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INTRODUCTION |
The engagement of the epidermal growth factor receptor by its
cognate ligand results in the generation of a number of intracellular signals (1). The initial changes induced by ligand binding are receptor
dimerization, activation of the kinase activity of the receptor, and
autophosphorylation of the receptor on tyrosine residues (2, 3).
Autophosphorylation of the receptor results in the creation of docking
sites for a number of secondary signaling proteins bearing specific
protein interaction domains such as the Src homology 2 domain, which
interact specifically with phosphorylated tyrosine residues (4). As a
consequence of this interaction, these secondary signaling proteins may
themselves become activated and trigger a number of downstream signals.
These signaling cascades result in the activation of a number of
transcription factors such as AP-1 and STATS (5).
The NF-
B1 family of
transcription factors plays an important role in inflammatory responses
(6). A diverse number of stimuli including cytokines such as TNF
and
IL-1, UV irradiation, and lipopolysaccharide are known to activate
NF-
B. In unstimulated cells NF-
B is sequestered in the cytoplasm
by the I
B family of proteins (7). Binding of I
B to NF-
B masks
nuclear localization signals on NF-
B and prevents its translocation
to the nucleus (8). Stimulation of cells with a diverse array of
stimuli results in phosphorylation of I
B
on both serines 32 and
36. This results in the ubiquitination and degradation of I
B
,
allowing NF-
B to translocate to the nucleus and activate
transcription (9-12). Considerable progress has been made in our
understanding of how the TNF receptor activates NF-
B. The 55-kDa
TNFR1 is thought to be the more important receptor type in the
activation of NF-
B. Binding of TNF to the TNFR1 results in
trimerization of the receptor and the recruitment of adaptor proteins
such as TRADD to the receptor (13). TRADD in turn recruits receptor
interacting protein (RIP) and TRAF2 to the receptor (14, 15). TRAF2
binds to the NF-
B-inducing kinase (NIK) (16). This results in the
activation of NIK, although the mechanism of this activation remains
unclear. NIK phosphorylates and activates the IKKs (17), which appear
to exist in a large multiprotein complex termed the signalosome (18,
19). The IKKs in turn phosphorylate I
B
on serines 32 and 36. NIK
appears to selectively target IKK
(20, 21), whereas MEKK1 and
atypical protein kinase Cs may activate both IKK
and IKK
(19,
22).
Protein kinase B/Akt also appears to be involved in NF-
B activation
induced by PDGF and TNF
. Activation of Akt requires the
phosphorylation of Akt on residues Ser-473 and Thr-308 (23). Akt is
activated by both TNF
and PDGF. Recent studies have indicated NF-
B activation induced by these cytokines is inhibited by both wortmannin (a PI 3-kinase inhibitor) and by a kinase inactive Akt
mutant, while expression of a constitutively activated myristylated Akt
mutant was sufficient for NF-
B activation (24, 25). Akt has been
shown to enhance the degradation of I
Bs (26). Furthermore, activated
Akt can associate with the IKK complex and may activate IKK
by
phosphorylating it at Thr-23 (24, 25).
The epidermal growth factor receptor has also been shown to activate
NF-
B. EGF has been shown to activate NF-
B in smooth muscle cells,
in A431 cells, in fibroblasts, and in several estrogen receptor-negative EGF-overexpressing breast cancer cell lines (27-29).
A previous study has shown that EGF stimulation of A431 cells leads to
degradation of I
B
(28). This study also showed that wild type
I
B
transfected into COS cells undergoes degradation upon
treatment with EGF, whereas an I
B
S32/36 mutant fails to undergo
degradation, suggesting that activation of the EGFR leads to
phosphorylation of I
B
on serines 32 and 36 and subsequent activation of NF-
B in a manner similar to the TNF receptor. A requirement for intracellular free calcium was also noted in this study. However, the proximal signals generated by the EGFR that lead to
the activation of NF-
B remain unknown.
In this study we present evidence showing that the EGFR activates
NF-
B by mechanisms similar to but not identical with the TNF
receptor. We identify components of the signalosome involved in
EGFR-induced NF-
B activation and suggest a mechanism of NF-
B activation at the level of protein-protein interaction. We show that
the EGFR-induced NF-
B activation involves some of the proteins previously identified as key proximal elements in the TNFR pathway of
NF-
B activation, but unlike for the TNFR we find that the protein
kinase Akt is not involved in EGFR-mediated activation of NF-
B in
the cell types we have studied.
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EXPERIMENTAL PROCEDURES |
Cell Lines, Reagents, and Transfection--
MDA-MB-468 and MCF7
cells were obtained from ATCC. R1hER cells were obtained from Dr.
Michael Weber (University of Virginia, Charlottesville, VA).
293-EBNA cells were obtained from Invitrogen. A RIP-Myc construct was
obtained from Dr. Brian Seed (Massachusetts General Hospital, Boston,
MA) and cloned into pcDNA 3.1 vector (Invitrogen) using standard
molecular techniques. Wild type pFLAG-NIK and NIK-AA were obtained from
Dr. Joe DiDonato (Cleveland Clinic Foundation, Cleveland, OH). A
Myc-TRADD construct was obtained from Dr. David Goeddel (Tularik Inc.,
South San Francisco, CA, Tularik, CA). An anti-phospho-ERK antibody was
obtained from Promega (catalog no. V6671). EGFR (sc-03), Myc (9E10
sc-40), I
B
(sc-203), Akt (sc-8312), and ERK2 (sc-154) antibodies
were obtained from Santa Cruz Biotechnology. Antibodies against p65
(sc-372X), p50 (sc-114X), c-Rel (sc-70X), and Rel B (sc-226) were also
obtained from Santa Cruz Biotechnology. Anti-RIP antibodies were
obtained from PharMingen (65591A). Anti-phospho-Akt antibodies (Ser-473 (06-801) and Thr-308 (06-678)) were obtained from Upstate Biotechnology Inc. Wortmannin was purchased from Calbiochem. Poly(dI-dC) was obtained
from Amersham Pharmacia Biotech.
Transfections were done using the calcium phosphate technique using
standard protocols, and expression of transfected genes was confirmed
by Western blotting. For transient transfection experiments, cells were
harvested 24-48 h after transfection. For stable transfection, an
HA-tagged I
B
S32/36 mutant cloned into pcDNA 3.1 or the empty
vector were transfected into MDA-MB-468 cells and mass populations of
zeocin-resistant cells were screened by Western blotting.
Western Blotting and Immunoprecipitation--
Standard protocols
were used for immunoprecipitation and Western blotting (30, 31).
Quantitation of proteins was performed by using a Bio-Rad
detergent-compatible protein assay kit. For immunoprecipitation, cells
were lysed in a modified radioimmune precipitation buffer (150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate, 1 mM EGTA, 1 mM NaF, 50 mM Tris-HCl,
1 mM phenylmethylsulfonyl fluoride, and 2 mM
orthovanadate), and equal amounts of protein were incubated with the
primary antibody for 90 min. Protein A-agarose or Protein G-agarose
beads were subsequently added to the lysates and incubated overnight at
4 °C. The beads were subsequently washed and solubilized in SDS
sample buffer and then boiled and analyzed by SDS-polyacrylamide gel
electrophoresis, followed by transfer to nitrocellulose. Western blots
were developed with ECL reagents (Amersham Pharmacia Biotech). In
experiments where cell lysates were examined directly, cells were lysed
in SDS sample buffer, boiled, and subjected to SDS-polyacrylamide gel
electrophoresis followed by Western blotting.
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared as previously described (32). Double-stranded oligonucleotide
containing the consensus sequence for binding of NF-
B was filled by
the Klenow fragment with [
-32P]dCTP and the three
other nonradiolabeled dNTPs. The sequence of the probe is as below.
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For binding reactions, nuclear extracts (5-10 µg of protein)
were incubated with 1 µg of poly(dI-dC) in 10 mM Tris-HCl
buffer (pH 7.5) containing 0.1 mM EDTA, 3 mM
dithiothreitol, 100 mM KCl, and 1% glycerol for 15 min at
room temperature. The 32P-labeled oligonucleotide probe
(20,000 cpm) was then added and incubated for 30 min at room
temperature. In competition assays, excess oligonucleotide competitor
was preincubated with nuclear extracts for 15 min at room temperature.
In supershift assays, antibodies were added 30 min before
32P-labeled oligonucleotide. At the end of the incubation,
10× DNA loading buffer was added and the sample was electrophoresed in a native 4.5% polyacrylamide gel at a constant voltage (10 V/cm). Gels
were dried and exposed to x-ray film with intensifying screens overnight at
80 °C.
Luciferase Assays--
1 × 105 R1hER cells
were transfected using the calcium phosphate method. A dual-luciferase
reporter assay system was used according to the instructions of the
manufacturer (Promega). 30 ng of a pRL vector was cotransfected along
with 300 ng of a SV40
B-luc reporter plasmid (42) and 4 µg of
either empty vector or a dominant negative NIK mutant. Firefly
luciferase activity was measured in a luminometer and normalized on the
basis of Renilla luciferase activity.
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RESULTS |
A High Level of EGF Receptor Expression Is Optimal for EGF-induced
NF-
B Activation--
It has been reported that EGF induces NF-
B
in aortic smooth muscle cells (27), in A431 cells, transiently in mouse
embryo fibroblasts (28), and in several breast cancer cell lines (29), but not in human omental microvascular endothelial cells (33). The
activation of NF-
B in the various cell types may be a consequence of
high level of EGF receptor expression. To determine whether the level
of EGF receptor expression influences the activation of NF-
B, we
used Rat-1 fibroblasts and compared them to R1hER cells, which are
Rat-1 fibroblasts expressing a high level of the EGFR. Electrophoretic
mobility shift assays showed that EGF stimulation failed to activate
NF-
B in Rat-1 cells (Fig.
1A, lanes
1 and 2). However, exposure of R1hER cells to EGF
resulted in a robust activation of NF-
B (Fig. 1A,
lanes 3 and 4). Furthermore, in R1hER
cells we also detected constitutive activation of NF-
B compared with
untransfected Rat-1 cells. R1hER cells express about 7.5 × 105 EGF receptors, which is a nearly 7-fold increase over
untransfected cells (34). There is constitutive phosphorylation of the
EGFR in R1hER cells, suggesting that the receptor is active in these cells even in the absence of ligand. Interestingly, the increase in the
level of EGF receptor expression leads to a selective rather than a
general amplification of intracellular signaling pathways. For example,
ERK activation in response to EGF (100 ng/ml) was not increased in
R1hER cells compared with Rat-1 cells (Fig. 1D). This
observation also holds true at lower EGF concentrations (ranging from
0.1 to 1 ng/ml), which result in submaximal levels of ERK activation in
both cell lines (data not shown). We did not detect an increase in ERK
activation in R1hER cells compared with Rat-1 cells at any EGF
concentration tested.

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Fig. 1.
EGF-induced activation of
NF- B is shown in panels
A-C. Cultures of MDA-MB-468 cells, MCF7 cells,
Rat-1 fibroblasts, or R1hER cells growing in 100-mm tissue culture
dishes were serum-starved overnight prior to the addition of EGF (100 ng/ml) for 1 h. Nuclear extracts were prepared, and NF- B
binding activities were examined by EMSA as described under
"Experimental Procedures." A shows NF- B activation in
Rat-1 fibroblasts (lanes 1 and 2) and
R1hER cells (lanes 3 and 4).
B shows NF- B activation in MCF7 cells (lanes
1 and 2) and MDA-MB-468 cells, (lanes
3 and 4). Lanes 2 and
4 show the effect of EGF stimulation (+). C shows
a supershift assay in MDA-MB-468 cells demonstrating that p50 and p65
are the major subunits of NF- B activated by EGF. Lane
1, control; lane 2, antibodies against p65;
lane 3, p50; lane 4, c-Rel;
lane 5, Rel B. Lanes 6 (5-fold) and
7 (25-fold) show the effects of excess unlabeled nucleotide.
D, EGF-induced ERK2 activation is similar in Rat-1 and R1hER
despite the higher level of expression of the EGFR expression in R1hER
cells, suggesting that high levels of EGFR expression result in a
selective rather that a general amplification of signals. The
upper panel shows immunoblotting with a
phospho-ERK2 antibody. The blot was stripped and reprobed with an ERK2
antibody to show protein loading (lower
panel).
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To determine whether the relationship between EGFR levels and NF-
B
activation was a consistent finding between cell types, we studied
EGF-mediated NF-
B activation in two breast cancer cell lines
expressing different levels of the EGFR. The MDA-MB-468 cell lines
expresses a high level of EGF receptors (1-3 × 106),
while the MCF7 cell line expresses a lower level of EGFR, about 2.4 × 103 (35). EGF stimulation failed to induce a
significant activation of NF-
B in MCF7 cells, whereas a robust
activation of NF-
B was observed in MDA-MB-468 cells (Fig.
1B). On the other hand, activation of ERKs in response to
EGF was similar in MCF7 and MDA-MB-468 cells (data not shown). Thus,
our data suggest that, in the cell types used, a high level of EGFR
expression is optimal for the activation of NF-
B.
The NF-
B family is composed of several subunits including, p50, p65,
c-Rel, and Rel B. Supershift experiments were performed on MDA-MB-468
cells to identify which NF-
B subunits were induced by EGF. In
agreement with previous studies (28), we found that NF-
B in
MDA-MB-468 cells stimulated with EGF consists of p65 and p50 (Fig.
1C).
Expression of a Dominant Negative I
B
S32/36 Mutant Blocks
EGFR-induced NF-
B Activation--
NF-
B is regulated by its
interaction with a group of cytoplasmic inhibitory proteins termed
I
B. The major species of this family of proteins is designated
I
B
. Phosphorylation of I
B
on serines 32 and 36 targets it
for degradation via the ubiquitin-proteasome pathway. This releases
NF-
B, which translocates to the nucleus and activates transcription.
It has been shown previously that EGF stimulation of cells results in
the degradation of I
B
. To test whether an I
B
mutant that
cannot be phosphorylated at Ser-32/36 would block EGFR-induced NF-
B
activation, we stably transfected MDA-MB-468 cells with this mutant in
a plasmid expression vector. Mass populations of zeocin-resistant cells
were pooled and tested for expression of the HA-tagged I
B
mutant
by Western blot analysis (Fig.
2A). Having established that
these cells expressed the transfected mutant, electrophoretic mobility
shift assays were performed and compared with MDA-MB-468 cells
expressing the empty vector. As can be seen in Fig. 2B,
expression of a dominant negative I
B
mutant blocks EGFR-induced
NF-
B activation, demonstrating that the EGFR utilizes elements of
the canonical pathway of NF-
B activation.

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Fig. 2.
The effect of stable expression of
I B M on EGFR-induced
NF- B activation. A shows
stable expression of HA-tagged I B with serines 32 and 36 mutated
to alanine in MDA-MB-468 cells (lane 2) but not
in cells transfected with vector alone (lane 1).
The upper arrow points to HA-tagged I B
mutant while the lower arrow points to endogenous
I B . B shows that induction of basal as well as
EGF-mediated NF- B induction is blocked in MDA-MB-468 cells
transfected with I B mutant but not in vector-transfected
cells.
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The EGFR Associates with RIP but Not TRADD--
The activation of
NF-
B by TNF is mediated by a specific signalosome composed of
distinct proteins. TRADD is an adaptor protein, which associates with
the TNFR when the receptor is activated (13). It has a domain of about
80 amino acids in the carboxyl terminus, which is termed the death
domain. The death domain of TRADD binds to a similar death domain on
the TNFR. TRADD then recruits RIP and also TRAF2 to the TNFR (14, 15).
RIP may also bind directly to the TNFR, but this has been reported to be a weak interaction. To identify the proteins constituting the receptor tyrosine kinase signalosome at the EGF receptor, we
transfected EGFR cDNA in a plasmid expression vector along with
Myc-tagged TRADD into 293 EBNA cells to determine whether the two
proteins would associate into a physical complex. Cells transfected
with the two plasmids were subjected to immunoprecipitation with an EGFR antibody followed by immunoblotting with a Myc antibody. We failed
to detect Myc-TRADD in EGFR immunoprecipitates in repeated experiments
(Fig. 3A). We also failed to
detect an interaction between the EGFR and Fas-associated death
domain protein (FADD), another protein that interacts with the TNFR and
Fas receptors and is involved in apoptosis (data not shown). However,
when we cotransfected the EGFR with Myc-tagged RIP, which is a key
protein in TNFR-induced NF-
B activation, we detected a physical
association between the two proteins. This interaction was detected by
immunoprecipitating with an EGFR antibody and immunoblotting with Myc
antibodies (Fig. 3B, middle panel). We estimate
that 5-10% of the transfected RIP becomes associated with the EGFR in
these cells as shown in Fig. 3C. The association can also be
detected by immunoprecipitating with a Myc antibody and immunoblotting
with the EGFR antibody (Fig. 3D). The kinase activity of the
EGFR is required for association with RIP since a kinase-inactive EGFR
mutant fails to bind RIP (Fig. 3D). Also, addition of
tyrphostin AG 1478, a specific inhibitor of the EGFR kinase, blocks the
association between wild type EGFR and RIP (Fig. 3D). 293 EBNA cells express very low levels of endogenous EGFR, and we did not
detect EGF-induced ERK activation in these cells (data not shown). We
found that the endogenous level of EGFR expression in 293 EBNA cells is
not sufficient to mediate a detectable association with RIP. However,
when we transfected increasing amounts of EGFR DNA into these cells
along with a constant amount of RIP, an interaction between the two
proteins can be detected with increasing levels of EGFR expression
(Fig. 3B). This suggests that RIP may play a role in
EGF-mediated NF-kB activation since RIP is known to be a key component
of TNFR-NF-
B signaling, and also correlates with our observation
that in certain cell types a high level of EGFR expression is required
to activate NF-
B. It should be noted that addition of EGF to cells
did not increase the EGFR-RIP interaction in these cells. This is
because ectopic expression of the EGFR leads to a substantial
constitutive increase in the level of EGF receptor tyrosine
phosphorylation in 293 EBNA cells as shown in Fig. 3F, where
addition of EGF did not result in any further increase in tyrosine
phosphorylation of the EGFR. This suggests maximal activation of the
receptor in these cells even in the absence of ligand. This phenomenon has also been noted previously in 293T cells (36).

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Fig. 3.
The EGFR forms a complex with RIP.
A shows that the EGFR does not coimmunoprecipitate with
TRADD. 293 EBNA cells were transfected with Myc-TRADD (8 µg) and EGFR
(3 µg) (all lanes). Lane
1 shows expression of the transfected Myc-TRADD
(arrow) that cannot be detected in EGFR immunoprecipitates
(IP) even with EGF stimulation (lane
3). In B, 293 EBNA cells were cultured in 100-mm
dishes and transfected using the calcium phosphate method with 8 µg
of Myc-tagged RIP plus the EGFR (µg) in the amounts indicated. In
lane 7, cells were transfected with RIP plus
empty vector. Appropriate amount of vector DNA were added to keep the
total transfected DNA amount constant. 24 h after transfection
lysates were immunoprecipitated with with an EGFR antibody
(lanes 2-7) or with an isotype-matched control
antibody in lane 1. Immunoblotting was performed
with a Myc antibody as shown in the middle panel. As can be
seen in the middle panel, increasing the level of EGFR
expression results in an increased association of RIP with the EGFR.
The blot was stripped and reprobed with an EGFR antibody. This is shown
in the upper panel. 2% of the lysate from each
lane was immunoblotted with a Myc antibody to ascertain levels of
transfected RIP as shown in the lower panel,
while 90% of the lysate was used for immunoprecipitation. C
shows that 5-10% of transfected RIP binds to the EGFR. Cells were
transfected with RIP and EGFR as above and immunoprecipitated with an
EGFR antibody in lane 1 (90% of the cell lysate)
or control antibody in lane 2. 10% of the cell
lysate was loaded into lane 3. The
arrow points to RIP. D shows that a
kinase-inactive EGFR fails to bind RIP. 293 EBNA cells were transfected
with 8 µg of RIP-Myc (all lanes), 3 µg of
EGFR (wild type in lanes 1, 5, and
6 and kinase-inactive EGFR in lanes 2 and 3) and empty vector in lane 4.
Cells in lanes 3 and 6 were exposed to
tyrphostin AG 1478 (250 nM) for 30 min prior to
immunoprecipitation. Cell lysates were immunoprecipitated with anti-Myc
(9E10) antibodies in lanes 1-4 and
lane 6 and with an isotype-matched control
antibody in lane 5 and immunoblotted with
anti-EGFR antibodies. E shows expression of wild type
(lane 1) and kinase-inactive (lane
2) and endogenous (lane 3,
vector-transfected cells) EGFR in 293 EBNA cells. F shows
that ectopic expression of the EGFR in 293-EBNA cells results in a high
level of tyrosine phosphorylation of the EGFR even in the absence of
EGF (lane 1). Addition of EGF to transfected
cells does not result in further increases in tyrosine phosphorylation.
293-EBNA cells were transfected with 3 µg of EGFR in cells shown in
both lanes. EGF was added to cells in lane 2, and
lysates were immunoprecipitated with an EGFR antibody followed by
immunoblotting with phosphotyrosine antibodies (upper
panel). The blot was stripped and reprobed with anti-EGFR
antibodies (lower panel).
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A physical association between the EGFR and endogenous RIP in
untransfected MDA-MB-468 cells can also be detected. Cell lysates were
immunoprecipitated with a EGFR antibody and immunoblotted with a RIP
antibody. As can be seen in Fig.
4A, RIP coimmunoprecipitates with the EGFR in these cells. It should be noted that MDA-MB-468 cells
express high levels of the EGFR and that there is considerable basal
activation of the receptor in these cells even in the absence of
exogenous EGF. This is shown in Fig. 4B, which demonstrates a significant autophosphorylation of the receptor in the absence of
EGF. Although we do not see an increase in EGFR-RIP association when
these cells are exposed to EGF (data not shown), consistent with our
earlier observation in 293 cells, treating the cells with tyrphostin AG
1478 reverses the association between the two proteins (Fig.
4A). Taken together, this indicates that the protein complex
responsible for signaling by the two receptors, namely, TNFR and the
EGFR, are distinct.

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Fig. 4.
The EGFR associates with RIP in MDA-MB-468
cells. A shows coimmunoprecipitation of the EGFR with RIP in
MDA-MB-468 cells (upper panel). Cells were grown
in 100-mm dishes, and lysates were immunoprecipitated (IP)
with an EGFR antibody in lanes 2 and 3 and with an isotype-matched control antibody in lane
1 and immunoblotted with a RIP antibody. Cells in
lane 2 were pretreated with tyrphostin AG 1478 (250 nM) for 30 min, and cells in lanes
1 and 3 were treated with vehicle
(Me2SO). The arrow points to RIP. This blot was
stripped and reprobed with an EGFR antibody (shown in the
lower panel). This blot was developed after a 3-s
exposure, and the intensity of the EGFR bands reflects the high level
of EGFR in these cells. B shows that the high level of
endogenous EGFR expression in MDA-MB-468 cells results in a substantial
constitutive tyrosine phosphorylation of the EGFR even in the absence
of EGF (lane 1). Addition of EGF results in an
additional increase in tyrosine phosphorylation of the receptor
(lane 2). Cells were serum-starved, followed by
immunoprecipitation with anti-EGFR antibodies and immunoblotting with
phosphotyrosine (PTyr) antibodies. The blot was stripped and
reprobed with anti-EGFR antibodies (lower
panel).
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The EGFR Associates with NIK--
We next addressed the issue of
whether NIK coimmunoprecipitates with the EGFR in vivo since
NIK appears to be downstream of RIP in the TNFR NF-
B pathway and a
dominant negative NIK mutant blocks RIP-induced NF-
B activation
(16). 293 EBNA cells were cotransfected with FLAG-tagged NIK cDNA
along with the EGFR. Concurrent with our observation with RIP, NIK
associated with the EGFR with increasing levels of EGFR expression
(Fig. 5A). This interaction was detected by immunoprecipitating cell lysates with an EGFR antibody
and immunoblotting with anti-FLAG antibodies (Fig. 5A, middle panel), and also by immunoprecipitating with
anti-FLAG antibodies and immunoblotting with an EGFR antibody (Fig.
5B). The relationship between increasing level of EGFR
expression and association with NF-
B-inducing protein seems to be
more linear with Nik than with RIP. Similar to RIP, addition of EGF did
not increase the EGFR-NIK association in these cells. In the absence of
exogenous EGFR, we did not detect coprecipitation of the two proteins
suggesting that the endogenous level of EGFR expression is too low in
293 EBNA cells to allow a significant interaction between the two
proteins (Fig. 5, A and B).

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Fig. 5.
The EGFR associates with NIK. In
A, 293 EBNA cells were cultured in 100-mm dishes and
transfected using the calcium phosphate method with 8 µg of
FLAG-tagged NIK plus the EGFR (µg) in the amounts indicated. In
lane 1, cells were transfected with NIK plus
empty vector. Appropriate amounts of vector DNA were added to keep the
total amount of transfected DNA constant. Cell lysates were
immunoprecipitated (IP) with an EGFR antibody in
lanes 1-6 or with an isotype-matched control
antibody in lane 7. Immunoblotting was performed
with anti-FLAG antibodies shown in the middle
panel. As can be seen in the middle
panel, increasing the level of EGFR expression results in
increasing association with NIK. The blot was stripped and reprobed
with an EGFR antibody, which is shown in the upper
panel. An aliquot of each lysate was immunoblotted with a
FLAG antibody to show expression levels of transfected NIK. This is
shown in the lower panel. B shows that
the EGFR can be detected in FLAG (NIK) immunoprecipitates. Cells were
transfected with Nik (8 µg) in all lanes and
with 5 µg of EGFR in lanes 1 and 2 or empty vector in lane 3. In lane
2 cells were stimulated with EGF (100 ng/ml) for 5 min.
Cells were lysed and immunoprecipitated with anti-FLAG antibodies
(all lanes) and immunoblotted with EGFR
antibodies.
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The EGFR May Form Multiprotein Complexes with RIP and NIK--
It
is increasingly apparent that signal transduction involves the physical
assembly of individual components of a pathway into multiprotein
complexes. In the case of NF-
B, a number of proteins such as NIK,
IKKs, and I
B
exist in a complex termed the signalosome. To
investigate the formation of complex formation between the EGFR, RIP,
and NIK, we cotransfected Myc-tagged RIP, Flag-tagged NIK, and the EGFR
along with appropriate controls into 293 EBNA cells. The data shown in
Fig. 6 suggest that multiprotein complexes may form among the three proteins. This was demonstrated by
immunoprecipitating with an EGFR antibody and immunoblotting with a Myc
antibody to detect the presence of RIP (Fig. 6A). The same
blot was stripped and reprobed with anti-FLAG antibodies to detect the
presence of NIK (Fig. 6C). Similarly, in another independent
experiment, anti-FLAG antibodies were used to immunoprecipitate the
complex which was analyzed by immunoblotting with an EGFR antibody
(Fig. 6B). This blot was later stripped and reprobed with a
Myc antibody (Fig. 6D) to identify the presence of RIP. Thus, we detect both Rip and Nik in EGFR immunoprecipitates and also
find both EGFR and Rip in Nik immunoprecipitates. This experiment suggests that the signaling complex at the EGFR may involve all three
proteins. However, we have found that RIP can bind to NIK in the
absence of exogenous EGFR in 293 EBNA cells, which express a low level
of endogenous EGFR. Thus, finding both RIP and NIK in EGFR
immunoprecipitates could also be a consequence of pairwise interactions
between the three proteins. Our coimmunoprecipitation studies are
suggestive of a direct physical association between the EGFR and RIP
and NIK, although the presence of other proteins and their role in the
formation of the complex remains as a possibility.

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Fig. 6.
The EGFR forms complexes with RIP and
NIK. 293 EBNA cells were cultured in 100-mm dishes and transfected
with EGFR (3 µg), RIP-Myc (5 µg), and NIK-FLAG (5 µg), using the
calcium phosphate method, in lanes 1 and
3. Cells in lane 2 were transfected
with an empty vector. Cells were lysed 24 h after transfection. In
A, cell lysates were immunoprecipitated (IP) with
an EGFR antibody (lanes 1 and 2) or
isotype-matched control antibody (lane 3) and
immunoblotted with anti-Myc antibodies. Arrow points to
RIP-Myc This blot was stripped and reprobed with anti-FLAG antibodies
shown in C. Arrow points to FLAG-NIK. In
B, cell lysates were immunoprecipitated with anti-FLAG
(FL) antibodies (lanes 1 and
2) or with an isotype-matched control antibody (lane
3) and immunoblotted with anti-EGFR antibodies. Arrow
points to EGFR. The lower band probably represents the precursor form
of the receptor. This blot was stripped and reprobed with anti-Myc
antibodies as shown in D. Arrow points to
RIP-Myc.
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A Dominant Negative NIK Mutant Blocks EGFR-mediated NF-
B
Activation--
NF-
B activation by cytokines involves signal
transduction cascades, which involve a number of key intermediate
signaling proteins. NIK was originally identified as a
TRAF2-interacting protein (16). When overexpressed, NIK activates
NF-
B and kinase-inactive mutants of NIK behave as dominant negative
inhibitors that block NF-
B activation by TNF and RIP.
The observation that NIK forms a complex with the EGFR suggests that
NIK may play a role in EGFR-induced NF-
B activation. We tested this
hypothesis in R1hER cells. As noted previously, EGF induces a
substantial activation of NF-
B in these cells. There is also a
significant constitutive activation of NF-
B in these cells compared
with untransfected Rat-1 fibroblasts. A NIK kinase domain mutant
(NIK-K429A/K430A) was transfected into R1hER cells along with an
NF-
B promoter linked to a luciferase construct along with
appropriate controls. As can be seen in Fig.
7A, expression of a dominant
negative NIK mutant significantly blocks the ability of the EGFR to
activate NF-
B-dependent transcription. A dominant negative NIK mutant blocks both the constitutive as well as the EGF-dependent increase in NF-
B activity in R1hER cells.
This suggests that NIK is required for EGFR-induced NF-
B activation. Fig. 7 (B and C) shows EGFR tyrosine
phosphorylation and protein levels in transfected cells shown in Fig.
7A. Although expression of a dominant negative NIK blocks
EGFR-induced NF-
B activation, it does not influence EGFR expression
or tyrosine phosphorylation.

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Fig. 7.
A dominant negative NIK mutant blocks
NF- B activation by the EGFR shown in
A. 6 × 104 cells were plated in
24-well tissue culture dishes. Following an overnight incubation, cells
were transfected with plasmids as described under "Experimental
Procedures." NF- B transcriptional activity is low in Rat-1
fibroblasts and significantly higher in R1hER cells transfected with
empty vector, both with and without EGF stimulation Expression of a
dominant negative NIK mutant leads to a significant suppression of
EGFR-induced NF- B activity in these cells. Cells were treated with
EGF for 90 min. Error bars represent sample S.D.
The experiment shown is representative of three independent experiments
done in triplicate. B (upper panel)
shows tyrosine phosphorylation of the EGFR in cells shown in
A, as determined by immunoprecipitation with EGFR antibodies
and immunoblotting with phosphotyrosine antibodies. The lanes
correspond to those shown in A. As seen with MDA-MB-468
cells, there is constitutive tyrosine phosphorylation of the EGFR in
R1hER cells (lanes 3 and 5). In the
lower panel, the immunoblot was stripped and
reprobed with anti-EGFR antibodies to show loading. IP,
immunoprecipitation; PTyr, phosphotyrosine.
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Akt Does Not Play a Role in EGF-induced NF-
B
Activation--
Previous studies have demonstrated that protein kinase
B/Akt is involved in NF-
B activation by cytokines such as PDGF and TNF (24-26). The epidermal growth factor is also known to activate Akt. To investigate the role of Akt in EGFR-induced NF-
B activation, the following experiments were undertaken. First, we confirmed that EGF
stimulation leads to Akt activation in R1hER cells using a
phosphospecific (Ser-473) antibody (Fig.
8A). However, EGF-induced Akt
activation can also be detected in Rat-1 cells and since these cells do
not appear to activate NF-
B significantly in response to EGF,
activation of Akt may not be an intermediary step in the activation of
NF-
B.

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Fig. 8.
Activation of Akt is not required for
EGFR-induced NF- B activation.
A, stimulation of both Rat-1 and R1hER cells with EGF (100 ng/ml) for 5 min leads to activation of Akt in Rat-1 and R1hER cells.
The upper panel shows a Western blot stained with
pAkt (Ser-473) antibodies while the lower blot
shows the same blot stripped and reprobed with Akt antibodies.
B, pretreatment of cells with wortmannin (W; 100 nM for 30 min) fails to block NF- B activity in R1hER
cells (error bars represent sample S.D.). Cells
were treated with EGF for 90 min. The experiment shown is
representative of three independent experiments done in triplicate.
However, the same exposure to wortmannin completely blocks Akt
activation in R1hER cells as shown in C. The
upper panel shows a Western blot stained with a
pAkt (Ser-473) antibody (arrow points to pAkt), which was
stripped and reprobed with anti-Akt antibodies to show loading
(lower panel). w, wortmannin.
D shows that wortmannin also blocks Akt activation in
MDA-MB-468 cells. The upper panel shows a Western
blot stained with pAkt (Thr-308) antibodies, which was stripped and
reprobed with Akt antibodies (lower panel). A
similar result was found with pAkt Ser-473 antibodies in these cells
(data not shown).
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To address this definitively, we examined the effects of the PI
3-kinase inhibitor wortmannin since activation of Akt is mediated primarily through the PI 3-kinase pathway. The data presented in Fig.
8B clearly show that incubation of cells with wortmannin had
no effect on EGF-induced NF-
B activation in R1hER cells. Wortmannin
also failed to block EGFR-induced NF-
B activation in MDA-MB-468
cells (data not shown). Although wortmannin had no effect on
EGF-induced NF-
B activation, it completely blocked EGF-induced Akt
activation in R1hER cells (Fig. 8C) as determined by
immunoblotting EGF-treated cell lysates with phosphospecific Akt
antibodies. A similar result was evident in MDA-MB-468 cells using both
a phospho-Ser-473 antibody as well as a phospho-Thr-308 antibody (Fig.
8D). The observation that blocking EGF-induced Akt
activation does not hinder EGF-induced NF-
B activation argues strongly against a role of Akt in EGF-induced NF-
B activation. However, a recent report has shown that EGF-induced NF-
B activation can be blocked by inhibition of PI 3-kinase (29). The apparent discrepancy between our work and this study may be explained by the
different time points of activation investigated by the two groups. The
previous study has observed the effect at much later time points than
we have investigated.
 |
DISCUSSION |
The response of mammalian cells to extrinsic stimuli involves the
activation of signaling cascades with discrete specificity. There is
increasing evidence that generation of a specific signal involves the
formation of specific multiprotein complexes (37). The formation of
such complexes involves selective recruitment of subcomplexes of
signaling proteins to an activated receptor. This is followed by
activation of signaling proteins through mechanisms such as
phosphorylation and/or other protein-protein interaction. Identification of the composition of signal-specific multiprotein complexes is the initial step in understanding the mechanisms that
underlie the generation of a specific signal. The EGF receptor is a
prototypical tyrosine kinase that assembles a multiprotein complex to
induce mitogenic signaling. In this work we have identified a novel
receptor tyrosine kinase (EGF receptor)-bound complex, which mediates
transactivation of NF-
B.
Activation of the EGF receptor results in activation of a number of
transcription factors including AP-1, STATs, and NF-
B (28, 38, 39).
Although EGF stimulation of cells has previously been shown to lead to
degradation of I
B
and NF-
B activation, the proximal signaling
events that lead to EGFR-induced activation of NF-
B have not been
elucidated. In this study we demonstrate an interaction between RIP and
NIK, which are two key components of TNF-induced NF-
B signal, and
the EGFR. Our studies suggest a role for these proteins in NF-
B
activation by the EGFR and indicate that mechanisms of NF-
B
activation by the EGFR and TNF
are similar, but not identical.
RIP was initially identified as a CD95-interacting protein (40).
Overexpression of RIP activates NF-
B in cells (41, 42). Jurkat cell
lines, which lack RIP, are unable to activate NF-
B in response to
TNF
. Introduction of RIP into these cells restores the ability of
TNF to activate NF-
B (42). Finally, cells from mice lacking RIP are
unable to activate NF-
B in response to TNF, establishing RIP as a
key component of the TNF-induced NF-
B signal (43). The mechanism of
action of RIP remains obscure, but a recent study suggests that RIP
binds to NEMO, another component of the TNFR NF-
B pathway (44). We
show that RIP physically associates with the EGFR when the two proteins
are coexpressed in cells, and that increasing the level of EGF receptor
expression in cells promotes this association. The kinase activity of
the EGFR appears to be required for EGFR-RIP association since a
kinase-inactive EGFR fails to bind RIP. We also show an association
between the EGFR and RIP in untransfected cells, and show that this
association is reversed by tyrphostin AG 1478, a specific inhibitor of
the EGFR kinase. It is intriguing that the kinase activity of the receptor is required for its association with RIP. We have not observed
RIP to be tyrosine-phosphorylated, and it is possible that other
protein(s) may be required for this interaction in a
phosphorylation-dependent manner. The finding that the
EGFR-RIP association requires the kinase activity of the receptor also supports our hypothesis that the EGFR-RIP association is an important step in NF-
B activation since the EGFR activates NF-
B in a
ligand-dependent manner.
RIP is recruited to the activated TNFR via an adaptor protein, TRADD
(14). TRADD and RIP interact with each other via their death domain,
which is a conserved sequence of amino acids also present in the TNF
receptor. Although the EGFR associates with RIP, it does not bind to
TRADD (or to another death domain-bearing protein, FADD). This
suggests RIP may associate directly with the EGFR through a
heterologous interaction or via some other protein(s) of the complex.
Although the mechanism of action of RIP is not well understood,
recruitment of RIP to the TNFR is likely to be a key step in NF-
B
signaling. Similarly, RIP binding to the EGFR may be a key step in the
activation of NF-
B by EGF.
NIK was originally identified as a TRAF2-binding protein. Ectopic
expression of NIK in cells results in NF-
B activation. A dominant
negative NIK mutant blocks NF-
B activation by a number of stimuli
including TNF
, IL-1, and RIP, suggesting that NIK functions
downstream of RIP in the signaling cascade. Although the mechanism of
activation of NIK itself remains unclear, NIK appears to activate the
IKK enzymes leading to the phosphorylation of I
B
on both of its
regulatory serines at residues 32 and 36, which leads to I
B
degradation. We find that NIK associates with the EGFR when the two
proteins are coexpressed and, as in the case of RIP, increasing the
level of EGFR in cells promotes the association between NIK and the
EGFR. We were unable to demonstrate an interaction between the EGFR and
NIK in untransfected cells because of lack of a good NIK antibody. We
show that a dominant negative NIK mutant blocks EGFR-induced NF-
B
activation, demonstrating that the interaction between the two proteins
has functional significance.
Our data also suggest that EGFR, NIK, and RIP may form multiprotein
complexes, indicating that EGFR-induced NF-
B activation also
involves the formation of multiprotein complexes like the signalosome
described in TNFR-mediated NF-
B signaling (18, 44). We also show
that transfection of a dominant negative I
B
mutant that cannot be
phosphorylated on serine at residues 32 and 36 blocks EGFR-induced
NF-
B activation, demonstrating again that the EGFR uses signals that
are similar but not identical to the TNF receptor to activate
NF-
B.
Previous studies have invoked a role for the protein kinase Akt in
NF-
B activation by both the PDGF receptor and TNF
. Both cytokines
activate Akt primarily through activation of PI 3-kinase. A
constitutively active Akt mutant activates NF-
B. Wortmannin, a PI
3-kinase inhibitor, blocks NF-
B activation by both cytokines as does
introduction of a dominant negative Akt mutant. Our study, however,
does not support a role for Akt in NF-
B activation by the EGFR. We
find that, although wortmannin blocks EGF-induced phosphorylation of
Akt in MDA-MB-468 and in R1hER cells, it has no effect on EGF-induced
NF-kB activation. This suggests that, unlike the TNFR and the PDGF
receptor, Akt activation is not required for EGFR-mediated NF-
B
activation, at least in the cell types we have studied. Our results are
in agreement with another recent study, which shows that inhibiting Akt
activation in human endothelial cells does not block NF-
B activation
in response to TNF or IL-1 (45).
Previous studies have indicated that EGF induces a slow activation of
NF-
B in primary smooth muscle cells, a transient activation of
NF-
B in primary embryo fibroblasts, and a more robust activation in
A431 cells and in several endoplasmic reticulum-negative breast cancer
cell lines that express high levels of the EGFR. Our experiments also
suggest that a high level of EGFR expression is optimal for EGF-mediated NF-
B activation. This hypothesis is supported by our
finding that there is a correlation between the level of EGFR expression and the recruitment of key NF-
B signaling proteins (RIP
and NIK) to the EGFR. It is also important to note that EGF stimulation
of cells with high levels of EGFR expression (which are common in human
tumors) leads to a selective rather than a general amplification of
signals. For example, EGF stimulation of both Rat-1 fibroblasts and
R1hER cells leads to increases in ERK activation, whereas Rat-1
fibroblasts fail to activate NF-
B. Similar results are seen when
comparing MCF7 cells to MDA-MB-468 cells.
The EGF receptor generates mitogenic signals in cells and a large
number of human tumors express high levels of the EGFR (46). In
addition, increased expression of the EGFR correlates with the
malignant phenotype. NF-
B, in addition to its role in inflammatory responses, is also involved in the control of cell growth and transformation. EGF is known to induce expression of genes involved in
cell growth and some of these genes, such as c-myc, contain NF-
B promoter binding sites (47, 48). It has also been shown that
activated forms of Ras and Raf activate reporter genes controlled by
NF-
B sites (49, 50). EGF is known to activate both Ras and Raf (3).
A dominant negative I
B
mutant has been shown to block Ras
transformation and decreased levels of I
B
promote transformation
(51, 52). EGF-induced NF-
B activation has also been implicated in
cyclin D1-dependent cell cycle progression (29).
Taken together, this suggests that EGFR-mediated NF-
B activation is
likely to influence cell growth and proliferation. We have elucidated
the proximal steps in EGF-mediated activation of NF-
B. Our study
indicates that the activated EGFR recruits RIP and NIK to initiate the
signaling cascade via I
B
to localize NF-
B to the nucleus for
transactivation. Recent studies have shown that NF-
B also plays a
key role in regulating apoptosis (53-56). This is of particular
interest since cells such as A431 and MDA-MB-468, which express high
levels of the EGFR and show the most robust NF-
B activation are
known to undergo apoptosis in response to EGF (57, 58). Further studies
are needed to elucidate the role of NF-
B in EGF-induced apoptosis
and growth control and are likely to be relevant to a better
understanding of the biology of human tumors as well as EGF-induced
normal growth. Our identification of a novel complex at the EGF
receptor distinct from the complex required for mitogenic signaling and
from the complex assembled by the TNFR opens up new lines of
investigation pertaining to EGF receptor-mediated signaling.