From the Lineberger Comprehensive Cancer Center,
§ Curriculum in Genetics and Molecular Biology and
¶ Department of Biology, University of North Carolina School of
Medicine, Chapel Hill, North Carolina 27599
Received for publication, February 5, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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The serine/threonine kinase Akt/PKB is a potent
regulator of cell survival and has oncogenic transformation potential.
Previously, it has been shown that Akt can activate the transcription
factor NF- Akt/PKB is a serine/threonine kinase that is activated in response
to certain growth factors and cytokines (1, 2). Consistent with its
activation by growth factors, Akt has transforming potential (3). Akt
isoforms have been shown to be overexpressed in breast cancer cell
lines, in ovarian and pancreatic cancers, and amplified in gastric
adenoma (4, 5). Additionally, Akt is a downstream activator for
oncogenic Ras and Src and for the proto-oncoprotein HER-2/neu
(6-8). Importantly, Akt provides a potent cell survival signal that is
likely involved in its transformation and growth-promoting properties
(3). Mechanisms associated with the ability of Akt to suppress
apoptosis include the phosphorylation and inactivation of many
proapoptotic proteins (9-15). Additionally, Akt has been shown to
activate the transcription factor NF- Classic nuclear factor- Although the induced nuclear translocation of NF- Reports describing processes whereby Akt stimulates NF- In this study, we show that the ability of Akt to stimulate the
transactivation potential of the RelA/p65 subunit of NF- Cell Culture and Reagents--
Murine NIH 3T3 fibroblasts were
grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% calf serum (Hyclone Laboratories, Logan, UT) and
penicillin/streptomycin unless otherwise indicated. Human 293T kidney
cells and IKK null mouse embryo fibroblasts were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
(Hyclone Laboratories, Logan, UT) and penicillin/streptomycin unless
otherwise indicated. The specific p38 MAPK pharmacological inhibitor
SB203580 and the MEK inhibitor PD98059 (Calbiochem) were used at a
working concentration of 600 nM and 2 µM,
respectively, unless otherwise indicated. Recombinant human IL-1 Plasmid Constructs and Mutagenesis--
3x- Transfection and Luciferase Reporter Assays--
NIH3T3 cells at
60-80% confluency were transiently transfected using the Superfect
reagent (Qiagen, Valencia, CA) according to the manufacturer's
instructions. Briefly, plasmid constructs (1-2 µg of DNA total) were
diluted in serum-free medium and mixed with the Superfect
reagent. Complexes were allowed to form for 10 min before
serum-containing medium was added to the mixture. The cells were washed
once with 1× phosphate-buffered saline, and Superfect-DNA
complexes were added to the cells and placed in a humidified incubator
at 37 °C with 5% CO2. Three hours after the addition,
cells were washed with 1× phosphate-buffered saline and replenished
with fresh serum-containing medium. Human 293T cells at 70-80%
confluency were transfected by the calcium phosphate protocol. 24-48 h
post-transfection, cells were washed once with 1× phosphate-buffered
saline and lysed in Reporter Lysis Buffer (Promega, Madison, WI) for 10 min at room temperature. Extracts were collected and cleared by
centrifugation at high speed. Protein concentration was determined with
the Bio-Rad protein assay dye reagent. D-Luciferin was used
as a substrate, and relative light units were measured using an
AutoLumat LB953 luminometer (Berthold Analytical Instruments). Results
were normalized to an internal Western Blot Analysis and Kinase Assays--
Western blot
analysis was performed by preparing whole cell extracts in the presence
of protease inhibitors or by lysing cells in 2× SDS sample buffer. The
indicated primary antibodies were incubated, washed, and visualized by
incubation with horseradish peroxidase-conjugated secondary antibodies
(Promega, Madison, WI) and ECL chemiluminescent reagents (Amersham
Pharmacia Biotech). Western blotting antibodies were obtained from the
following companies: Akt and p38 antibodies (New England Biolabs,
Beverly, MA); M2 FLAG epitope and Akt Stimulates the Transactivation Domain I of RelA/p65 by
Utilizing the I
Since Akt expression alone does not induce nuclear localization of
NF- IL-1
Since IKK IL-1 Akt Utilizes p38 and CBP/p300 for Efficient Stimulation of the
RelA/p65 Subunit of NF- The results presented here are consistent with the idea that
IL-1B and that this functions to block apoptosis induced by
certain stimuli. The mechanism whereby Akt activates NF-
B has been
controversial, with evidence supporting induction of nuclear
translocation of NF-
B via activation of I
B kinase activity and/or
the stimulation of the transcription function of NF-
B. Here we
demonstrate that Akt targets the transactivation function of NF-
B by
stimulating the transactivation domain of RelA/p65 in a manner that is
dependent on I
B kinase
activity and on the mitogen-activated
protein kinase p38 (p38). Activation of RelA/p65 transactivation
function requires serines 529 and 536, sites shown previously to be
inducibly phosphorylated. Consistent with the requirement of p38 in the activation of NF-
B transcriptional function, expression of activated Akt induces p38 activity. Furthermore, the ability of IL-1
to activate NF-
B is known to involve Akt, and we show here that IL-1
induces p38 activity in manner dependent on Akt and I
B kinase
activation. Interestingly, activated Akt and the transcriptional co-activators CBP/p300 synergize in the activation of the RelA/p65 transactivation domain, and this synergy is blocked by p38 inhibitors. These studies demonstrate that Akt, functioning through I
B kinase and p38, induces the transcription function of NF-
B by stimulating the RelA/p65 transactivation subunit of NF-
B.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B to provide cell survival
functions (7, 16-19).
B (NF-
B) is a heterodimer composed of the
p50 and the RelA/p65 subunits. NF-
B is activated by a variety of
stimuli including cytokines and oncoproteins (20, 21). In unstimulated
cells, the majority of NF-
B is found in the cytoplasm associated
with a family of inhibitory molecules known as the I
Bs. The
canonical NF-
B activation mechanism involves the phosphorylation of
I
B on two critical serine residues by the I
B kinase
(IKK)1 signalsome complex
(22-25). Phosphorylated I
B is then targeted for ubiquitination and
subsequent degradation by the 26 S proteosome, which allows liberated
NF-
B to translocate to the nucleus, where it activates transcription
of NF-
B-responsive genes (20, 21).
B has been highly
regarded as the principal method to activate
NF-
B-dependent gene expression, an alternate mechanism
of NF-
B activation is emerging that involves the phosphorylation of
the RelA/p65 transactivation subunit. For example, it has been shown
that the proinflammatory cytokines tumor necrosis factor and IL-1
lead to the phosphorylation of RelA/p65 and the subsequent stimulation
of NF-
B transactivation through pathways distinct from induced
nuclear translocation (18, 26, 27). The catalytic subunit of protein
kinase A (PKAc) has also been shown to phosphorylate RelA/p65, which
leads to the association of RelA/p65 with the CREB-binding protein/p300 (CBP/p300) transcriptional co-activator (28, 29). Recently, the
generation of GSK3 and T2K (TBK1) knockout mice has highlighted the
physiological importance of modulating transactivation functions of
NF-
B, because cells generated from these animals are capable of
inducing NF-
B nuclear translocation but are deficient in their ability to stimulate transactivation functions of NF-
B (30, 31). In
addition, evidence has been presented that the stress-activated kinase
p38 is involved in the regulation of NF-
B transcription function at
a level distinct from the induction of nuclear translocation (32, 33).
These studies indicate that dual controls exist for NF-
B with
mechanisms controlling induction of nuclear translocation of NF-
B as
well as regulating the inherent transcriptional activity of
NF-
B.
B activity
have indicated different mechanisms whereby this process may occur. Two
studies indicated that Akt, either in the context of tumor necrosis
factor signaling or in response to growth factor stimulation,
stimulated NF-
B nuclear translocation via the activation of the IKK
complex (16, 17). Another study indicated that Akt alone could not
induce nuclear translocation of NF-
B but synergized with PMA to
induce this response (19). Arguments against the involvement of Akt in
controlling nuclear accumulation of NF-
B have been recently reported
(34). Others, including ourselves, have provided evidence that Akt
signaling involves the stimulation of the transcription function of
NF-
B (7, 18). Thus, we showed that the ability of oncogenic Ras to
activate NF-
B transcriptional activity is dependent on Akt activity
(7). In a separate study, Sizemore et al. showed that
IL-1
induces phosphorylation of the RelA/p65 subunit in an
Akt-dependent manner and that IL-1
activated the
RelA/p65 transcriptional activation domain (18).
B requires
IKK and p38. Expression of activated Akt in IKK
null mouse embryo
fibroblasts (MEFs) significantly reduces transcriptional activity of
NF-
B. In addition, mutation of serine 529 and the IKK
phosphorylation site serine 536 (24, 35, 36) within the RelA/p65
transactivation domain, sites previously shown to be inducibly
phosphorylated, decreases the activation of NF-
B-mediated transcription in response to Akt. Since p38 has been determined to
activate NF-
B by targeting the transactivation function of NF-
B,
we examined the role of p38 in Akt-mediated NF-
B transactivation. Treatment of cells with IL-1
or expression of an activated form of
Akt stimulates the phosphorylation and activation of p38.
Interestingly, IL-1
-induced phosphorylation of p38 requires Akt and
IKK. Furthermore, inhibition of p38 kinase activity by the p38
inhibitor SB203580 significantly reduces Akt- and IL-1
-induced
NF-
B activation. The stimulation of the RelA/p65 transactivation
domain is most likely not due to a direct phosphorylation event on
RelA/p65 by p38 (32); rather, we provide evidence that p38 activates
RelA/p65 in response to Akt through cooperation with the CBP/p300
transcriptional co-activator. These studies define new mechanisms for
Akt-mediated NF-
B transactivation and demonstrate that Akt utilizes
IKK and the p38 MAPK to stimulate the transactivation potential of the RelA/p65 subunit of NF-
B.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Promega Corp., Madison, WI) or recombinant mouse IL-1
(Life
Technologies) was used at a concentration of 5 ng/ml.
B luciferase
reporter constructs contain 4
B DNA binding consensus sites from the
MHC class I promoter fused upstream to firefly luciferase. The Gal4
luciferase constructs (Gal4-Luc) contain five Gal4 DNA consensus
binding sites derived from the yeast GAL4 gene upstream of luciferase,
and Gal4-p65 constructs have the yeast Gal4 DNA binding domain fused to
the carboxyl-terminal transactivation domain of p65 (37). Activated Akt
as well as dominant negative constructs have been described previously
(38, 39). Wild type, dominant negative p38, MAPK kinase 6 (MKK6), and
IKK constructs have been described previously (25, 40). Mutagenesis was
performed per the manufacturer's suggestions (Stratagene, La Jolla,
CA). Briefly, oligonucleotides were made corresponding to the sequence
in RelA/p65 overlapping the serines 529 and 536. PCR was performed, and
products were digested with DpnI and subsequently transformed into the XL-1 Blue strain of Escherichia coli.
All mutants were verified by sequencing.
-galactosidase-expressing plasmid
(PCMV-LacZ) by a
-galactosidase colorimetric assay followed by
spectrophotometric quantitation (Promega, Madison, WI). In addition,
cells transfected with Akt, p38, or IKK mutant constructs were
co-transfected with pCMV-LacZ and assayed for transfection efficiency
and/or cell death by counting
-galactosidase-positive cells as
previously described (41).
-tubulin antibodies (Sigma), and
HA epitope antibody (Babco, Berkeley, CA). The p38 kinase assays were
performed as per the manufacturer's instructions (Cell Signaling
Technology, Inc., Beverly, MA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B Kinase
--
Various cellular stimuli can
activate NF-
B-dependent transcription, at least in part,
through mechanisms independent of signaling pathways that influence
nuclear translocation. These signaling pathways stimulate the
transactivation domain of the RelA/p65 subunit of NF-
B, presumably
by targeting basal or induced levels of NF-
B in the nucleus (7, 26).
Previous work in our laboratory has established that Akt activates
NF-
B by stimulating the carboxyl-terminal transactivation domain I
of RelA/p65. In addition, expression of a dominant negative mutant of
IKK
inhibited the ability of Akt to activate NF-
B (7). Our
results imply that Akt requires IKK to efficiently stimulate the
transactivation function of NF-
B. To further address this question,
we were interested in determining if Akt could activate
NF-
B-dependent transcription in a setting where
endogenous IKK is absent. The multisubunit IKK complex contains two
primary catalytic subunits, IKK
and IKK
, and each has been shown
to be involved in NF-
B activation at the level of nuclear accumulation (20). We utilized nullizygous IKK
and IKK
mouse embryo fibroblasts (MEFs) and performed transient transfection experiments with M-Akt or full-length wild type RelA/p65 and an NF-
B
reporter construct (3X-
B Luc). As seen in Fig.
1A, wild type RelA/p65
activated the NF-
B-responsive promoter equally in the wild type,
IKK
/
, or IKK
/
cells as expected. However, in response to
activated Akt, the IKK
/
cells were deficient in
NF-
B-dependent transcriptional activation, and,
furthermore, reintroduction of IKK
by transfection restored this
activity. Consistent with the role of Akt in regulating NF-
B
transcriptional activity, IKK
/
cells were inhibited in their
ability to activate a Gal4-dependent luciferase reporter
when transfected with activated Akt and a fusion (Gal4-p65) between the
Gal4 DNA binding domain and the transactivation domain I of RelA/p65
(data not shown). To determine if the reduction of NF-
B activity was
due to aberrant expression of these proteins, the extracts were
reanalyzed for M-Akt, IKK
, and IKK
expression by Western blot,
and both the IKK
/
and IKK
/
MEFs show equal levels of
M-Akt protein (data not shown). This result demonstrates that Akt
requires endogenous IKK
for efficient stimulation of
NF-
B-dependent transcription in a manner distinct from
the induction of nuclear translocation of NF-
B.
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Fig. 1.
Akt-mediated NF- B
activation requires IKK
and serine 536 of
RelA/p65. A, wild type, IKK
, and IKK
nullizygous
mouse embryo fibroblasts were transfected with 3X-
B luciferase,
vector control, M-Akt (2 µg each), p65 (100 ng), or wild type IKK
and IKK
(for reintroduction experiments). B, NIH 3T3
cells were transiently co-transfected with Gal4-Luc, Gal4-p65 mutants
S529A and S536A (100 ng each), and activated myristylated Akt (M-Akt)
or a vector control plasmid (VC) (2 µg each). 48 h
post-transfection, luciferase levels were assayed, normalized, and
expressed as -fold induction over the vector control ± S.D. of
three independent experiments.
B and because Akt requires IKK for activation of NF-
B (7, 16,
17, 19), we were interested in determining whether IKK utilizes a
specific site in RelA/p65 for Akt-mediated stimulation of RelA/p65.
Since IKK
has been reported to phosphorylate RelA/p65 on serine 536 in vitro and in vivo, it was important to
determine if this site is essential for Akt-mediated RelA/p65
transactivation (24, 36). To address this question, we utilized a
plasmid encoding the Gal4-p65 fusion protein, where sequences encoding the DNA-binding domain of Gal4 have been joined with sequences encoding the transactivation domain of RelA/p65 (37). This plasmid, when co-transfected with a Gal4-Luc reporter, allows us to determine whether cellular signals up-regulate gene expression by specifically targeting the transactivation domain of the RelA/p65 subunit of NF-
B. To examine the importance of the reported IKK
phosphorylation site, Gal4-p65 was mutated at serine 536 and for
control purposes at serine 529 (Gal4-p65 S536A and S529A). The Gal4-p65
S529A mutant was examined, because serine 529 has been previously shown
to be important for transactivation of NF-
B in response to tumor necrosis factor (26). NIH 3T3 cells were transiently co-transfected with Gal4-Luc, wild type Gal4-p65, Gal4-p65 S529A, or Gal4-p65 S536A in
the presence (M-Akt) or absence (VC) of activated Akt. As shown in Fig.
1B, M-Akt stimulates the transactivation function of
RelA/p65 but has little or no effect on gene expression when serine 529 or serine 536 are mutated to alanine. In addition, stimulation of cells
with IL-1
also required serines 529 and 536 for efficient activation
of NF-
B transactivation function (data not shown). These results
demonstrate that a known IKK
phosphorylation site, serine 536, and a
known tumor necrosis factor-inducible site, serine 529, are each
required for Akt to efficiently stimulate the transactivation domain of
the RelA/p65 subunit of NF-
B.
Requires Akt and IKK for Efficient Stimulation of p38
Activity--
Previous data from our laboratory and from others have
suggested that the stress-activated kinase p38 is required for
activation of NF-
B by expression of oncogenic Ras or by IL-1
treatment (33, 42). Additionally, p38 and IL-1
have been shown to
modulate the transactivation potential of NF-
B independent of
signals that induce nuclear accumulation and DNA binding (18, 33, 43).
Therefore, we were interested in determining if p38 was required by
IL-1
and Akt to modulate the transactivation potential of NF-
B,
since it has also been demonstrated that Akt modulates the
transactivation functions of NF-
B (7, 18). First, it was important
to determine if Akt can stimulate the phosphorylation of p38 and
therefore lead to its activation. To address this question, FLAG-tagged
wild type p38 (FLAG-p38) and activated Akt were transiently transfected
into human 293T cells. 48 h post-transfection, extracts were
prepared and analyzed for the presence of phosphorylated p38 using a
p38 phosphospecific antibody. As shown in Fig.
2A, activated Akt induces the
phosphorylation of FLAG-p38, suggesting that p38 is a downstream target
of Akt signaling. The activity of p38 in response to M-Akt was also
measured by p38 kinase assay, and, as shown in Fig. 2A,
M-Akt stimulated the activity of p38 to phosphorylate the transcription
factor ATF-2, a known p38 substrate. These extracts were also analyzed
for equivalent FLAG-p38 and
-tubulin (to control for loading)
expression (Fig. 2A, bottom panels).
Interestingly, IL-1
has been shown to activate both Akt and p38;
however, it has not been demonstrated that p38 is a target of
IL-1
-Akt signaling (18, 44). Fig. 2B demonstrates that
stimulation of cells with IL-1
leads to the activation of p38 as
previously reported (44). Interestingly, this activation requires
functional Akt because concomitant expression of a dominant negative
mutant of Akt (DN-Akt) effectively abolished the ability of IL-1
to
activate p38. To control for equivalent expression of FLAG-p38 and
DN-Akt, we reanalyzed these extracts with FLAG- and HA-specific
antibodies, respectively (Fig. 2B, bottom
panels). Taken as a whole, these results suggest that Akt
signals to activate p38 and that IL-1
requires Akt for efficient
stimulation of p38.
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Fig. 2.
IL-1 requires Akt
and IKK for efficient stimulation of p38 activity. A,
human 293T cells were transiently transfected with constructs encoding
VC, M-Akt, or FLAG-tagged p38 (FLAG-p38) (2 µg each). 48 h
post-transfection whole cell extracts were isolated and assayed for
phosphorylated p38 using a phosphospecific p38 antibody or p38 kinase
activity by assaying phosphorylated ATF-2. Extracts were reanalyzed for
equivalent expression of FLAG-p38 and
-tubulin for control purposes.
NS, nonspecific bands. B, 293T cells were
transfected with constructs encoding FLAG-p38 and/or a dominant
negative Akt mutant, Akt K179A (HA-DN-Akt) (2 µg each). 48 h
post-transfection, cells were stimulated with recombinant human IL-1
(5 ng/ml) for 30 min and assayed for p38 phosphorylation status.
Extracts were reanalyzed for expression of FLAG-p38 and HA-DN-Akt
(NS indicates nonspecific products). C, 293T
cells were transfected with vector control (VC), FLAG-p38,
DN-MKK6, or FLAG-DN-IKK
(FLAG-IKK
SS-AA) by the calcium phosphate
transfection protocol. 48 h post-transfection, cells were
stimulated with recombinant murine IL-1
(5 ng/ml) for 30 min and
assayed for p38 phosphorylation status. The arrows indicate
proteins detected, FLAG-p38 and
-tubulin as a loading control and
nonspecific products (NS). D, wild type
(WT) and IKK
(IKK
/
) and IKK
(IKK
/
) null
MEFs were plated in equal cell numbers, stimulated with IL-1
(5 ng/ml) for 30 min, and assayed for p38 phosphorylation status.
Bottom panel, blots were stripped and reprobed
for total endogenous p38. Results are representative of three
independent experiments.
is required for Akt-mediated NF-
B activity (Fig.
1A), we were also interested in determining if IL-1
stimulation of p38 required the IKK pathway. To address this question,
293T cells were transiently transfected with constructs encoding a vector control plasmid (VC), FLAG-p38, dominant negative MKK6 (DN-MKK6), and FLAG-tagged dominant negative IKK
(F-DN-IKK
(SS-AA)). The dominant negative IKK
construct affects both
endogenous IKK
and IKK
function, because the dominant negative
IKK
dimerizes with and inactivates both IKK subunits (23). 24 h
post-transfection, the cells were stimulated with IL-1
for 30 min
and assayed for p38 phosphorylation status. As shown in Fig.
2C, DN-MKK6 blocked the ability of IL-1
to stimulate p38
phosphorylation, as expected (lane 4).
Interestingly, expression of DN-IKK
also blocked IL-1
-mediated stimulation of p38 phosphorylation (lane 5). In
addition, Western analysis was performed to control for proper
expression of the transfected constructs and equal loading (Fig.
2C, bottom panels). However, these
results do not distinguish between the IKK subunits relative to which
one may be required for this process. To address this question, we
performed similar experiments utilizing the IKK
and IKK
null
MEFs. These cells were treated with IL-1
for 30 min and assayed for
phosphorylated endogenous p38 as described above. As shown in Fig.
2D, cells expressing wild type IKK
and IKK
(WT) and cells lacking the IKK
subunit (IKK
/
)
retain the ability of IL-1
to stimulate phosphorylation of p38
(lanes 1 and 2 and lanes
5 and 6), but interestingly, the IKK
null MEFs are defective in IL-1
-mediated p38 phosphorylation (lanes
3 and 4). This observation suggests that IL-1
signaling requires IKK
, but not IKK
, for stimulation of p38 and
potentially places p38 downstream of the IL-1
-Akt-IKK pathway (see
"Discussion").
and Akt Utilize the Mitogen-activated Protein Kinase p38
for Transactivation of NF-
B--
To test whether p38 mediates the
stimulation of NF-
B transcriptional activity by Akt and IL-1
, we
suppressed p38 activity in cells expressing M-Akt and determined the
effect on NF-
B transactivation. NIH3T3 cells were co-transfected
with plasmids encoding M-Akt, Gal4-Luc, and Gal4-p65 and then were
treated with the pharmacological inhibitor of p38 (SB203580), or, for
control purposes, the pharmacological inhibitor of the MEK pathway
(PD98059) was added for 24 h. Fig. 3A shows that in the presence
of the p38 inhibitor, the ability of Akt to activate Gal4-p65 was
reduced, while the MEK inhibitor showed only a marginal reduction in
Gal4-Luc activity. In addition, Fig. 3B shows that IL-1
stimulation of 293T cells similarly showed a reduction in NF-
B
transactivation potential in the presence of SB203580. The SB203580
compound has been shown to inactivate additional MAPK signaling
pathways and has been suggested to inhibit Akt kinase activity at high
concentrations (45). The effects of the p38 inhibitor shown in Fig. 3,
A and B, are unlikely to be due to reduced Akt
kinase activity or other nonspecific effects, since the concentration
of SB203580 used in our experiments (600 nM) is
considerably lower than reported concentrations (3-5 µM) affecting Akt or other signaling pathways (45). Additionally, analysis
of extracts from cells treated with SB203580 or PD98059 shows that
these inhibitors do not affect M-Akt expression levels as determined by
Western blot (Fig. 3A, bottom panel).
Nevertheless, additional experiments were performed to confirm the
results obtained with the SB203580 compound. Constructs encoding a
dominant negative mutant of p38 (DN p38) were transiently transfected
into NIH 3T3 cells with activated Akt and assayed for 3X-
B Luc and
Gal4-Luc/Gal4-p65 activity. The dominant inhibitory mutant of p38
blocked the ability of Akt to stimulate NF-
B dependent transcription
and showed a similar inhibition on Gal4-Luc/Gal4-p65 activity
corroborating the effects seen with the SB203580 compound (Fig.
4, A and B). Importantly, these effects were not due to aberrant expression of M-Akt
or p38 as assayed by Western blot (Fig. 4C). These results taken in whole demonstrate that IL-1
requires Akt and IKK to stimulate p38 and that p38 activity is required for IL-1
and Akt to
activate the transcription function of NF-
B. Interestingly, the
activation of p38 alone is apparently insufficient for the activation
of p65 transactivation function, since MKK3, an upstream inducer of p38
function, was unable to activate Gal4-p65 (data not shown).
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Fig. 3.
Akt requires the p38 MAPK for efficient
stimulation of NF- B transactivation.
A, NIH 3T3 cells were transiently transfected with plasmids
encoding Gal4-Luc and Gal4-p65 (100 ng each) and VC or M-Akt (2 µg
each). 24 h post-transfection, SB203580 (600 nM) and
PD98059 (2 µM) were added for an additional 24 h.
B, human 293T cells were transiently transfected with
plasmids encoding Gal4-Luc and Gal4-p65 (1 µg each). 24 h
post-transfection, SB203580 (600 nM) and PD98059 (2 µM) were pretreated for 90 min followed by human
recombinant IL-1
(5 ng/ml) or vehicle (NA) for an
additional 20 h. Luciferase levels were assayed, normalized, and
expressed as -fold induction over vector control ± S.D. of three
experiments.
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Fig. 4.
Dominant negative p38 mutants block
Akt-mediated NF- B transactivation.
A, NIH 3T3 cells were transiently transfected with plasmids
encoding 3X-
B Luc (0.5 µg) or VC or M-Akt (2 µg each).
B, NIH 3T3 cells were transiently transfected with plasmids
encoding Gal4-Luc and Gal4-p65 (100 ng each) and VC, M-Akt, or dominant
negative p38 (DN-p38) (2 µg each). 48 h post-transfection, whole
cell extracts were isolated. Luciferase was normalized to total
protein, and
-galatosidase staining was performed to ensure equal
transfection efficiency and to ensure equal cell viability between
conditions. Data is presented as -fold activation, where the values
obtained for the vector control group were normalized to 1. Results
represent the mean ± S.D. of three independent experiments.
C, whole cell extracts were reanalyzed for equivalent
protein expression by Western blot. Activated HA-tagged Akt (HA
M-Akt), FLAG-tagged DN p38, and
-tubulin-specific antibodies
were used as described under "Experimental Procedures."
B--
The mechanism of p38-mediated NF-
B
activation is most likely not directly at the level of NF-
B, because
p38 does not activate RelA/p65 by a direct phosphorylation event (32).
In addition, RelA/p65 does not contain a consensus MAPK/p38
phosphorylation site.2
However, NF-
B interacts with the basal transcription machinery and
requires co-activators for efficient stimulation of transcriptional activity (46, 47). One co-activator that is essential for NF-
B-dependent transcription is CBP/p300 (28).
Therefore, we hypothesized that in response to Akt, p38 may utilize
CBP/p300 to stimulate NF-
B transcription. To answer this question,
constructs encoding activated Akt, CBP, or p300 were transiently
co-transfected into NIH 3T3 cells along with Gal4-Luc and Gal4-p65. As
shown in Fig. 5A, CBP and p300
synergistically activate RelA/p65 in conjunction with activated Akt.
Importantly, these effects were not due to elevated levels of HA-M-Akt
in the transfections (Fig. 5A, bottom
panel). Interestingly, as shown in Fig. 5B, the
SB203580 compound blocked this synergy by approximately 2-fold,
consistent with the data presented in Fig. 3A. This effect
is specific to p38, since the MEK inhibitor PD98059 does not
appreciably reduce this synergy (Fig. 5B). Together, these
data support the notion that Akt activates p38 to indirectly stimulate
the transactivation domain I of the RelA/p65 subunit of NF-
B through
a functional interaction with the co-activator CBP/p300.
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Fig. 5.
Akt and p38 require the co-activator CBP/p300
for efficient stimulation of the RelA/p65 transactivation domain.
A, NIH 3T3 cells were transiently co-transfected with
constructs encoding Gal4-Luc and Gal4-p65 (100 ng of each) and VC,
M-Akt, CBP, or p300 (2 µg of each). B, NIH 3T3 cells were
transiently transfected with plasmids encoding Gal4-Luc and Gal4-p65
(100 ng each) and VC, M-Akt, or CPB (2 µg of each). 24 h
post-transfection, SB203580 (600 nM), PD98059 (2 µM), or Me2SO (DMSO; vehicle
control) were added for an additional 24 h. Luciferase levels were
assayed, normalized, and expressed as -fold induction over vector
control ± S.D. of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Akt activate NF-
B by stimulating the transactivation domain I of RelA/p65. In Fig. 6, we
present a model to explain how Akt transcriptionally activates the
RelA/p65 subunit of NF-
B. We propose two mechanisms for Akt-mediated
NF-
B activation: 1) Akt expression alone utilizes IKK
in a
p38-dependent manner, which requires serines 529 and 536 of
RelA/p65 to directly stimulate NF-
B activity, and 2) Akt signaling
in response to IL-1 exposure stimulates NF-
B by activating p38 in a
manner dependent on IKK
. It is presently unclear why different
Akt-dependent events differentially target the two forms of
IKK. Additionally, how Akt may activate p38 is presently unclear, but
it may involve utilization of the p38-activating kinases known as the
MKKs, or the activation of p38 by Akt may involve
MKK-independent mechanisms. In addition, Ras expression or integrin
ligation of cells has been shown to activate p38 by a linear pathway
requiring Rac, Pak, and MKK3 (48, 49). Interestingly, Ras-induced
activation of Pak required Akt kinase activity, indicating that Akt may
be a Pak kinase leading to p38 phosphorylation. However, in a
separate study, IL-1 stimulation of cells activated p38 by a
Ras-dependent, but Rac-independent mechanism, indicating
that IL-1 stimulation may not require the Rac/MKK pathway (50). Recent
data suggest that MKK6 interacts with IKK
, suggesting that p38 may
be activated by the MKK6·IKK
complex (51). However, our
results presented here demonstrate that IKK
is dispensable for the
ability of IL-1
to activate p38. IL-1 signaling is known to involve
many pathways in addition to the described Akt-IKK pathway, and this
report highlights the complexity of IL-1 signaling. This observation
and the data presented in Fig. 2, C and D,
suggest that p38 lies downstream of IKK activity. However, these
observations do not rule out the possibility that IKK and p38 are on
separate but parallel pathways for NF-
B activation and that the
inhibition of IKK inhibits a parallel but not upstream event required
for p38 induction. Regulation of p38 activation is complex, and the
mechanisms whereby Akt and IL-1 regulate this kinase require further
investigation.
View larger version (17K):
[in a new window]
Fig. 6.
Model:
IL-1 /Akt stimulates the transactivation
potential of RelA/p65 by targeting IKK and p38. In this model, we
propose that 1) Akt expression alone utilizes IKK
in a
p38-dependent manner that requires serines 529 and 536 of
RelA/p65 to directly stimulate NF-
B activity and 2) Akt signaling in
response to IL-1 exposure stimulates NF-
B by activating p38 in a
manner dependent on IKK
.
In contrast to the canonical role of IKK to induce phosphorylation
of I
B and to subsequently induce nuclear translocation of NF-
B,
we find that IKK
can also modulate the transactivation function of
NF-
B in response to Akt. The function of IKK
in the activation of
NF-
B by Akt expression alone is probably associated with activation
of NF-
B transcriptional function and may not be associated with its
well defined role in transient NF-
B activation. However, we cannot
rule out the possibility that Akt functions to induce nuclear
translocation in response to certain physiological stimuli.
Additionally, our findings provide a function for the direct
phosphorylation event seen on RelA/p65 by IKK
that has been
previously reported (24, 36), and they likely explain, at least partly,
previous reports showing that Akt utilizes IKK for activation of
NF-
B.
Our findings also demonstrate that p38 is activated by Akt to utilize
the CBP/p300 co-activator for transactivation of NF-B. The p38
pathway is well established in stimulating the transactivation properties of NF-
B independent of signals that induce the nuclear accumulation and DNA binding. Our work suggests that p38 utilizes co-activators to stimulate NF-
B, and it corroborates previously published reports demonstrating that p38 modulates NF-
B by an indirect mechanism (32, 42). Together, these findings represent novel
functions associated with IKK
, IKK
, and p38 in IL-1
- and
Akt-mediated NF-
B activation.
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ACKNOWLEDGEMENTS |
---|
We thank Phillip Hawkins (The Babraham Institute, Cambridge, United Kingdom) for providing dominant negative Akt constructs, Anke Klippel (Chiron Corp., Emeryville, CA) for providing the other phosphatidylinositol 3-kinase and Akt constructs, Michael Karin (University of California San Diego) for providing IKK constructs and IKK nullizygous MEFs, Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Worchester, MA) for providing the p38 constructs, and Jiahuai Han (The Scripps Research Institute, La Jolla, CA) for MKK6 constructs used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported by NCI, National Institutes of Health, Grants K01 78595 (to M. W. M.); CA72771, AI35098, and CA73756 (to A. S. B.); CA75080 (to A. S. B. and M. W. M.); and predoctoral training grant T32GM07092 (to L. V. M.).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.
To whom correspondence should be addressed. Tel.:
919-966-3652; Fax: 919-966-8212; E-mail: jhall@med.unc.edu.
Present Address: Dept. of Biochemistry and Molecular Genetics,
University of Virginia, Charlottesville, VA 22908.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M101103200
2 L. V. Madrid and A. S. Baldwin Jr., unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
IKK, IB kinase;
IL, interleukin;
CBP, CREB-binding protein;
MEF, mouse embryo
fibroblasts;
MAPK, mitogen-activated protein kinase;
MKK, MAPK kinase;
MEK, mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase;
HA, hemagglutinin.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Downward, J. (1998) Curr. Opin. Cell Biol. 10, 262-267[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Datta, S. R.,
Brunet, A.,
and Greenberg, M. E.
(1999)
Genes Dev.
13,
2905-2927 |
3. |
Aoki, M.,
Batista, O.,
Bellacosa, A.,
Tsichlis, P.,
and Vogt, P. K.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14950-14955 |
4. |
Cheng, J. Q.,
Ruggeri, B.,
Klein, W. M.,
Sonoda, G.,
Altomare, D. A.,
Watson, D. K.,
and Testa, J. R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
3636-3641 |
5. | Cheng, J. Q., Godwin, A. K., Bellacosa, A., Taguchi, T., Franke, T. F., Hamilton, T. C., Tsichlis, P. N., and Testa, J. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9267-9271[Abstract] |
6. |
Datta, K.,
Bellacosa, A.,
Chan, T. O.,
and Tsichlis, P. N.
(1996)
J. Biol. Chem.
271,
30835-30839 |
7. |
Madrid, L. V.,
Wang, C. Y.,
Guttridge, D. C.,
Schottelius, A. J.,
Baldwin, A. S., Jr.,
and Mayo, M. W.
(2000)
Mol. Cell. Biol.
20,
1626-1638 |
8. |
Zhou, B. P.,
Hu, M. C.,
Miller, S. A., Yu, Z.,
Xia, W.,
Lin, S. Y.,
and Hung, M. C.
(2000)
J. Biol. Chem.
275,
8027-8031 |
9. | Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve] |
10. |
Cardone, M. H.,
Roy, N.,
Stennicke, H. R.,
Salvesen, G. S.,
Franke, T. F.,
Stanbridge, E.,
Frisch, S.,
and Reed, J. C.
(1998)
Science
282,
1318-1321 |
11. |
del Peso, L.,
Gonzalez-Garcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689 |
12. |
Dudek, H.,
Datta, S. R.,
Franke, T. F.,
Birnbaum, M. J.,
Yao, R.,
Cooper, G. M.,
Segal, R. A.,
Kaplan, D. R.,
and Greenberg, M. E.
(1997)
Science
275,
661-665 |
13. | Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857-868[Medline] [Order article via Infotrieve] |
14. | Kops, G. J., de Ruiter, N. D., De Vries-Smits, A. M., Powell, D. R., Bos, J. L., and Burgering, B. M. (1999) Nature 398, 630-634[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Tang, E. D.,
Nuñez, G.,
Barr, F. G.,
and Guan, K. L.
(1999)
J. Biol. Chem.
274,
16741-16746 |
16. | Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D. B. (1999) Nature 401, 82-85[CrossRef][Medline] [Order article via Infotrieve] |
17. | Romashkova, J. A., and Makarov, S. S. (1999) Nature 401, 86-90[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Sizemore, N.,
Leung, S.,
and Stark, G. R.
(1999)
Mol. Cell. Biol.
19,
4798-4805 |
19. | Kane, L. P., Shapiro, V. S., Stokoe, D., and Weiss, A. (1999) Curr. Biol. 9, 601-604[CrossRef][Medline] [Order article via Infotrieve] |
20. | Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve] |
21. | Baldwin, A. S. (1996) Annu. Rev. Immunol. 14, 649-681[CrossRef][Medline] [Order article via Infotrieve] |
22. | DiDonato, J., Mercurio, F., Rosette, C., Wu-Li, J., Suyang, H., Ghosh, S., and Karin, M. (1996) Mol. Cell. Biol. 16, 1295-1304[Abstract] |
23. |
Zandi, E.,
and Karin, M.
(1999)
Mol. Cell. Biol.
19,
4547-4551 |
24. |
Mercurio, F.,
Zhu, H.,
Murray, B. W.,
Shevchenko, A.,
Bennett, B. L.,
Li, J.,
Young, D. B.,
Barbosa, M.,
Mann, M.,
Manning, A.,
and Rao, A.
(1997)
Science
278,
860-866 |
25. | Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve] |
26. |
Wang, D.,
and Baldwin, A. S., Jr.
(1998)
J. Biol. Chem.
273,
29411-29416 |
27. |
Bird, T. A.,
Schooley, K.,
Dower, S. K.,
Hagen, H.,
and Virca, G. D.
(1997)
J. Biol. Chem.
272,
32606-32612 |
28. | Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell 1, 661-671[Medline] [Order article via Infotrieve] |
29. | Zhong, H., SuYang, H., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1997) Cell 89, 413-424[Medline] [Order article via Infotrieve] |
30. | Hoeflich, K. P., Luo, J., Rubie, E. A., Tsao, M. S., Jin, O., and Woodgett, J. R. (2000) Nature 406, 86-90[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Bonnard, M.,
Mirtsos, C.,
Suzuki, S.,
Graham, K.,
Huang, J.,
Ng, M.,
Itie, A.,
Wakeham, A.,
Shahinian, A.,
Henzel, W. J.,
Elia, A. J.,
Shillinglaw, W.,
Mak, T. W.,
Cao, Z.,
and Yeh, W. C.
(2000)
EMBO J.
19,
4976-4985 |
32. |
Wesselborg, S.,
Bauer, M. K. A.,
Vogt, M.,
Schmitz, M. L.,
and Schulze-Osthoff, K.
(1997)
J. Biol. Chem.
272,
12422-12429 |
33. |
Bergmann, M.,
Hart, L.,
Lindsay, M.,
Barnes, P. J.,
and Newton, R.
(1998)
J. Biol. Chem.
273,
6607-6610 |
34. | Delhase, M., Li, N., and Karin, M. (2000) Nature 406, 367-368[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Wang, D.,
Westerheide, S. D.,
Hanson, J. L.,
and Baldwin, A. S., Jr.
(2000)
J. Biol. Chem.
275,
32592-32597 |
36. |
Sakurai, H.,
Chiba, H.,
Miyoshi, H.,
Sugita, T.,
and Toriumi, W.
(1999)
J. Biol. Chem.
274,
30353-30356 |
37. | Schmitz, M. L., and Baeuerle, P. A. (1991) EMBO J. 10, 3805-3817[Abstract] |
38. | Klippel, A., Kavanaugh, W. M., Pot, D., and Williams, L. T. (1997) Mol. Cell. Biol. 17, 338-344[Abstract] |
39. |
Welch, H.,
Eguinoa, A.,
Stephens, L. R.,
and Hawkins, P. T.
(1998)
J. Biol. Chem.
273,
11248-11256 |
40. |
Enslen, H.,
Raingeaud, J.,
and Davis, R. J.
(1998)
J. Biol. Chem.
273,
1741-1748 |
41. |
Mayo, M. W.,
Wang, C. Y.,
Cogswell, P. C.,
Rogers-Graham, K. S.,
Lowe, S. W.,
Der, C. J.,
and Baldwin, A. S., Jr.
(1997)
Science
278,
1812-1815 |
42. |
Norris, J. L.,
and Baldwin, A. S., Jr.
(1999)
J. Biol. Chem.
274,
13841-13846 |
43. |
Vanden Berghe, W.,
Plaisance, S.,
Boone, E.,
De Bosscher, K.,
Schmitz, M. L.,
Fiers, W.,
and Haegeman, G.
(1998)
J. Biol. Chem.
273,
3285-3290 |
44. |
Raingeaud, J.,
Gupta, S.,
Rogers, J. S.,
Dickens, M.,
Han, J.,
Ulevitch, R. J.,
and Davis, R. J.
(1995)
J. Biol. Chem.
270,
7420-7426 |
45. |
Lali, F. V.,
Hunt, A. E.,
Turner, S. J.,
and Foxwell, B. M.
(2000)
J. Biol. Chem.
275,
7395-7402 |
46. |
Carter, A. B.,
Knudtson, K. L.,
Monick, M. M.,
and Hunninghake, G. W.
(1999)
J. Biol. Chem.
274,
30858-30863 |
47. |
Sheppard, K. A.,
Rose, D. W.,
Haque, Z. K.,
Kurokawa, R.,
McInerney, E.,
Westin, S.,
Thanos, D.,
Rosenfeld, M. G.,
Glass, C. K.,
and Collins, T.
(1999)
Mol. Cell. Biol.
19,
6367-6378 |
48. |
Tang, Y.,
Zhou, H.,
Chen, A.,
Pittman, R. N.,
and Field, J.
(2000)
J. Biol. Chem.
275,
9106-9109 |
49. | Mainiero, F., Soriani, A., Strippoli, R., Jacobelli, J., Gismondi, A., Piccoli, M., Frati, L., and Santoni, A. (2000) Immunity 12, 7-16[Medline] [Order article via Infotrieve] |
50. |
Palsson, E. M.,
Popoff, M.,
Thelestam, M.,
and O'Neill, L. A.
(2000)
J. Biol. Chem.
275,
7818-7825 |
51. |
Craig, R.,
Larkin, A.,
Mingo, A. M.,
Thuerauf, D. J.,
Andrews, C.,
McDonough, P. M.,
and Glembotski, C. C.
(2000)
J. Biol. Chem.
275,
23814-23824 |