
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B degradation, which results in NF
B induction
(7). This implicates PKB in cytokine-mediated immunity and
anti-apoptotic signaling. On the other hand, Forkhead transcription
factors, members of the FOXO subfamily, are negatively regulated when
phosphorylated by PKB (for review, see Ref. 8). Phosphorylated FOXOs
are inactive and sequestered in the cytoplasm away from their
transcriptional targets, which prevents induction of apoptosis or cell
cycle arrest by these transcription factors. Furthermore, it was
reported recently that PKB phosphorylates Mdm2 and, thus, promotes its
nuclear translocation. This leads to increased ubiquitination of p53
and resistance to apoptosis in some cancer cells (9, 10).
, osmotic
stress, H2O2, UV radiation, and anisomycin can
induce phosphorylation of TCF through the MEK kinase (MEKK)-SEK1-JNK as
well as through the TAK1-MKK3-p38 pathways (for review, see Ref. 13 and
14). The mammalian TCFs, Elk1, Sap1, and Sap2/ERP/Net, regulate
transcription from the serum response element (SRE) (for review, see
Ref. 13). SRE is the major cis-element responsible for activation of
immediate-early gene transcription in response to mitogens (for review,
see Ref. 15). It functions as a target for the Ras-MAPK pathway; thus, SRE is also termed a Ras-responsive element. The c-fos SRE
is constitutively occupied by a serum response factor (SRF) dimer, which binds with high affinity to the CC(A/T)6GG motif
within the SRE. SRF dimer recruits monomeric TCF, which forms a ternary complex with SRF on SRE and promotes transcription (for review, see
Ref. 13).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
and Myc-tagged ERK2 proteins were
immunoprecipitated from 100 µg of cell extracts as described
previously (3). The immune complexes were washed with lysis buffer
containing 0.5 M NaCl followed by lysis buffer and finally
with kinase buffer (50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol). In vitro kinase assays were
performed for 60 min at 30 °C in 50 µl of reaction mixture
containing 30 µl of immunoprecipitate in kinase buffer, 3 µg of
glutathione S-transferase-ElkC as a substrate, 10 mM MgCl2, 1 µM protein kinase A
inhibitor peptide (Bachem), and 100 µM
[
-32P]ATP (Amersham Biosciences; 1000-2000 cpm/pmol).
All reactions were stopped by adding Laemmli sample buffer, resolved by
SDS-10% PAGE, and transferred to Immobilon P membranes (Millipore).
Phosphorylated proteins were visualized by autoradiography and
quantified with ImageQuant software (Molecular Dynamics).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activity increases after
I
K-mediated cytokine and platelet-derived growth factor signaling,
and the SRE is activated by mitogen-induced ERK/JNK pathways.
B-driven transcription, respectively, whereas the CRE pathway was
not significantly affected by m/pPKB.
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Fig. 1.
PKB specifically reduces
transcription from the serum response element. A, HEK 293 cells were co-transfected with 100 ng/ml empty vector or a vector
encoding constitutively active PKB (m/pPKB)
together with 100 ng/ml SEAP reporter vectors under the control of AP1,
CRE, NF B, and SRE, respectively. SEAP activity from the cells
co-transfected with an empty vector was taken as 1. The experiment was
performed three times, and a representative experiment is shown. PKB
expression (inset) was confirmed by immunoblot analysis
using an anti-HA-epitope antibody. RLU, relative
light units. B, structure of the SRE luciferase reporter
containing binding sites for TCF, SRF, and AP1 (upper
panel). HEK 293 cells were co-transfected with an empty vector or
a vector encoding either wild-type (WT) PKB or a
kinase-defective mutant (PKB-KD) together with 100 ng/ml
SRE-luciferase reporter. Luciferase activity from the cells
co-transfected with an empty vector was taken as 1 and was calculated
from four independent experiments. PKB expression was confirmed as in
A. C, HEK 293 cells were co-transfected with 100 ng/ml empty vector or a vector encoding PKB, m/pPKB, or constitutively
active PKB-DD or PKB-KD. Luciferase activity was assayed as in Fig.
1B, and the average values of four independent experiments
are shown.
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Fig. 2.
PKB down-regulates transcription from human
c-fos promoter. A, structure of the human
c-fos promoter, containing Sis-inducible enhancer (SIS),
SRE, and CRE. B, HEK 293 cells were co-transfected with an
empty vector or a vector encoding either PKB or PKB-KD together with
100 ng/ml luciferase reporter construct under the control of the
c-fos promoter. Luciferase activity was determined as
described in Fig. 1B, and the average values from four
independent experiments are shown. C, HEK 293 cells were
transfected and assayed for luciferase activity as in Fig.
1C. The experiment was performed four times. WT,
wild type.
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Fig. 3.
Ras-Raf-MEK pathway is a target of PKB
regulation. HEK 293 cells were co-transfected with an empty vector
or an m/pPKB expression vector together with the vector encoding either
constitutively active RasV12 (A), Raf BXB (B), or
EE-MEK (C). The luciferase reporter under control of the
c-fos promoter was used as a readout, and luciferase
activity was assayed as in Fig. 1B. PKB expression was
confirmed by Western blotting. Each experiment (A-C) was
performed four times with similar results.
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Fig. 4.
PKB reduces TCF-dependent
transcription. A, HEK 293 cells were co-transfected with 100 ng/ml empty vector or a vector encoding PKB together with 500 ng/ml
expression vectors for ETS1p68, Elk1, Sap1a, or Net (not shown) and 500 ng/ml pGL2(Pal)8-TK-luc reporter. Luciferase activity was determined as
described in Fig. 1B, and the average values from four
independent experiments are shown. B, HEK 293 cells were
transfected with 100 ng/ml empty vector or a vector encoding PKB
together with 250 ng/ml Gal4.ElkC and 250 ng/ml (Gal4)5-TK-luc
reporter. Luciferase activity was determined as described in Fig.
1B, and the average values from four independent experiments
are shown. WT, wild type.
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Fig. 5.
PKB regulates ERK activity. A, HEK
293 cells were transfected with 1 µg/ml HA-ERK2 together with 100 ng/ml HA-PKB and/or 1 µg/ml RasV12. In vitro kinase assays
were performed as described under "Experimental Procedures."
Quantification of three independent experiments is shown. The total
levels of PKB and ERK on the beads were analyzed by immunoblotting by
HA-antibody followed by stripping and blotting with pERK antibody.
B, HEK 293 cells were stimulated with 100 ng/ml TPA or with
50 ng/ml IGF-1 for the indicated times. In vitro kinase
assays were performed as described under "Experimental Procedures,"
and the average values from three independent experiments are
shown.
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Fig. 6.
PKB regulates protein levels of Elk1.
HEK 293 cells were co-transfected with 500 ng/ml HA-Elk1 together with
increasing amounts (25, 50, 100, and 250 ng/ml) of HA-tagged PKB
constructs or empty vector (pCMV) as indicated. Cell lysate (20 µg)
was assayed for protein expression by immunoblotting. A representative
experiment of three is shown. WT, wild type.
by its upstream kinases on Ser-473 and somewhat less on Thr-308
(21). These findings identify CTMP as a specific negative regulator of
PKB, which prompted us to test whether CTMP can also reverse the effect
of PKB on the c-fos promoter. As shown in Fig. 7A, co-transfection of CTMP
rescued the inhibitory effect of PKB and restored c-fos
transcription. The control vector for CTMP (pSG5) did not change the
inhibition promoted by PKB. The restoration of transcription by CTMP
was dose-dependent and was observed at the level of both
basal and serum-induced transcription. Moreover, CTMP even increased
c-fos transcription above its initial level, suggesting that
CTMP inhibits endogenous PKB, consistent with our previous observations
(21).
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Fig. 7.
Negative regulators PTEN and CTMP are able to
revert the effect of PKB on c-fos in vitro
and in vivo. A, HEK 293 cells
were transfected with 100 ng/ml empty vector or a vector encoding PKB
together with 100 ng/ml c-fos luciferase reporter. Increased
concentrations (0.01, 0.05, 0.25, and 1.25 µg) of an empty vector or
vector encoding CTMP were co-transfected where indicated. Luciferase
activity was determined as described in Fig. 1B, and the
average values from four independent experiments are shown.
B, U343MG, U87MG, and LN229 cells were grown until
confluency and then starved for 24 h before lysis. Indicated
endogenous proteins were detected by direct Western blotting of
equivalent amounts of total cell lysates. A representative experiment
is shown. WT, wild type.
/
cells also have high ERK activity (for review, see Ref. 33). As
shown in Fig. 7B, low PKB activity in LN229 co-relates with
increased levels of c-Fos protein, as demonstrated by Western blotting.
In contrast, PKB was phosphorylated and, thus, activated in both U87MG
and U343MG cells, whereas the levels of c-Fos were low (Fig.
7B). These data provide evidence that in the glioblastoma
cells studied c-Fos levels are inversely correlated with PKB activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Proposed mechanism for down-regulation of
SRE-dependent transcription by PKB. A, in
response to Ras signaling, ERK is activated by MEK-mediated dual
phosphorylation (P) in the activation loop. Active ERK
phosphorylates its substrates, one of which is the transcription factor
Elk1. Phosphorylated Elk1 accumulates in the nucleus and strongly
promotes transcription from SRE-regulated genes, such as
c-fos. B, active PKB reduces ERK activity either
directly by phosphorylating ERK at a site distinct from TEY in the
activation loop or by phosphorylating a yet unidentified inhibitor of
ERK (1). Low ERK activity results in decreased phosphorylation and
shifts the equilibrium toward unphosphorylated Elk1 (2).
Unphosphorylated Elk1 is inactive, unstable, and prone to degradation
(3). It is also possible that reduction of ERK activity (1) and decline
of Elk1 protein levels (3) involve distinct mechanisms, both regulated
by PKB. However, each of these events could separately account for
down-regulation of SRE-dependent transcription (4).
We show that co-transfection of PKB inhibits the activity of ERK and, thus, its ability to phosphorylate Elk (Fig. 5A). Constant levels of TEY phosphorylation in the activation loop (Fig. 5A) rule out the likelihood that PKB signals through a phosphatase and, thus, inactivates ERK. Our data indicate that PKB negatively regulates ERK activity either by direct phosphorylation at a site distinct from TEY in the activation loop or indirectly, by phosphorylating a yet unidentified inhibitor of ERK. Results from in vitro kinase assays (Fig. 5A) exclude the possibility that PKB activates a phosphatase that dephosphorylates Elk. Regulation of ERK activation is rather complex, and it depends on two phosphorylations in the activation loop and subsequent re-ordering of the N- and C-terminal domains of the kinase (36). It is known that ERKs are phosphorylated on tyrosine before threonine in two separate reactions, both performed by MEK (37). The first phosphorylation of Tyr-185 (in ERK2) induces a conformational change that forms the C-terminal part of the substrate binding site (38, 39). Once this is completed, the second phosphorylation on Thr-183 occurs, leading to the orientation of N-terminal part of the active site (38, 39). Thus, ERK becomes activated only upon completion of both phosphorylation events. Our time course experiments (Fig. 5B) with the endogenous kinases suggest that PKB delays the activation of ERK without affecting the TEY phosphorylation. This could reflect the different rates of phosphorylation of Tyr-185 and Thr-183, suggesting that phosphorylation of Tyr-185 happens relatively fast (37) and could already be detected by the polyclonal phosphospecific antibody, but it is not sufficient for the enzymatic activity of ERK toward its substrates. Further experiments are required to elucidate the precise mechanism for ERK down-regulation by PKB.
PKB-mediated decrease in ERK activity would lead to lower levels of
Elk-1 phosphorylation, resulting in a reduction in transcriptional activity. Phosphorylation regulates protein targeting for degradation. In some cases, phosphorylation is required before ubiquitination (IB,
-catenin), whereas in others, phosphorylation protects proteins from degradation (c-Jun, p53, ATF2) (for review, see Ref. 30).
Stress-activated kinases regulate stability of transcription factors,
the best example being JNK/c-Jun (31). Thus, phosphorylation on Ser-73
by JNK protects c-Jun from degradation, whereas unphosphorylated c-Jun
is efficiently ubiquitinated upon binding to JNK (31). It is tempting
to speculate that phosphorylation protects Elk from degradation. To our
knowledge, little is known about the regulation of Elk stability. In
contrast to c-Jun, JNK is able to phosphorylate Elk, but it neither
associates with nor promotes Elk1 ubiquitination (40). Our preliminary
data show that decrease of Elk protein was not
proteasome-dependent (data not shown) but relies rather on
the activity of other cellular proteases. On the other hand, reduction
of Elk protein levels may be indirect and due to activation of another
enzyme or a specific protease by PKB. For this reason, we cannot
exclude the possibility that the two events (decrease in ERK activity
and decrease in Elk protein levels) that lead to the inhibition of
SRE-dependent transcription are actually uncoupled. We did
not investigate whether PKB is also able to regulate the activity
of other MAP kinases but, rather, focused on the consequences of
lowered ERK activity on down-stream signaling. Nevertheless, because
Elk represents a convergence point for all three MAPK pathways, the
observed decreased Elk protein could alone account for the inhibition
of SRE-dependent transcription.
By its action on Elk, PKB drastically decreases levels of immediate-early gene transcription. Based on several reports suggesting that continuous c-fos expression precedes apoptosis in vivo, at least in the tissues that require de novo protein synthesis for programmed cell death (for review, see Ref. 41), the anti-apoptotic role of PKB becomes apparent at this level. Moreover, under some conditions, Elk is very efficient in causing cell death (42). In addition, by regulating the levels of Elk, PKB controls not only transcription of c-fos gene but also reduces the level of AP1 transcription factor. This may lead to down-regulation of other genes containing AP-binding sites within their promoters that are preferably occupied by a Fos-containing heterodimers. Moreover, TCFs can act independently of SRF via direct binding to PEA3 elements in some promoters (43). Therefore, our data (Fig. 4A) might also indicate a role for PKB in the expression of those SRE/AP1-independent genes.
The mechanism (Fig. 8) we propose here is consistent with the
anti-apoptotic role of PKB. PKB promotes cell survival by inhibition of
an intrinsic cell death machinery in the cytoplasm (BAD, caspase 9) as
well as in the nucleus (Forkhead transcription factors, NFB, p53)
(for review, see Ref. 4). Although it seems contradictory at first
glance that PKB inhibits Ras-induced MAPK activity, this can be
explained given the dual role of Ras in apoptosis. Namely, regarding
its oncogenic potential, Ras is generally considered in the context of
activation of cellular proliferation and suppression of apoptosis.
However, this is only partially correct because Ras also induces
apoptosis (for review, see Ref. 44). Generally, Ras signals mostly
through Raf-MEK-ERK but also via the PI3K/PKB pathway. This
differential signaling seems to mirror opposing effects of Ras-induced
pathways on apoptosis (for review, see Ref. 44). Although signaling
through PI3K/PKB always promotes cell survival, proapoptotic Ras
signals exclusively through the Raf-MEK-ERK cascade (for review, see
Ref. 44). In this context, Elk is not the only transcription factor
differentially regulated by Ras and PKB. It was shown that
transformation of fibroblasts by Ras causes inhibition of NF
B
transcriptional activity, which leads to apoptosis (45, 46). In
contrast, PKB regulates the levels of NF
B inhibitor I
B through
activation of I
K (7). When phosphorylated by I
K, I
B is
targeted for degradation, which enables NF
B to translocate to the
nucleus and promote transcription of anti-apoptotic genes. Thus
Ras-transformed cells require activation of PKB to survive (46).
However, the role of the Raf-MEK-ERK pathway in apoptosis can be
completely opposite depending on the particular cellular setting. This
has to be taken into account when investigating the interplay between
the two pathways. Based on data from many reports addressing these
issues it can be concluded that the specific physiological outcome
results from a number of parameters including concentration of the
particular component, nature and duration of the signal, specific
interaction between signaling molecules, differentiation status, and
distinct cellular machinery in a particular tissue. Therefore, the type
of SRE regulation by PKB that we observed in HEK 293 cells and
confirmed in NIH 3T3 and COS-1 cells (data not shown) may not be
extended to all cell lines and tissues.
It is very well established that PKB activity can be regulated by PTEN, a tumor-suppressor frequently mutated in cancers (for review, see Ref. 32). Recently, we discovered another PKB inhibitor, which we named C-terminal modulator protein (CTMP). Like PTEN, CTMP is also able to revert transforming phenotypes of PKB and, thus, acts as a potential tumor suppressor (21). Here, we demonstrate that CTMP can revert the effect of PKB on c-fos transcription (Fig. 7A). Moreover, our data suggest that the proposed regulation of SRE-dependent transcription by PKB might occur in vivo. We reported previously the differential expression of CTMP in glioblastoma multiforme cell lines with different PTEN status (21). Thus, in cells with compromised PTEN function, CTMP is also expressed at very low levels, which explains why PKB is constitutively active in these cells. Accordingly, in cells with normal PTEN status, the level of CTMP is also higher (21). Moreover, in addition to its activity toward PI3K, PTEN is also able to down-regulate the MAPK pathway (for review, see Ref. 33). Consequently, one would expect that both the PI3K/PKB and MAPK pathways are active in PTEN-negative cells. However, we demonstrate here in U87MG and U343MG cells, which are both PTEN-negative and have high PKB activity, that the ERK pathway was apparently down-regulated, as measured by the low levels of Fos protein.
In summary, we have identified two new points of convergence in the
cross-talk between the MAPK and PKB pathways, (i) PKB reduces the
activity of ERK and (ii) indirectly decreases the amount of the
transcription factor Elk1. In combination these regulatory responses
lead to drastic inhibition of SRE-dependent transcription.
Our findings suggest a novel role for PKB in the fine-tuning of the
signals between the cell surface and nucleus, leading to differential
regulation of gene expression. This shows how the same or similar
stimuli induce differential responses in certain cells/tissues to
regulate various biological processes. Because PKB is a major mediator
of insulin and IGF-1 signaling, the identification of new PKB effectors
in cell survival may provide more specific targets for therapy of
malignancies without affecting metabolism.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Johannes Bos, Bohdan Wasylyk, Yoshikuni Nagamine, Melanie Cobb, Andrew Sharrocks, and Peter Shaw for providing plasmids, Dr. Adrian Merlo for the glioblastoma cell lines, Drs. Michelle Hill, Nancy Hynes, and Amir Faisal for critical comments on the manuscript, and Ralph Tiedt and Jongsun Park for help with formatting of the figures.
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FOOTNOTES |
---|
* The work was supported in part by a Schweizerische Krebsliga grant (to M. A.) and a European Molecular Biology post-doctoral fellowship (S.-M. M.). The Friedrich Miescher Institute is part of the Novartis Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Oncology, Novartis Pharma AG, CH-4057
Basel, Switzerland.
§ Present address: Dept. of Vascular and Metabolic Diseases, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland.
¶ To whom correspondence should be addressed: Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Tel.: 41-61-697-40-46; Fax: 41-61-697-39-76; E-mail: hemmings@fmi.ch.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M210578200
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ABBREVIATIONS |
---|
The abbreviations used are: PKB, protein kinase B; m/pPKB myristoylated/palmitoylated PKB, CTMP, C-terminal modulator protein; ERK, extracellular signal-regulated kinase; IGF-1, insulin-like growth factor; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein (MAP) kinase; MEK, MAPK/ERK kinase; JNK, c-Jun NH2-terminal kinase; HA, hemagglutinin; TK, thymidine kinase; PI3K, phosphoinositide 3-kinase; SEAP, secreted alkaline phosphatase; SRE, serum response element; SRF, serum response factor; TCF, ternary complex factor; TPA, phorbol 12-myristate 13-acetate; HEK cells, human embryonic kidney cells; CRE, cAMP-response element; UO126, MEK inhibitor 1,4Diamino-2,3-dicyano-1,4bis[2-aminophenylthio]butadiene; TEY, Thr-Glu-Tyr.
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