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INTRODUCTION |
The transcription factor
NF
B1 is activated by
interleukin-1 (IL-1), tumor necrosis factor (TNF), and a variety of
other stress-inducing stimuli (1-3). In addition to its role in
inflammation, NF
B has also been implicated in cellular survival,
transformation, and oncogenesis (1-3). Predominantly a heterodimeric
complex of two polypeptides (p65/RelA and p50), NF
B is physically
confined to the cytoplasm through its interactions with inhibitors
belonging to the I
B family of proteins (1-3). When phosphorylated
on serine 32 and serine 36, I
B
is marked and degraded by the
ubiquitin/26 S proteasome pathway liberating the NF
B heterodimer so
that it may translocate to the nucleus. The signaling cascade
that induces I
B degradation and thus leads to NF
B activation has
recently been delineated (3). There is compelling evidence that
phosphorylation of the regulatory serines on I
B
is mediated by a
300-500-kDa multisubunit I
B protein kinase (IKK) (4-10). This
kinase complex was purified to apparent homogeneity and shown to be
composed primarily of the protein kinases IKK
and IKK
as well as
a protein that lacks a catalytic kinase domain known as IKK
(4-10).
The phosphorylation and degradation of I
B may not, however, be
sufficient to activate NF
B. Using two different phosphoinositide 3-kinase (PI 3-kinase) inhibitors, we have previously shown that the PI
3-kinase signaling pathway is also required for NF
B activation (11).
Whereas wortmannin efficiently blocked IL-1-induced increases in the
DNA binding activity of NF
B, a dominant-negative mutant of the p85
regulatory subunit of PI 3-kinase inhibited the ability of IL-1 to
induce an NF
B-dependent reporter gene (11). More recently, Marmiroli et al. (12) have shown that tyrosine 479 on the type I IL-1 receptor (IL-1RI) is required for receptor interaction with PI 3-kinase. When tyrosine 479 was replaced with phenylalanine, the mutant IL-1RI lost its ability to interact with PI
3-kinase and was deficient in signaling for the activation of both PI
3-kinase and NF
B.
Our recent studies have shown that TNF-induced NF
B activation also
requires PI 3-kinase and that, when inhibited, PI 3-kinase potentiates
TNF-induced apoptosis (13). Consistent with a role for PI 3-kinase in
NF
B activation and the antiapoptotic properties of NF
B, p65/RelA
protected cells from apoptosis induced by TNF in combination with
wortmannin (13). Various other studies have shown the
involvement of PI 3-kinase and/or its mediator Akt kinase in NF
B
activation induced by IL-1, TNF, phorbol myristate acetate, platelet-derived growth factor, bradykinin, hypoxia, oncogenic ras, and SV40 small t antigen (14-21). The precise
role of PI 3-kinase in NF
B activation is, however, still uncertain
as there is evidence in support of (15, 16) and against (14, 20) a role
for it in I
B degradation and NF
B nuclear translocation. Evidence has been presented that, rather than being involved in DNA binding, PI
3-kinase and Akt are instead critical for the transcriptional activity of NF
B (p65/RelA) (14). The reasons for the discrepancies between the various studies are not clear, but they could be related to
the inhibitors used and/or the molecular characteristics of the cell
lines employed.
Although these studies have implicated the PI 3-kinase/Akt pathway in
NF
B activation, much ambiguity about its role remains. Indeed, there
is a need for approaches that would more clearly reveal the function of
PI 3-kinase in NF
B activation. One approach would be to analyze mice
with targeted deficiencies in the relevant individual molecules, and
another would be to study somatic cell lines with lesions in the PI
3-kinase/Akt pathway. In this study, we utilized a glioma cell
line that lacks MMAC/PTEN, a natural antagonist of the PI 3-kinase/Akt
pathway, to investigate the function of PI 3-kinase in cytokine-induced
NF
B activation. The lipid products of PI 3-kinase that are critical
for the activation of downstream protein kinases such as Akt are
specifically dephosphorylated at the 3'-OH position by the lipid
phosphatase activity of MMAC/PTEN (22, 23). This function of MMAC/PTEN,
which appears to be responsible for its tumor suppressor properties, is
important for regulating PI 3-kinase activity in vivo (24,
25). The use of MMAC/PTEN as a specific inhibitor could therefore be
advantageous for studying the role of PI 3-kinase in NF
B activation.
Our results indicate that PI 3-kinase is involved in the regulation of
DNA binding activity and trans-activation potential of NF
B through a
phosphorylation-dependent mechanism that is parallel to but distinct from the I
B degradation pathway.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Reagents--
U251 human glioblastoma cells
(ATCC) were cultured in Dulbecco's modified Eagle's medium/F12
medium containing 5% fetal bovine serum and antibiotics in a
humidified atmosphere containing 5% CO2 at 37 °C.
Antibodies were obtained from either Santa Cruz Biotechnology, Santa
Cruz, CA (MMAC/PTEN, p65/RelA, Jun N-terminal kinase (JNK), and
I
B
antibodies), New England Biolabs, Beverly, MA
(anti-phospho-Akt/protein kinase B serine 473), or Imgenex, San
Diego, CA (anti-IKK
). LY294002 was obtained from Biomol Research Laboratories Inc., Plymouth Meeting, PA.
Retrovirus Gene Construction--
pLNCX retroviral vector
(CLONTECH) derived from Moloney murine leukemia
virus was utilized for retroviral gene delivery and expression.
A full-length MMAC/PTEN retroviral construct was generated by ligating
a NotI-SalI fragment from pBluescript-MMAC/PTEN
into the multiple cloning site of pLNCX.
Stable Expression of MMAC/PTEN in the U251 Glioma Cell
Line--
PT67 retrovirus producer cells were grown in Dulbecco's
modified Eagle's medium/F12 containing 10% fetal calf serum, 1000 units/ml penicillin-streptomycin, and 2 mM glutamine and
transfected with the wild-type MMAC/PTEN construct by calcium phosphate
precipitation. The human glioma cell line U251 was infected with 48-h
supernatants from the transfected PT67 cells. After 14 h of
incubation, infected cells were selected with 400 µg/ml G418.
Drug-resistant colonies were expanded to generate clonal cell lines and
screened for MMAC/PTEN expression by immunoblotting.
Immunoblotting--
Cells were washed with ice-cold
phosphate-buffered saline and lysed in a buffer containing 50 mM HEPES, pH 7.5, 1.5 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 20 mM NaF, 10 mM Na4P2O7 (sodium
pyrophosphate), 10% glycerol, 1% Triton X-100, 3 mM
benzamidine, 1 mM Na3VO4
(sodium orthovanadate), 1 µM pepstatin, 10 µg/ml
aprotinin, 5 mM iodoacetic acid, and 2 µg/ml leupeptin to
prepare whole-cell lysates. Lysates were clarified by centrifugation at
14,000 × g for 5 min. Proteins were resolved by
SDS-PAGE and electroblotted to polyvinylidene difluoride
membranes (Millipore), and then they were probed with various primary
antibodies (MMAC/PTEN, I
B, IKK, and phospho-Akt). For the
detection of p65/RelA, nuclear extracts were used instead of whole-cell
lysates. Specific proteins were detected by chemiluminescence (ECL)
(Amersham Pharmacia Biotech) following incubation with
horseradish peroxidase-conjugated secondary antibodies.
Electrophoretic Mobility Shift Assay--
Parental U251 or
MMAC/PTEN-expressing U251(MMAC) cells were treated with IL-1 (1 nM) or TNF (1 nM) for various periods of time.
2.5 µg of nuclear extracts that were prepared as described previously
(11) were incubated for 15 min at room temperature with radiolabeled
NF
B-binding probe. For supershift assays, anti-p65/RelA antibody or
IgG was added to the incubation mixtures for 5 min before the
addition of the radiolabeled probe. Where indicated, IL-1-treated U251
nuclear extracts were incubated with 10 units/ml of the catalytic
subunit of protein phosphatase 2A (homogeneity determined by silver
staining, a gift of Dr. Zahi Damuni) for 10 min at 37 °C before
incubation with the radiolabeled probe. The protein-DNA complexes were
resolved on 5% nondenaturing polyacrylamide gels and visualized by autoradiography.
Reporter Assays--
U251 and U251(MMAC) cells were plated in
6-well tissue culture plates and transfected the following day with an
(NF
B)4/luciferase reporter along with an I
B
expression plasmid or an empty vector control using the
FuGeneTM reagent (Roche Molecular Biochemicals) according
to the manufacturer's protocol. After 24 h, cells were treated
with IL-1, lysed, and assayed with the enhanced luciferase assay kit
(PharMingen). HEK 293 and Hep3B cells were treated in an identical
manner except that the (NF
B)4/luciferase reporter was
cotransfected with empty pCMV-Flag2 vector or with
pCMV-Flag2-MMAC/PTEN.
Immunoprecipitations--
Whole-cell lysates were prepared as
described earlier and incubated for 1 h with appropriate
antibodies to immunoprecipitate either Akt or phospho-Akt (serine 473).
Immune complexes were precipitated with a 50% slurry of protein
A-Sepharose beads (Pierce), washed, and eluted by boiling in SDS sample
buffer. Eluted proteins were then resolved by SDS-PAGE and probed by
Western blotting analysis with anti-IKK
or anti-I
B
antibodies.
Proteins were visualized by ECL (Amersham Pharmacia Biotech).
JNK Assay--
Whole-cell lysates prepared from IL-1-treated
cells were incubated with anti-JNK antibodies. Immune complexes were
washed extensively with lysis buffer and assayed for JNK activity with 2 µg of GST-c-Jun-(1-79) as substrate. Assay mixtures, which
included 0.2 mM [
-32P]ATP and 10 mM MgCl2, were incubated for 5 min at 30 °C,
after which reactions were stopped by adding SDS sample buffer. Protein phosphorylation was visualized by autoradiography.
In Vivo Labeling of Cells--
U251 or U251(MMAC) cells were
seeded in 100-mm culture dishes and incubated overnight in serum- and
phosphate-free medium. On the following day, cells were washed and
radiolabeled for 3 h in
[32P]orthophosphate-containing medium (0.1 mCi/ml). After
treatment with IL-1 for 30 min, whole-cell lysates were prepared and
clarified by centrifugation. To extract protein from any intact nuclei
that remained in the insoluble fraction, we incubated the pellets with buffer containing 0.4 M NaCl (11) and mixed this extract
with whole-cell lysate. Equal amounts of protein from the mixture were used for the immunoprecipitation of NF
B with anti-p50 antibodies. After coupling to protein A-Sepharose beads, immune complexes were
washed several times and resolved by SDS-PAGE. Radiolabeled protein
bands were visualized by autoradiography.
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RESULTS |
Stable Expression of MMAC/PTEN in U251 Glioma Cells Inhibits Serum
and IL-1-induced Akt Phosphorylation--
MMAC/PTEN is frequently
mutated or deleted in a wide variety of human cancers (26, 27). We
examined various cell lines for MMAC/PTEN expression to identify those
cells that might be suitable for studying the role of PI 3-kinase in
NF
B activation. Among others, the U251 glioma cell line
lacked MMAC/PTEN expression and was highly responsive to IL-1 and TNF
stimulation. These cells were previously reported to have a mutated
MMAC/PTEN gene (27). Both IL-1 and TNF were able to stimulate the
activation of PI 3-kinase and NF
B very potently in U251 cells (data
not shown) with kinetics that were similar to those induced by them in
other cell lines (11, 13). We proceeded to generate stable
MMAC/PTEN-expressing clones by infecting parental U251 cells with
supernatants from retrovirus producer cells transfected with wild-type
MMAC/PTEN. Drug-resistant U251(MMAC), but not parental U251 cells,
expressed MMAC/PTEN as confirmed by immunoblotting analysis (Fig.
1).

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Fig. 1.
MMAC blocks Akt phosphorylation in U251
glioma cells. U251 and U251(MMAC) cells were serum-starved for
24 h before treatment with either serum (10%) or IL-1 (1 nM) for 15 min. Cells were then lysed, and whole-cell
extracts were analyzed by Western blotting with anti-phospho-Akt
(serine 473), anti-MMAC, and anti-Akt antibodies. Specific bands were
detected by ECL (Amersham Pharmacia Biotech).
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The lipid products of PI 3-kinase are known to target the
serine/threonine protein kinase Akt to the plasma membrane, where it is
fully activated through phosphorylation on serine 473 and threonine 308 (28-30). MMAC/PTEN dephosphorylates the lipid products of PI
3-kinase at the 3'-OH position and prevents the phosphorylation and
activation of Akt (22, 24). We therefore examined U251 and U251(MMAC)
cells to compare and contrast the phosphorylation status of Akt. The
basal levels of phosphorylated Akt, which were clearly detectable in
U251 cells, were significantly higher than those in the
MMAC/PTEN-expressing cell line (Fig. 1). In addition, whereas serum-
and IL-1-inducible levels of Akt phosphorylation were profound in U251
cells, they were barely detectable in the U251(MMAC) cells (Fig. 1).
Because MMAC/PTEN did not affect the protein levels of Akt (Fig. 1),
its effect on Akt phosphorylation is presumably attributable to its
lipid phosphatase function in the PI 3-kinase pathway as demonstrated
previously (for example, see Refs. 24, 31, 32). We conclude that when
MMAC/PTEN is reintroduced into U251 cells, it is able to block
IL-1-induced phosphorylation and activation of Akt through the
inhibition of PI 3-kinase-generated signals.
MMAC/PTEN Inhibits IL-1- and TNF-induced NF
B
Activation--
Both PI 3-kinase and Akt have been shown to be
required for the activation of NF
B (11-21). Because IL-1- induced
Akt phosphorylation was efficiently inhibited in U251(MMAC) cells, we
investigated whether MMAC/PTEN expression would also affect NF
B
activation. Both IL-1 and TNF strongly induced the DNA binding activity
of NF
B (30-120 min) in parental U251 cells as determined by gel shift assays (Fig. 2). Cytokine-inducible
DNA-protein complexes contained NF
B (p65/p50 heterodimer) because
they could be supershifted with anti-p65/RelA antibodies but not by
nonspecific IgG. The ability of IL-1 and TNF to activate NF
B was,
however, inhibited in U251(MMAC) cells, suggesting that MMAC/PTEN had
the potential to regulate the DNA binding activity of NF
B (Fig. 2).
It is interesting to note that the cytokine-mediated induction of a
second DNA-protein band, which was probably the p50/p50 homodimeric
complex of NF
B, was also inhibited in U251(MMAC) cells (Fig.
2).

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Fig. 2.
MMAC inhibits cytokine-induced DNA binding
activity of NF B. U251 and U251(MMAC)
cells were treated with IL-1 (A) or TNF (B) for
various periods of time before harvesting. Nuclear extracts were
prepared and incubated for 15 min at room temperature with a
radiolabeled probe that contained an NF B binding site. For
supershift assays, antibodies were added to incubation mixtures before
the probe was added. Protein·DNA complexes were resolved on 5%
polyacrylamide gels and visualized by autoradiography. NF B refers to
the p65/p50 heterodimer. These data are representative of three
independent experiments.
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To confirm that MMAC/PTEN inhibits cytokine-induced NF
B activation,
we examined its effects on the induction of an NF
B/luciferase reporter gene. The expression of this gene, which could be strongly induced (~10-fold) in IL-1-treated U251 cells (Fig.
3A), required NF
B
activation because it was inhibited in the presence of the I
B
inhibitor. Consistent with the gel mobility shift assays (Fig. 2A), IL-1-induced expression of the NF
B/luciferase
reporter gene was inhibited in U251(MMAC) cells (Fig. 3A).
Furthermore, transient transfection of MMAC/PTEN into human embryonic
kidney 293 and hepatoma Hep3B cells also sufficed to inhibit
NF
B-dependent gene expression (Fig. 3B).
Taken together, these results would strongly support a role for the PI
3-kinase pathway in the DNA binding and transcriptional activities of
NF
B.

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Fig. 3.
IL-1-induced expression of an
NF B/luciferase reporter is inhibited by
MMAC. A, NF B activation is inhibited in U251(MMAC)
cells. An (NF B)4/luciferase reporter gene was
cotransfected into U251 or U251(MMAC) cells with empty vector or I B
expression plasmid by the FuGeneTM method (Roche Molecular
Biochemicals). Transfected cells were left untreated or incubated with
IL-1 for 16 h before lysis. Cell lysates were assayed for
luciferase activity. All activities were normalized to untreated
controls. B, transient expression of MMAC inhibits NF B
activation in 293 and Hep3B cells. The NF B/luc reporter was
cotransfected into HEK 293 or Hep3B cells with a control or
with pCMV-Flag2-MMAC vector. Cells were then treated with IL-1 as
described above before extracts were prepared to assay for luciferase
expression. luc, luciferase.
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PI 3-Kinase Is Not Required for IL-1-induced JNK
Activation--
Because expression of MMAC/PTEN in U251 cells blocked
the activation of both Akt and NF
B (Figs. 1-3), we investigated
whether it generally inhibited various other IL-1-induced signals as
well. To evaluate this possibility, we examined the effects of
MMAC/PTEN on the ability of IL-1 to stimulate the serine/threonine
protein kinase JNK. JNK was immunoprecipitated from IL-1-treated
U251 and U251(MMAC) cell extracts and assayed for activity with
GST-Jun as the substrate (Fig. 4). The
stimulation of JNK activity was observed within 15 min of treatment of
U251 cells with IL-1 and persisted for about 2 h. Interestingly,
the pattern of activation and the level of induction of JNK in these
cells were similar to those observed in the U251(MMAC) cells (Fig. 4).
It is therefore unlikely that MMAC/PTEN and, by inference, the PI
3-kinase pathway are involved in regulating JNK activation in IL-1
signaling.

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Fig. 4.
JNK activity is induced by IL-1 through a PI
3-kinase-independent mechanism. Anti-JNK antibodies were used to
immunoprecipitate JNK from whole-cell extracts that were prepared from
IL-1-treated U251 and U251(MMAC) cells. Immune complexes were incubated
with GST-Jun in protein kinase assays for 5 min. Phosphorylated
protein was resolved by SDS-PAGE and visualized by
autoradiography.
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In a manner similar to IL-1, TNF was also able to stimulate JNK
activity equally well in both U251 and U251(MMAC) cells (data not
shown). Consistent with these results, two other PI 3-kinase inhibitors, wortmannin and a dominant-negative mutant of PI 3-kinase (p85DN), did not affect cytokine-induced JNK activation either (data
not shown). PI 3-kinase-independent pathways have previously been shown
to be involved in the activation of JNK, such as in platelet-derived
growth factor signal transduction (33). On analysis, these
results would therefore strongly argue that the inhibitory effects of
MMAC/PTEN on Akt and NF
B activation are relatively specific.
MMAC/PTEN Does Not Interfere with IL-1-induced I
B
Degradation
or the Nuclear Translocation of p65/RelA--
The site-specific
phosphorylation of I
B
induces its degradation and facilitates the
nuclear translocation of the p65/p50 heterodimer. Because IL-1- and
TNF-induced NF
B activation was inhibited in U251(MMAC) cells (Figs.
2 and 3), we investigated whether this occurred because
MMAC/PTEN interfered with either of these obligatory steps in NF
B
activation. To our surprise immunoblotting analysis revealed that IL-1
induced the degradation of I
B efficiently and with nearly identical
kinetics in both U251 and U251(MMAC) cells (Fig.
5A). Furthermore, NF
B
(p65/RelA) was found to translocate normally to the nucleus after
cytokine treatment with no apparent differences between parental and
MMAC/PTEN-expressing cells (Fig. 5B). Similar results were
observed when the PI 3-kinase inhibitors wortmannin and LY294002 were
employed (data not shown). These data would suggest that MMAC/PTEN does
not regulate any of the steps that lead to I
B degradation and NF
B
nuclear translocation, and this indicates that the PI 3-kinase
pathway modulates the DNA binding activity of NF
B through an
alternative mechanism.

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Fig. 5.
PI 3-kinase and Akt are
not required for I B degradation and
NF B nuclear translocation. Whole-cell or
nuclear extracts were prepared from IL-1-treated U251 and U251(MMAC)
cells. Whole-cell extracts were analyzed by Western blotting with
anti-I B antibodies (A), whereas nuclear extracts were
probed with anti-p65/RelA (B) antibodies. Specific bands
were detected by ECL (Amersham Pharmacia Biotech).
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Physical Interaction of Akt with I
B
and IKK--
Our results
suggest that the PI 3-kinase/Akt pathway regulates the DNA binding
activity of NF
B. To identify the underlying mechanisms, we first
tested the possibility that the signaling proteins of the PI 3-kinase
pathway might physically interact with proteins that are known to
function in IL-1-induced NF
B activation. Physical interactions
between Akt and IKK were recently reported to be important in TNF- and
platelet-derived growth factor-induced NF
B activation (15, 17). We
therefore used specific anti-Akt antibodies to immunoprecipitate Akt
and any interacting proteins from IL-1-treated U251 and U251(MMAC) cell
extracts. Western blotting analysis of the immune complexes from
IL-1-treated U251 cell extracts showed that I
B
coprecipitated
with Akt and suggested that the two proteins physically interact with
each other (Fig. 6A).
Consistent with its degradation in stimulated cells, little or no
I
B
was detectable in complex with Akt about 15 min after
treatment with IL-1. I
B
did, however, reappear in Akt immune
complexes from U251 cells after about 60 min of IL-1 treatment. In
addition to I
B
, the I
B kinase IKK
was also detectable in
Akt immune complexes, and interactions between the three proteins were
observed even when anti-phospho-Akt (serine 473) antibodies were used
for immunoprecipitation (Fig. 6B). The association of Akt
with I
B
and IKK
was found to be inducible in a time- and
IL-1-dependent manner (Fig. 6B).

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Fig. 6.
Physical interaction of Akt with
I B and IKK.
Anti-Akt (A) or anti-phospho-Akt (Ser-473)
(B) antibodies were used to immunoprecipitate Akt
from IL-1-treated U251 and U251(MMAC) cells. Immune complexes were
analyzed by Western blotting with anti-I B or anti-IKK
antibodies. C, U251 cells were left untreated or stimulated
with IL-1 for 5 or 15 min before immunoprecipitation with
anti-phospho-Akt antibodies. In parallel, one set of cells was
preincubated with the PI 3-kinase inhibitor LY294002 (10 µM) for 30 min before treatment with IL-1 for 5 min. Akt
immune complexes were then analyzed by immunoblotting for the presence
of I B .
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Interestingly, physical interactions between Akt and I
B
(Fig. 6,
A and B) or Akt and IKK
(Fig. 6B)
were inhibited in the U251(MMAC) cells. Because IL-1-induced Akt
phosphorylation was also strongly inhibited in these cells (Fig. 1), it
is possible that phosphorylation of Akt is required for its
interactions with I
B
and IKK
. Consistent with this
possibility, IL-1-induced interactions between Akt and I
B
were
also inhibited in U251 cells that were pretreated with the PI 3-kinase
inhibitor LY294002 (Fig. 6C).
IL-1-induced Phosphorylation of NF
B Might Be Required for Its
DNA Binding Activity and Can Be Inhibited by MMAC/PTEN--
The p50
and p65/RelA subunits of NF
B have been shown to be phosphorylated
(34-38). Because our results suggested that PI 3-kinase regulates the
DNA binding activity of NF
B without involving the I
B degradation
pathway, we assessed the possibility that the underlying mechanism
could involve NF
B phosphorylation. IL-1-treated U251 nuclear
extracts were incubated with near homogeneous preparations (purity
determined by silver staining, data not shown) of the serine/threonine
protein phosphatase 2A (PP2A). Phosphatase treatment of nuclear
extracts resulted in a significant reduction of IL-1-induced DNA
binding activity of NF
B (Fig.
7A), as determined by gel
mobility shift assays. Immunoblotting analyses confirmed that the
PP2A preparations did not contain any I
B and that the inhibitory
effect of PP2A was not attributable to any degradation of the NF
B
proteins (data not shown). The effect of PP2A on NF
B is therefore
similar to that observed with alkaline phosphatase (23), and it
supports a role for phosphorylation in the DNA binding activity of
NF
B.

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Fig. 7.
Involvement of NF B
phosphorylation in DNA binding activity. A, nuclear
extracts prepared from IL-1-treated U251 cells were incubated with or
without PP2A for 10 min before incubation with the radiolabeled NF B
probe. Protein·DNA complexes were resolved by electrophoresis on
nondenaturing polyacrylamide gels. B, radiolabeled U251 and
U251(MMAC) cells were treated with IL-1 for the indicated times. p50
NF B was immunoprecipitated from cell extracts with specific
antibodies and resolved by SDS-PAGE. Protein bands were detected by
autoradiography.
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It is possible that MMAC/PTEN inhibits the DNA binding activity of
NF
B by interfering with the steps that lead to NF
B
phosphorylation. To assess this possibility, we examined the effect of
MMAC/PTEN on the in vivo phosphorylation of p50, which is
primarily responsible for the DNA binding activity of NF
B. IL-1 was
found to strongly induce the phosphorylation of a 50-kDa polypeptide
immunoprecipitated from radiolabeled U251 cells with anti-p50
antibodies (Fig. 7B). The phosphorylated 50-kDa band was
judged to be p50/NF
B based on Western blotting analyses, in
parallel, of immune complexes from unlabeled cells and through the use
of a nonspecific control antibody for immunoprecipitation (data not
shown). Although still detectable, phosphorylation of p50 NF
B
was significantly inhibited in U251(MMAC) cells. This observation would
support the possibility that MMAC inhibits the DNA binding activity of
NF
B by interfering with its phosphorylation on specific
IL-1-inducible sites.
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DISCUSSION |
We have investigated the role of PI 3-kinase in cytokine-induced
NF
B activation. Our results show that both IL-1- and TNF-induced DNA
binding and the transcriptional activities of NF
B were potently inhibited in a glioma cell line that stably expressed MMAC/PTEN. The
inhibition of NF
B activation was deemed to be relatively specific
because MMAC/PTEN did not interfere with other IL-1-induced signals,
such as those that lead to I
B
degradation, NF
B nuclear translocation, or JNK activation. Consistent with its role as a PI
3-kinase antagonist, MMAC/PTEN inhibited the ability of IL-1 to induce
Akt phosphorylation in the stable cell line. These observations would
suggest that the PI 3-kinase pathway is essential for the activation of
NF
B. Various PI 3-kinase inhibitors such as wortmannin, LY294002,
and dominant-negative mutants of PI 3-kinase and/or Akt have been used
previously to implicate PI 3-kinase in NF
B activation (11, 14, 15,
17, 20).
Whereas PI 3-kinase and Akt are required for NF
B activation, they
induce NF
B-dependent reporter gene expression
poorly when compared with IL-1 or TNF (11, 13, 14). Our previous
studies showed, however, that NF
B-dependent gene
expression was synergistically activated when PI
3-kinase-overexpressing cells were stimulated with IL-1 or TNF (11,
13). We had therefore suggested that PI 3-kinase must synergize with
other IL-1- or TNF-inducible signals to activate NF
B (11, 13). The
results presented in this report and elsewhere (14) indicate that PI
3-kinase does not participate in the pathway that leads to I
B
degradation, which may explain its failure to activate NF
B by
itself. Furthermore, the synergism between PI 3-kinase and IL-1 or TNF
for the induction of NF
B-dependent gene expression could
be attributable to the ability of PI 3-kinase-generated signals to
cooperate with the I
B degradation pathway. A mechanism of this type,
involving the convergence of two or more signals, is unlikely to be
involved in the IL-1 signaling pathway for the activation of other
transcription factors such as AP-1 (11).
Using two different assays, we have shown that MMAC/PTEN can prevent
IL-1 and TNF from activating NF
B. Whereas gel mobility shift assays
revealed that MMAC/PTEN blocks IL-1- and TNF-induced increases in the
DNA binding activity of NF
B, transient transfection assays showed
that MMAC/PTEN inhibited NF
B-dependent gene expression. Because the expression of the reporter gene used in our transient transfection experiments is driven by the NF
B-binding consensus sequences, it cannot be trans-activated in the absence of NF
B or
when NF
B binds poorly. Indeed, a mutant reporter gene that did not
bind NF
B was unresponsive to IL-1 in our previous studies (11). We
are, therefore, unable to evaluate those mechanisms that are sensitive
to inhibition by MMAC/PTEN but that regulate only the trans-activation
potential of NF
B. The phosphorylation of serine 529 on p65/RelA is
an example of a mechanism that exclusively regulates the
transcriptional activity of NF
B (36).
Although MMAC/PTEN inhibited the cytokine-induced activation of NF
B
in our studies, it did not interfere with the degradation of I
B nor
with the nuclear translocation of p65/RelA, which are two obligatory
steps in NF
B activation. These results would underscore the
insufficiency of the I
B degradation pathway and indicate that
additional PI 3-kinase-dependent signals are required for the ability of NF
B to bind DNA and to trans-activate genes. The identity of such signals, which cooperate with the I
B
degradation pathway for NF
B activation, is not entirely clear. There
is evidence to suggest, however, that PI 3-kinase-mediated signals
might induce site-specific phosphorylation of the p65 and/or p50
NF
B proteins. First, IL-1 and TNF have both been shown to induce the
phosphorylation of NF
B (14, 35, 36). Second, the IL-1-induced
phosphorylation of p65/RelA and the expression of an
NF
B-dependent reporter gene are both inhibited by the PI
3-kinase inhibitor LY294002 (14). Third, the mutation of serine
residues 276 and 529, which are inducibly phosphorylated on p65/RelA,
caused the inhibition of NF
B-dependent gene expression
(36-38). Whereas serine 529 was shown to be required only for the
transcriptional activity of NF
B, the phosphorylation of serine 276 by protein kinase A greatly enhanced the DNA binding affinity of NF
B
in in vitro experiments (38). The phosphorylation of serine
276 also facilitates the physical interaction of p65/RelA with
CREB-binding protein and constitutes a mechanism by which the
phosphorylation of NF
B regulates transcriptional activity (38).
In this study, the treatment of nuclear extracts from IL-1-treated
cells with the serine/threonine protein phosphatase PP2A drastically
reduced the DNA binding activity of NF
B. A similar effect was
reported by Naumann and Scheidereit (35), who showed that alkaline
phosphatase treatment abolished the DNA binding activity of NF
B.
Because recombinant NF
B is capable of binding DNA without
modification (for example, see Ref. 38), phosphorylation might serve to
enhance the affinity of NF
B for the binding site. Indeed, the
phosphorylation of serine 276 greatly increases the binding affinity of
NF
B for DNA (38). The incomplete inhibition that we observed in
cytokine-induced U251(MMAC) cells (Fig. 2) might therefore be
reflective of the DNA binding activity of NF
B in its
unphosphorylated or hypophosphorylated state.
The possibility that a phosphorylation-dependent mechanism
is induced by PI 3-kinase/Akt to regulate NF
B activation is
therefore well supported and deserves further investigation. We have
shown that the IL-1-induced phosphorylation of p50 NF
B was inhibited in MMAC/PTEN-expressing cells. Because LY294002 inhibits the
phosphorylation of p65/RelA (14), our data would suggest that the PI
3-kinase pathway induces the phosphorylation of both subunits of
NF
B. Although our studies have suggested that the phosphorylation of p50 is involved in the DNA binding activity of NF
B, a supporting role cannot be ruled out for p65/RelA phosphorylation. So far, however, the evidence has linked p65/RelA phosphorylation only to the
transcriptional activity of NF
B (14, 20, 36, 37). The logical next
step would be to identify the phosphorylation sites on p65/p50 that are
regulated by PI 3-kinase and that are involved in the DNA binding and
transcriptional activities of NF
B.
The PI 3-kinase/Akt-mediated signals that could lead to NF
B
phosphorylation have not been identified so far. Two different studies have recently shown that Akt interacts with IKK. Ozes et al. (15) have noted that whereas the interaction of Akt
with IKK is constitutive, the phosphorylation of Akt in IKK
immunoprecipitates increases with TNF stimulation. Romashkova and
Makarov (17) have also reported interactions between Akt and IKK,
although only in cells stimulated with platelet-derived growth factor. Akt phosphorylates threonine 23 on IKK
in vitro, and when
overexpressed, an IKK
mutant with alanine at position 23 was able to
inhibit the TNF-induced DNA binding activity of NF
B (15). The
interaction of IKK with Akt might be important for its activation (17). Our coimmunoprecipitation studies revealed that phosphorylated Akt
physically interacted with I
B as well as with IKK and that these
interactions were inhibited by MMAC/PTEN. It remains to be determined
if Akt directly interacts with either protein and whether these
interactions are critical for NF
B phosphorylation and activation.
Targeted gene disruption studies have shown that IKK
, but not
IKK
, is largely responsible for cytokine-induced I
B degradation and for the nuclear translocation of NF
B (39-42). However, when fibroblasts from mice lacking the IKK
gene were stimulated with IL-1
or TNF, there was a significant decrease in the DNA binding activity of
NF
B and in the ability of TNF to induce IL-6 and macrophage
colony-stimulating factor mRNA (42). These observations raise the
interesting possibility that whereas IKK
is dispensable for the
I
B degradation pathway, it might be required at another regulatory
step in NF
B activation, possibly in cooperation with Akt.
In addition to providing clues about the mechanisms that might be
induced by PI 3-kinase/Akt for NF
B activation, these studies have
implications for understanding how tumor suppressors, such as
MMAC/PTEN, and oncogenes, such as Akt or PI 3-kinase, play a decisive
role in cellular survival and oncogenesis.