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INTRODUCTION |
The epidermal growth factor receptor
(EGFR,1 ErbB1, HER1) is the
prototypical member of the ErbB family of receptors, which includes
ErbB2 (HER2, neu), ErbB3 (HER3), and ErbB4 (HER4) (reviewed in Refs. 1-3). The ErbBs mediate signaling by a large number of growth
factors that are structurally related to EGF such as transforming
growth factor-
or amphiregulin. This family of receptors plays
critical roles in the proliferation, migration, survival, and
differentiation of target cells, and dysregulation of signaling by
ErbBs has been implicated in the pathogenesis and progression of human
cancers (2, 4). ErbBs are activated via a process of receptor homo- and
heterodimerization, which is initiated by engagement of ligand by the
extracellular domain of an ErbB, and almost every possible dimeric
combination of ErbBs have now been observed (reviewed in Refs. 1-3,
5). Unlike EGFR, ErbB3, and ErbB4, no ligand has been identified for
ErbB2 to date. Recent work has demonstrated that binding of EGF to
EGFRs initiates a ligand-independent lateral propagation of receptor
activation in the plasma membrane (6).
ErbBs consist of an extracellular ligand binding domain, a
transmembrane spanning segment, an intracellular tyrosine kinase domain, and a C-terminal region that contains multiple tyrosines, which
upon phosphorylation provide docking sites for signal transducers such
as GRB2 and SHC (7). Unlike other family members, ErbB3 does not
possess kinase activity (8). Early research on the EGFR indicated that
the kinase activity was required for EGF-induced biological responses
(9, 10). However, subsequent studies have demonstrated that
kinase-inactive forms of the EGFR have the capacity to signal in an
EGF-dependent manner. EGF-stimulated kinase-inactive EGFRs
activate MAPK, c-fos gene expression, and/or DNA synthesis
(11-16). Targeted inactivation of the EGFR gene in mice
results in strain-dependent phenotypes that range from
death in utero to postnatal abnormalities in skin, lung,
kidney, gastrointestinal tract, and brain (17-19). Waved-2
(wa-2) mice, which harbor a V743G mutation in the EGFR
kinase domain exhibit mild skin and eye abnormalities but are healthy
and fertile (20, 21). Interestingly, the V743G mutation results in a
kinase-defective EGFR whose ability to phosphorylate exogenous
substrate has been reduced by >90% relative to its wild-type counterpart (20). The homologous mutation in the human EGFR (V741G)
significantly impairs EGFR kinase activity, and ectopic expression of
V741G EGFR in myeloid BaF/3 cells confers the ability for EGF to induce
survival but not proliferation (22, 23). Conversely, kinase-impaired
V741G EGFR can elicit EGF-dependent mitogenesis when
expressed in NIH 3T3 fibroblasts (21). Taken together, these findings
raise the possibility that the EGFR kinase activity may not be required
for certain aspects of EGFR function.
To better understand the role of the EGFR kinase activity in mediating
EGF-stimulated cellular responses we studied signaling by a
kinase-inactive EGFR in which a lysine to methionine mutation (K721M)
abrogates ATP binding. Because most cell types express one or more
endogenous ErbBs, an elucidation of the mechanism of EGF-induced
signaling by ectopically expressed kinase-inactive EGFRs has been
elusive. To circumvent this problem, we have utilized the ErbB-devoid
myeloid 32D cells as a model system into which we reconstituted K721M
signaling. Our studies demonstrate a specific requirement for ErbB2 in
K721M signaling and indicate that signaling is mediated by a
K721M-ErbB2 oligomer. The EGF-stimulated hetero-oligomer is a strong
activator of MAPK and the pro-survival kinase Akt, and EGFR kinase
activity is not required for this signaling.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The 32D cell line was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5% medium
conditioned by WEHI-3B cells (24). Cells stably expressing human EGFR
were provided by J. Pierce (NCI, National Institutes of Health; Ref.
25).
Antibodies and cDNAs--
Antibodies to EGFR and ErbB2 were
obtained from NeoMarkers. Biotinylated PY-20 and 4G10 antibodies were
purchased from ICN Biomedical and Upstate Biotechnology, respectively.
Antibodies directed against the phosphorylated activated forms of MAPK,
STATs, and Akt were obtained from New England BioLabs. Antibodies
against ErbB3 and Akt were obtained from Santa Cruz Biotechnology. ErbB cDNAs were provided by J. Pierce and subcloned into pcDNA3.1 or pcDNA3.1/Zeo. Mutations within cDNAs constructs were made using standard molecular biology techniques, and all constructs were DNA-sequenced.
Transfections and Generation of Stable Cell Lines--
Cells
were transfected with 20 µg of total DNA by electroporation (960 µF, 0.25 kV) using a Bio-Rad Gene Pulser II. Stable transfectants
were generated using the neomycin resistance marker in pcDNA3.1 or
the Zeocin resistance marker in pcDNA3.1/Zeo. Clonal populations
were generated by serial dilution and characterized by flow cytometry
and Western blotting.
Cell Proliferation Assays--
Assays were performed as
previously described (27). Briefly, cells were washed, resuspended into
60 × 15 mm dishes (100,000 cells/ml) in RPMI 1640 medium
containing 15% fetal bovine serum (basal medium) with or without 10 nM EGF. Cells were stained with trypan blue, and viable
cells were counted every day using a hemocytometer.
Immunoprecipitations--
Whole cell lysates were generated, and
protein concentration was determined as described (34). One µg of
antibody was added to 0.5 mg of lysate, incubated for 1 h at
4 °C and 5 µl of protein G-agarose was added. After 1 h at
4 °C, the immune complexes were centrifuged at 15,000 × g for 2 min and washed three times with ice-cold lysis
buffer. Bound proteins were released by boiling in SDS-PAGE sample
buffer for 4 min.
Western Blotting--
Proteins were separated on SDS-PAGE gels
and transferred to polyvinylidene difluoride membranes. Membranes were
probed with 0.3 µg/ml of primary antibody, and detection was
performed using the Vectastain ABC Elite Kit (Vector Labs), enhanced
chemiluminescence, and exposed to film.
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RESULTS |
Acquisition of Signaling by Kinase-inactive EGFR by Coexpression of
ErbB2 but Not ErbB3 or ErbB4--
To explore the signaling potential
of a kinase-inactive EGFR, we studied signaling by a mutant human EGFR
in which a lysine to methionine mutation has been introduced into the
ATP binding site (K721M). Work by us (data not shown) and others have
demonstrated that this mutation abolishes the kinase activity of the
EGFR (9, 10). To determine the intrinsic signaling capacity of K721M, we investigated EGF-dependent signaling by ectopically
expressed K721M in the interleukin-3 (IL-3)-dependent
myeloid 32D cell line (24). This ErbB-devoid cell line has proved to be
a powerful tool in the study of ErbB signaling (25-27). 32D cells were
transiently transfected with K721M cDNA and stimulated with EGF
(Fig. 1A). Lysates were
analyzed for MAPK activation using an antibody specific for
phosphorylated Thr-202/Tyr-204 in the activated form of MAPK (extracellular signal-regulated kinase 1 and 2; top panel).
No activation of MAPK was detected under these conditions even though expression of K721M was confirmed by Western blotting (lanes
1-2).

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Fig. 1.
Analysis of EGF-induced MAPK activation by
coexpression of K721M and ErbBs in 32D cells. A, 32D
cells were transfected with either K721M cDNA alone (lanes
1-2) or with ErbB2 (lanes 3 and 4), ErbB3
(lanes 5-6), or ErbB4 cDNA (lanes 7-8).
Serum-starved cells were stimulated for 5 min at 37 °C with 10 nM EGF and 15 µg of whole cell lysates were separated in
an 8% SDS-PAGE gel and transferred to a polyvinylidene difluoride
membrane. The activation of MAPK was monitored by Western blotting
(WB) using antibodies against the phosphorylated form of
MAPK (phosphoMAPK). Consistent loading of each lane was
confirmed by stripping the blot and reprobing for MAPK. Expression of
ErbBs was confirmed by probing the blot with appropriate
antibodies. B, cells were transfected with either EGFR
alone (lanes 1-5) or a combination of K721M and ErbB2
cDNA (lanes 6-10). Cells were stimulated with indicated
concentrations of EGF for 5 min at 37 °C.
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To test the hypothesis that kinase-devoid forms of the EGFR can signal
in the presence of additional human ErbB family members, we
simultaneously expressed K721M along with ErbB2, ErbB3, or ErbB4.
Interestingly, although ErbB2 by itself was unable to promote MAPK
phosphorylation in response to EGF (data not shown), coexpression of K721M with ErbB2 (lanes 3-4) resulted in EGF-induced
MAPK stimulation. In contrast, coexpression of K721M with either ErbB3
or ErbB4 could not reconstitute EGF-dependent signaling by
K721M (lanes 6 and 8). No differences in the
magnitude of MAPK activation were observed when the ratio of
transfected K721M:ErbB2 cDNAs was varied from 3 to 0.33 (data not
shown), suggesting that signaling to MAPK was independent of the
relative levels of the receptors. EGF dose-response studies (Fig.
1B) revealed that both EGFR or coexpressed K721M-ErbB2
activate MAPK at 10 pM EGF, with the EGFR-induced signal
being slightly stronger (compare lanes 2 and 7).
Maximal MAPK stimulation was observed at 100 pM EGF for
both EGFR and K721M-ErbB2 (lanes 3 and 8),
indicating that physiologically relevant concentrations of EGF can
activate MAPK in cells coexpressing K721M and ErbB2.
ErbB2 Kinase Activity but Not Its Tyrosine Phosphorylation Is
Essential for EGF-induced MAPK Activation--
To determine whether
ErbB2 tyrosine kinase activity was obligatory for this signaling, we
introduced an aspartic acid to asparagine mutation into the catalytic
domain of ErbB2 (D845N). This mutation abolished the ability of ErbB2
to support MAPK stimulation by coexpressed K721M (Fig.
2, lanes 3-4), demonstrating
the requirement for the ErbB2 kinase activity. The C-terminal region of
ErbB2 contains 6 potential tyrosine phosphorylation sites positioned at
1023, 1127, 1139, 1196, 1222, and 1249, which can function as docking
sites for signal-transducing proteins (1). To explore the role of ErbB2
tyrosine phosphorylation in supporting K721M signaling, a
C-terminal-truncated form of ErbB2 comprising residues 1-1026 was
coexpressed with K721M. This truncated ErbB2 was able to mediate
EGF-induced MAPK activation and tyrosine phosphorylation of K721M while
not being tyrosine phosphorylated itself (Fig. 2, lane 6).
Identical results were obtained when the remaining tyrosine at position
1023 was converted to phenylalanine in this truncated ErbB2 (data not
shown). Further, the D845N ErbB2 mutant did not elicit phosphorylation
of either K721M or itself in response to EGF (lane 4).

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Fig. 2.
The role of ErbB2 kinase activity and
tyrosine phosphorylation sites in EGF-stimulated MAPK activation by
coexpressed K721M-ErbB2. Cells were cotransfected with K721M and
either wild-type ErbB2 (WT) (lanes 1-2), ErbB2
D845N (lanes 3-4), or ErbB2-(1-1026) cDNA (lanes
5-6). Cells were stimulated with EGF, and MAPK activation was
evaluated by Western blotting (WB) as in Fig. 1.
Phosphotyrosine content (pY) and expression of ErbBs were
analyzed by immunoprecipitation (IP) followed by Western
blotting.
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A Requirement for the Mutated Kinase Domain of K721M but Not for
K721M Tyrosine Phosphorylation, in EGF-stimulated MAPK
Activation--
The C-terminal region of EGFR contains 6 potential
tyrosine phosphorylation sites positioned at 992, 1068, 1086, 1114, 1148, and 1173 (1). C-terminal truncations of K721M at either residue 1000 or 973 had no detectable effect on ErbB2-dependent
MAPK activation by EGF, but did eliminate tyrosine phosphorylation of
the truncated K721M receptors (Fig. 3,
compare lane 2 with 4 and 6). In
contrast, ErbB2 tyrosine phosphorylation was unaffected by the
truncation of K721M at residues 1000 or 973. However, truncation of the
EGFR on the cytoplasmic side of the transmembrane region obliterated all detectable signaling (lane 8). c-Src has been shown to
phosphorylate the EGFR at Tyr-845 (28). Nevertheless, mutation of
Tyr-845 to Phe within K721M-(1-973) had no detectable effect on
the EGF-induced MAPK activation, which is mediated by ErbB2 (data not
shown). Thus, whereas tyrosine phosphorylation of K721M is not a
requisite for the EGF-stimulated MAPK activation via ErbB2, the mutated kinase domain of K721M (residues 648-973) is required.

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Fig. 3.
EGF-dependent signaling by ErbB2
and truncated K721M. A, 32D cells were cotransfected
with ErbB2 cDNA and either K721M (lanes 1-2),
K721M-(1-1000) (lanes 3-4), K721M-(1-973) (lanes
5-6), or EGFR-(1-647) cDNA (lanes 7-8). Cells
were stimulated with EGF, and MAPK activation and phosphotyrosine
content (pY) were evaluated by Western blotting
(WB) as in Fig. 2.
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Tyrosine Phosphorylation of at least One of the Receptors Is
Necessary for Maximal MAPK Stimulation by EGF--
As shown in Figs. 2
and 3, removal of the C-terminal region of either K721M or ErbB2 did
not influence EGF-stimulated MAPK activation. Thus, we tested the
effect of simultaneous deletions of the C-terminal regions of K721M and
ErbB2 in this signaling system (Fig. 4).
Truncation of K721M at residue 973 resulted in a complete loss of its
tyrosine phosphorylation and a dramatic reduction in MAPK activation in
cells coexpresssing ErbB2-(1-1026) (lanes 2 and
4). The tyrosine residue in oncogenic neu (rat
ErbB2), which corresponds to position 1023 in human ErbB2, is a
negative regulator of neu-mediated transformation (29). We
tested whether mutation of Tyr-1023 to Phe in ErbB2-(1-1026) increased
EGF-induced MAPK stimulation by K721M-(1-973). However, this mutation
did not restore MAPK activation by EGF (lane 6). These data
demonstrate that tyrosine phosphorylation of either K721M or ErbB2 is
required for maximal MAPK activation by EGF.

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Fig. 4.
EGF-induced signaling by ErbBs lacking
tyrosine phosphorylation sites. 32D cells were cotransfected with
K721M and ErbB2-(1-1026) cDNA (lanes 1-2), or
K721M-(1-973) with either ErbB2-(1-1026) (lanes 3-4), or
[Y1023F]ErbB2-(1-1026) (lanes 5-6). Cells were
stimulated with EGF, and MAPK activation and phosphotyrosine content
(pY) were evaluated by Western blotting (WB) as
described in the legend to Fig. 2.
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EGF Is a Potent Activator of MAPK and Akt in 32D Cell Lines Stably
Coexpressing ErbB2 and Kinase-inactive EGFR--
To further
investigate the EGF-stimulated signaling by coexpressed K721M and
ErbB2, we generated cell lines that stably expressed these receptors.
32D cells were transfected with K721M cDNA, selected in G418, and
the clones obtained by serial dilution were screened for surface
expression of K721M by flow cytometry analysis (data not shown) and
Western blot-ting (Fig. 5A,
lower panel). Three independent clones, namely 9.10, 9.11, and 9.16, were selected, and consistent with the results obtained in
transient expression assays displayed in Fig. 1, these K721M clones
were incapable of activating MAPK in response to EGF (Fig. 5A,
lanes 6, 8, and 10). However,
transient expression of ErbB2 resulted in reconstitution of MAPK
stimulation by EGF in all three clones (lanes 12,
14, and 16), demonstrating that K721M in these
clones was functional. As a control for these experiments, we utilized
32D cells stably expressing human EGFR (lanes 1-2; Refs.
25-27).

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Fig. 5.
Evaluation of EGF-dependent
signaling in stable 32D cells expressing K721M alone or coexpressing
K721M and ErbB2. A, 32D cells (lanes 1-2)
or cells stably expressing wild-type EGFR (WT) (lanes
3-4) or clones expressing K721M (lanes 5-10) were
exposed to 10 nM EGF, and MAPK activation was evaluated as
described in the legend to Fig. 1. In lanes 11-16 clones
were transiently transfected with ErbB2 cDNA. Expression of EGFRs
was evaluated by Western blotting (WB: EGFR).
B, 32D cells (lanes 3-4) or cells stably
expressing EGFR (lanes 1-2), K721M (lanes 5-6)
or various clones coexpressing K721M and ErbB2 (lanes 7-16)
were stimulated with EGF. Akt activation was monitored by Western
blotting (WB) analysis of lysates using antibodies against
the phosphorylated form of Akt (phosphoS473 and
phosphoT308 Akt). Consistent loading of each lane was
confirmed by stripping the blot and reprobing for Akt, and expression
of ErbBs was confirmed by Western blotting.
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Clone 9.10 was then transfected with a vector containing ErbB2 cDNA
and a zeocin-resistance marker to generate 5 stable clones that
simultaneously expressed K721M and varying amounts of ErbB2 (Fig.
5B, lanes 7-16). EGF activated MAPK in all five
cell lines consistent with results obtained in transient expression
assays (Figs. 1-3). We then evaluated the EGF-induced activation of
the protein kinase Akt, which plays an important role in cell survival (30). Because activation of Akt results from phosphorylation at Thr-308
and Ser-473, we monitored activation using antibodies that specifically
recognize these two phosphorylated sites (Fig. 5B).
Treatment of all stable cell lines coexpressing K721M and ErbB2 with
EGF elicited phosphorylation of Akt on Thr-308 and Ser-473 to levels
comparable with cells stably expressing EGFR (lanes 2, 8, 10, 12, 14, and 16).
EGF Cannot Substitute for IL-3 in the Proliferation of 32D Cells
Coexpressing K721M-ErbB2 but Delays the Onset of
Apoptosis--
Withdrawal of IL-3 from 32D cells leads to the
accumulation of cells in G1 and rapid apoptosis (31).
However, as seen in Fig. 6A
and as previously demonstrated (25), expression of EGFR in 32D cells
allows EGF to supplant the IL-3 requirement. Thus, it was important to
evaluate the mitogenic and anti-apoptotic potential of EGF in cells
expressing K721M alone or in the presence of ErbB2. EGF had no
discernable effect on 32D cells expressing K721M alone (clone 9.10),
which, like the parental cells, underwent rapid apoptosis upon IL-3
deprivation (Fig. 6B). Identical findings were obtained with
clones 9.11 and 9.16, which also harbor K721M (data not shown).
Treatment of clone 7.1, which coexpresses K721M and ErbB2, with EGF
resulted in a delayed onset of apoptosis compared with untreated cells
(Fig. 6C). After 24 h of exposure to EGF, clone 7.1 cells were still viable, but underwent apoptosis during the
subsequent 24 h period. This finding was consistent in all five of the
32D clones, which express both K721M and ErbB2 (data not shown).
Therefore, unlike EGFR, the activated K721M-ErbB2 receptors are
incapable of generating a proliferative response in the 32D cell
context.

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Fig. 6.
Analysis of EGF-induced proliferation of
stable 32D cells. Stable 32D cells expressing either EGFR
(A), K721M (clone 9.10) (B) or K721M and ErbB2
(clone 7.1) (C) at a density of 100,000 cells/ml were
exposed to basal medium with or without 10 nM EGF.
Proliferation assays were performed as described under "Experimental
Procedures." The results represent the mean ± S.E. of
experiments performed in triplicate.
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One explanation for this difference could be that EGF induces the
expression and secretion of IL-3 in cells, which express EGFR, but not
in cells which express K721M and ErbB2. To test the hypothesis that
IL-3 may function as an autocrine mitogen for EGF-treated EGFR 32D
cells, we harvested conditioned medium from EGF-treated EGFR cells and
attempted to grow the parental ErbB-devoid 32D cells in this medium. We
observed no proliferative response in 32D cells to this conditioned
medium (data not shown). We also monitored the effect of anti-IL-3
neutralizing antibodies on the EGF-stimulated growth of EGFR 32D cells.
Whereas these antibodies blocked the mitogenic response of the EGFR
cells to exogenous IL-3, they had no effect on the EGF-induced growth
(data not shown). Therefore, the proliferation of the EGFR cells by EGF
is not because of an IL-3-mediated autocrine loop.
The Kinetics of MAPK and Akt Activation Is Virtually Identical in
Cells Expressing EGFR or K721M-ErbB2, but EGF Is Incapable of
Activating STATs in K721M-ErbB2 cells--
Two additional potential
explanations for the lack of an EGF-induced proliferative response in
K721M-ErbB2 cells were explored. First, it was possible that the
activation of MAPK and Akt in these cells was transient and thus, not
sufficient to drive cell division. Interestingly, a kinetic analysis in
cells expressing EGFR or cells expressing K721M and ErbB2 (clone 7.1)
revealed a strong and prolonged activation of Akt and MAPK by EGF that persisted for 24 h for MAPK and 8 h for Ser-473 of Akt (Fig.
7A). Phosphorylation of Akt on
Thr-308 was more transient and decreased to baseline levels after 30 min of exposure to EGF (lanes 1-4 and 11-14).
Within these parameters signaling by 32D cells possessing EGFR is
virtually indistinguishable from those harboring kinase-inactive K721M
simultaneously with ErbB2. Therefore, activation of MAPK and Akt does
not provide an explanation for the inability of K721M-ErbB2 cells to
grow in response to EGF.

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Fig. 7.
Kinetics of EGF-dependent MAPK
and Akt activation by EGFR and K721M-ErbB2 32D cells.
A, stable 32D cells expressing EGFR or K721M and ErbB2
(clone 7.1) were stimulated with 10 nM EGF. MAPK and Akt
activation were evaluated by Western blotting (WB) as
described in the legends to Figs. 1 and 5. B, STAT
activation by EGF in 32D cells. Cells stably expressing EGFR or
coexpressing K721M and ErbB2 (clone 7.1) were treated with
10 nM EGF for 10 min at 37 °C, lysed, and STAT
activation was monitored by Western blotting (WB) using
antibodies against the phosphorylated form of each STAT
(phosphoSTAT 1, phosphoSTAT 3, phosphoSTAT
5). Consistent loading of each lane was confirmed by stripping the
blot and reprobing for the respective STAT.
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Secondly, activation of STAT5 is critical to the survival and growth of
myeloid cells (32, 33). Previous work from our laboratory had
demonstrated that EGFR ligands activate STATs 1, 3, and 5 in an EGFR
kinase-dependent, Janus kinase (Jak)1-independent manner
(34). To test whether EGF was able to activate STATs in cells
simultaneously expressing K721M and ErbB2, we utilized antibodies
specific for the phosphorylated, activated forms of STAT1 (pY701),
STAT3 (pY705), and STAT5 (pY694/pY699) (Fig. 7B). Evaluation
of lysates derived from clone 7.1 cells treated with EGF for 10 min
indicated that the K721M-ErbB2 receptors were not capable of activating
any of the STATs (lane 4). Exposure of these cells to EGF
for 5 or 30 min also did not result in any detectable STAT activation,
and identical results were obtained using the clone 7.5 (data not
shown). Further, IL-3 was able to activate STATs 1, 3, and 5 in both
clone 7.1 and 7.5 (data not shown). Activation of STATs 1, 3, and 5 was
easily detected in the 32D cells expressing EGFR (lane 2).
Therefore, there was a correlation between EGF-induced proliferation
and activation of STATs in the cell lines expressing either EGFR or
K721M-ErbB2.
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DISCUSSION |
EGF-induced Signaling by Kinase-inactive EGFRs Is Mediated by
ErbB2--
The research presented here better defines the function and
requirement of the EGFR kinase activity in EGF signaling. Early work
indicated that the EGFR kinase was required for EGF-induced biological
responses (9, 10). In contrast, additional studies revealed that
kinase-inactive forms of the EGFR have the capacity to signal in an
EGF-dependent manner when ectopically expressed in certain
cells. Kinase-inactive EGFRs have been reported to stimulate MAPK,
c-fos gene expression, and/or DNA synthesis (11-16). Several possible explanations for this signaling have been proposed and
include the following. (i) The dimerized kinase-inactive EGFR alone has
the intrinsic ability to signal, possibly through the recruitment of
additional signaling proteins. (ii) Signaling by ectopically expressed
kinase-inactive EGFRs occurs through a process of signal amplification,
which is initiated by undetectable low levels of endogenous
kinase-active EGFRs, and (iii) signaling occurs because of an
interaction with another kinase, such as an ErbB-like protein. Our
findings indicate that in ErbB-devoid 32D cells, kinase-inactive EGFRs
have no intrinsic signaling capacity. Instead, we present strong
evidence that signaling by kinase-inactive EGFRs is mediated by
ErbB2 but not by the related ErbB3 and ErbB4. Further, using a
signaling model of EGFR-ErbB2 oligomers in the absence of kinase-active
EGFR homodimers, we demonstrate that the EGFR·ErbB2 complex is a
strong activator of MAPK and the pro-survival protein kinase Akt, and
that ErbB2, but not EGFR, kinase activity was a requisite for this process.
The Potential Physiological Relevance of Signaling by a
Kinase-inactive EGFR--
Targeted disruption of the murine
EGFR gene results in lethality (17-19), whereas
waved-2 mice, which possess an EGFR whose kinase activity is
significantly impaired, are healthy and viable (20). These findings
provide strong evidence that a significant component of EGFR signaling
is independent of EGFR kinase activity. Of all ErbBs, ErbB2 is
considered to be the most widely expressed and is almost always
coexpressed with EGFR in epithelial cells and fibroblasts. Our in
vitro experiments demonstrated that the K721M-ErbB2 oligomer is
exquisitely sensitive to ligand because physiological concentrations of
EGF (10 pM) readily activated MAPK (Fig. 1B).
Taken together, one might expect ErbB2 to play a significant role in
certain aspects of EGFR signaling in vivo.
EGF Cannot Drive Proliferation of 32D Cells Coexpressing K721M and
ErbB2--
Biochemical analysis of signaling through K721M-ErbB2
oligomers in 32D cells showed that EGF activated Ras (data not shown), MAPK and Akt, but not STATs 1, 3, and 5. However, EGF could not supplant the growth requirement of these cells for IL-3. Conversely, EGF was mitogenic in 32D cells expressing wild-type EGFR as has been
previously shown (25). The finding that the K721M-ErbB2 oligomer is
incapable of mediating proliferation of these cells extends previous
work demonstrating that overexpression of ErbB2 was not able to induce
proliferation of 32D cells, despite the fact that similar ErbB2
overexpression resulted in potent transformation of fibroblasts (26).
This difference in the ability of EGFR and ErbB2 to generate a
mitogenic signal in 32D cells was attributed to their respective kinase
domains, demonstrating cell context specificity for mitogenic signaling
by EGFR and ErbB2. In our studies, the kinetics and magnitude of
activation of the MAPK and Akt pathways by 32D cells expressing either
K721M-ErbB2 or EGFR were virtually indistinguishable (Fig.
7A) indicating that defects in these pathways could not
explain the differential growth response of the cells to EGF.
Additionally, the secretion of an EGF-induced autocrine growth factor,
such as IL-3, was ruled out as a reason for the proliferative response
in EGFR cells. The inability of the K721M-ErbB2 complex to activate
STAT transcription factors in response to EGF, however, may provide an
explanation for the lack of a mitogenic response in 32D cells. EGF
activates STATs 1, 3 and 5 via an EGFR kinase-dependent,
EGFR autophosphorylation- and Jak1-independent mechanism, which appears
to require c-Src (16, 34, 35). Activation of STAT5 is essential in the
survival and proliferation of myeloid cells (32). Expression of an
EGFR-Jak2 chimera in 32D cells results in EGF-dependent
tyrosine phosphorylation of STAT 5 and cell proliferation (33). Thus,
the inability of EGF-activated K721M-ErbB2 oligomers to elicit
proliferation in 32D cells may be related to the lack of STAT5 activation.
The Data Are Inconsistent with Signaling by a Kinase-inactive
EGFR-ErbB2 Heterodimer--
It is believed that transmodulation of
ErbB2 by EGF occurs via the generation of an EGFR-ErbB2 heterodimer
(1-3, 5). More recent data suggested that ErbBs may be activated by a
heterotetrameric mode of receptor kinase interaction (36, 37).
Biophysical studies of ligand-induced dimerization of the soluble
extracellular domains of ErbBs were unable to demonstrate the existence
of EGFR-ErbB2, ErbB2-ErbB3, or ErbB3-ErbB3 dimers, but were able to
observe EGFR-EGFR, ErbB4-ErbB4, and ErbB2-ErbB4 dimers (38) causing the
authors to question the prevailing heterodimerization model of ErbB
activation. Our findings are more consistent with a model of EGF-bound
K721M homodimers, which recruit and activate ErbB2 homodimers, and are inconsistent with signaling by K721M-ErbB2 heterodimers for several reasons. First, removal of all tyrosine phosphorylation sites in K721M
had no effect on EGF-stimulated MAPK activation. If
transphosphorylation of K721M by ErbB2 in a heterodimeric complex is
critical to transmodulation, as required in the heterodimer model, then
tyrosine phosphorylation acceptor sites should be required, at least to
some extent. Secondly, EGF-induced tyrosine phosphorylation of ErbB2
occurs in the absence of EGFR kinase activity. Both activation of MAPK
and tyrosine phosphorylation of ErbB2 by EGF requires ErbB2 kinase
activity. The simplest explanation for this observation is that a K721M homodimer presents an interface for an interaction with a monomeric ErbB2, which upon engagement, recruits an additional ErbB2 to form a
[K721M]2[ErbB2]2 heterotetramer. Within
this complex one ErbB2 can transphosphorylate the other in the
activated homodimer. Of course, we cannot rule out that an
EGF-activated heterologous kinase phosphorylates ErbB2, but this kinase
would require ErbB2 kinase activity for activation. Our data
demonstrate a requirement for the mutated kinase domain of K721M
(residues 648-973) in K721M-ErbB2 signaling. Assuming that the
truncated EGFR possessing the extracellular and transmembrane domains
can dimerize in response to EGF, it suggests that the mutated kinase
domains of dimerized K721M contribute to the formation of an interface
for ErbB2 recruitment. Tyrosine phosphorylation of at least one of the
ErbBs in the oligomeric complex is necessary for activation of MAPK,
presumably because this will provide docking sites for adaptor proteins
such as GRB2 and SHC, which can localize the guanine nucleotide
exchange factor, son-of-sevenless, to the plasma membrane where it can
activate Ras. Importantly, focal stimulation of cells with EGF results in an extensive propagation of ligand-independent receptor
phosphorylation over the entire cell surface (6). It is possible that
EGF-bound K721M homo-oligomers inititate a similar propagation of
receptor activation which involves ErbB2.
Conclusions--
The work reported here provides an explanation of
how a kinase-inactive EGFR can generate an EGF-dependent
signal and implies that certain aspects of EGF signaling may be EGFR
kinase-independent in vivo. Further, these results
underscore the importance of ErbB2 in EGF signaling and allow us to
attribute specific signaling events to the EGF-activated EGFR-ErbB2
oligomer. We found that the EGF-stimulated EGFR-ErbB2 complex is a
potent activator of MAPK and Akt, and ErbB2, but not EGFR, kinase
activity is required for this signaling.