From the
Although signaling by the epidermal growth factor (EGF) receptor
is thought to be dependent on receptor tyrosine kinase activity, it is
clear that mitogen-activated protein (MAP) kinase can be activated by
receptors lacking kinase activity. Since analysis of the signaling
pathways used by kinase-defective receptors could reveal otherwise
masked capabilities, we examined in detail the tyrosine
phosphorylations and enzymes of the MAP kinase pathway induced by
kinase-defective EGF receptors. Following EGF stimulation of B82L cells
expressing a kinase-defective EGF receptor mutant (K721M), we found
that ERK2 and ERK1 MAP kinases, as well as MEK1 and MEK2 were all
activated, and SHC became prominently tyrosine-phosphorylated. By
contrast, kinase-defective receptors failed to induce detectable
phosphorylations of GAP (GTPase-activating protein), p62, JAK1, or
p91STAT1, all of which were robustly phosphorylated by wild-type
receptors. These data demonstrate that kinase-defective receptors
induce several protein tyrosine phosphorylations, but that these
represent only a subset of those seen with wild-type receptors. This
suggests that kinase-defective receptors activate a heterologous
tyrosine kinase with a specificity different from the EGF receptor. We
found that kinase-defective receptors induced ErbB2/c-Neu enzymatic
activation and ErbB2/c-Neu binding to SHC at a level even greater than
that induced by wild-type receptors. Thus, heterodimerization with and
activation of endogenous ErbB2/c-Neu is a possible mechanism by which
kinase-defective receptors stimulate the MAP kinase pathway.
The epidermal growth factor receptor (EGF-R)
The
MAP kinase pathway is stimulated by activation of the EGF-R. It is
believed that upon EGF stimulation, both the receptor and SHC become
tyrosine-phosphorylated and bind to each other and the Grb2-Sos complex
(12, 14, 15). This brings Sos to the plasma membrane thus enabling it
to catalyze exchange of GTP for GDP on
p21
Early reports indicated that the kinase activity of the EGF-R is
necessary for all EGF-induced signaling. For example, it has been shown
that kinase-defective (K721A or K721M) EGF-Rs can no longer generate
EGF-induced mitogenesis or DNA synthesis, phospholipase C-
Because of the surprising
nature of this finding, and the fact that it runs counter to the normal
paradigm explaining EGF receptor signaling, we have investigated the
biochemical basis for signaling by a kinase-defective receptor. We have
considered four possible mechanisms by which MAP kinase might be
activated by kinase-defective EGF-Rs: 1) the kinase-defective receptors
possess a low level of residual kinase activity, but a level which is
sufficient to activate the MAP kinase pathway; 2) there are endogenous
wild-type EGF-Rs present in the parental cells that can dimerize with
the kinase-defective receptors, allowing amplification of endogenous
wild-type receptor signaling; 3) a novel kinase-independent signaling
pathway is stimulated by EGF binding and/or receptor dimerization; 4)
there is a heterologous tyrosine kinase that is activated upon EGF
binding and/or receptor dimerization, and it is the heterologous kinase
which is responsible for activating the MAP kinase pathway. We failed
to find evidence for the first three possibilities, but found
substantial EGF-dependent tyrosine phosphorylation and activation of
ErbB2/c-Neu in cells expressing kinase-defective EGF-Rs. We suggest
that transphosphorylation and activation of ErbB2/c-Neu or other
related receptor kinases is sufficient to explain signaling by
kinase-defective EGF receptors.
The following antibodies were kindly
provided: 3A and 4A, anti-human EGF-R monoclonal antibodies for
immunoblotting (D. McCarley, Syntex Research, Palo Alto, CA); 108, an
anti-human EGF-R monoclonal antibody for immunoprecipitation (Dr. J.
Schlessinger, New York University Medical Center, New York); anti-p125
GAP monoclonal (6F2) and polyclonal (P23) antibodies for
immunoprecipitation and immunoblotting (Dr. S. Parsons, University of
Virginia, Charlottesville, VA); anti-p91 polyclonal antibodies for
immunoprecipitation and immunoblotting (Dr. A. Larner, National
Institutes of Health, Bethesda, MD); and an anti-Jak1 polyclonal
antibody for immunoprecipitation and immunoblotting (Dr. A. Ziemiecki,
University of Berne, Berne, Switzerland). The following antibodies were
also used: an anti-phosphotyrosine rabbit polyclonal antibody and a
rabbit nonimmune serum generated in our laboratory; an anti-SHC rabbit
polyclonal and an anti-Jak2 rabbit polyclonal for immunoprecipitation
and immunoblotting (Upstate Biotechnology, Inc.); an anti-c-Neu
monoclonal antibody for immunoprecipitation and immunoblotting (Ab-3
from Oncogene Science); an anti-Grb2 monoclonal antibody and RC20H, a
horseradish peroxidase-linked anti-phosphotyrosine antibody, for
immunoblotting (Transduction Laboratories). All immunoblots (except
RC20H, which is already linked to horseradish peroxidase) were probed
with secondary antibodies (anti-rabbit or anti-mouse) conjugated to
horseradish peroxidase and immunoreactive bands were visualized by
enhanced chemiluminescence (ECL reagents and horseradish
peroxidase-linked antibodies were purchased from Amersham Corp.).
In the phosphotyrosine immunoblots of
Fig. 4A, 120- and 170-kDa phosphoproteins that
co-immunoprecipitate with SHC were observed. The identity of the
120-kDa protein is still unknown, but the phosphorylated 170-kDa
protein is probably the tyrosine-phosphorylated EGF-R, as shown in
Fig. 4B.
It is also known that EGF stimulation of
wild-type EGF-Rs induces SHC binding to Grb2 at the cell
membrane
(14) . By performing anti-Grb2 Western blot analysis on
SHC immunoprecipitates (Fig. 5) we determined that Grb2 was also
able to bind to SHC following stimulation of kinase-defective EGF-Rs.
As expected, both GAP and the p62 GAP
binding protein were clearly phosphorylated upon EGF stimulation of the
wild-type receptor. However, there was no induction of tyrosine
phosphorylation observed on either GAP or p62 following EGF stimulation
of kinase-defective EGF-Rs (Fig. 6A). These results
suggest that GAP and p62 phosphorylation is not required for
kinase-defective EGF-R signaling to the MAP kinase pathway.
We
hypothesize that there is an endogenous (murine) tyrosine kinase (other
than the human EGF-R itself) which associates with the receptor
following EGF stimulation. JAK family members are nonreceptor tyrosine
kinases which are known to become tyrosine phosphorylated and activated
by a variety of growth factor receptors, including EGF. Therefore, we
sought to investigate whether the 120-kDa band seen in Fig. 3and
Fig. 4
might be a JAK family member. It was observed
(Fig. 6, B and C) that JAK1, but not JAK2,
became tyrosine-phosphorylated upon EGF stimulation of wild-type
EGF-Rs, which is consistent with previously published
results
(13) . However, neither JAK1 nor JAK2 became
tyrosine-phosphorylated with stimulation of kinase-defective EGF-Rs.
Therefore, phosphorylation and presumably activation of the JAK-STAT
pathway does not appear to take place upon EGF stimulation of
kinase-defective EGF-Rs. Consequently, we hypothesize that MAP kinase
activation by the kinase-defective receptor occurs independently of
this signaling pathway.
A protein band that
corresponded specifically to the murine EGF-R appeared in the
anti-mouse EGF-R(1382) immunoprecipitation of Swiss 3T3 cells
(Fig. 7, A and B). However, this specific
murine EGF-R protein band did not appear in the 1382
immunoprecipitation of B82L parental cell lysates, even when
immunoprecipitations were done using as much as 24 mg of protein
(Fig. 7B). Hack et al.(40) suggested
that the murine EGF-R runs at a higher molecular weight than the human
EGF-R. To the contrary, we observed that the murine EGF-R
immunoprecipitated from Swiss 3T3 cells ran at a molecular weight
indistinguishable from the human EGF-R immunoprecipitated from B82L
cells overexpressing EGF-Rs (Fig. 7A). Data shown in
Fig. 7B also demonstrate that lysates from B82L (24 mg
of protein) and Swiss 3T3 (8 mg of protein) cells contained a protein
slightly higher in molecular weight than the EGF-R which was
immunoprecipitated using either the specific anti-EGF-R antibody(1382)
or the nonspecific nonimmune rabbit serum (NIS)
(Fig. 7B). Thus, we believe that the ``mouse
EGF-R'' detected in B82L cells by Hack et al.(40) is likely to have been a nonspecific protein that ran at a
molecular weight slightly higher than either the murine or human EGF-R.
In contrast to what was suggested by Hack et al.(40) ,
we found no detectable endogenous murine EGF-R present in the B82L
cells when blotting using either antibody 22 against the murine EGF-R
(Fig. 7) or RC20H against phosphotyrosine (data not shown).
One might analogously argue that very low levels of endogenous
EGF-Rs are responsible for the signaling detected in cells expressing
the kinase-defective receptor. However, our attempts to detect
endogenous receptors in B82L cells using immunological methods have
been entirely negative, which is consistent with previous reports of
Northern blot analysis and ligand binding studies
(37) . As
described under ``Results,'' we believe that the single
report
(40) to the contrary could be artifactual. Furthermore,
it is difficult to imagine how this hypothesis could be correct, since
the parental B82L cells (which do not express transfected human EGF-Rs)
do not respond detectably to EGF with respect to any signaling pathway
tested. For example, as demonstrated by Soler et
al.(46) , as few as 10
It is conceivable that the EGF receptor possesses a
signaling activity which does not rely on its kinase activity. For
example, EGF-induced phosphorylation and activation of phospholipase
C-
Furthermore, the relative activation of ERK1, ERK2, MEK1, and MEK2
is similar whether occurring by kinase defective or wild-type
receptors, consistent with the idea that the normal kinase-dependent
pathway is operative. By contrast, several reports indicate that
different signaling pathways preferentially activate different isoforms
of ERKs and MEKS. For example, thrombin preferentially activates ERK2
versus ERK1 in platelets
(52) , and v-ras preferentially activates MEK1 versus MEK2 in NIH 3T3
cells (53). Therefore, no evidence exists to support the notion that
kinase-defective EGF receptors signal via a novel, kinase-independent
pathway.
We feel it is most probable that kinase-defective EGF-Rs
signal by activating (directly or indirectly) a heterologous endogenous
tyrosine kinase. We found that the kinase-defective receptors do not
cause detectable phosphorylation of JAK1 or JAK2 and thus presumably do
not activate these nonreceptor tyrosine kinases. However, an
unidentified protein of similar M
We also have examined activation of
the Src family kinases, c-Src, c-Fyn, and c-Yes in response to
activation of EGF receptors. We found no consistent activation or
phosphorylation of any of these kinases in cells expressing either
wild-type or kinase-defective receptors (data not shown).
By
contrast, we found that endogenous ErbB2/c-Neu showed substantial
EGF-induced tyrosine phosphorylation and activation in cells expressing
the kinase-defective EGF receptor (Fig. 8, B and
C). It has been shown previously that ErbB2/c-Neu can cluster
with human kinase-defective (K721A) EGF-Rs
(45) and that the
kinase-defective EGF-Rs become tyrosine-phosphorylated following EGF
stimulation of cells that co-express ErbB2/c-Neu. It has been suggested
(54) that heterodimerization of ErbB2/c-Neu with the EGF-R occurs
preferentially over homodimerization of either ErbB family member in
the presence of EGF. In fact, kinase-defective ErbB2/c-Neu functions as
a dominant-negative inhibitor of EGF-R signaling
(55) .
Therefore, we hypothesize that upon EGF stimulation, the overexpressed
human EGF-R heterodimerizes with and activates endogenous B82L murine
ErbB2/c-Neu.
It is uncertain how kinase-inactive EGF receptors cause
the EGF-induced activation and tyrosine phosphorylation of ErbB2/c-Neu.
Although it is clear that activation of ErbB family receptors is
accompanied by transphosphorylation of dimerized or clustered
receptors, intramolecular phosphorylations have not been ruled
out
(5, 54, 56) . Alternatively, ErbB2/c-Neu
could be phosphorylated by transphosphorylation in receptor trimers or
higher order oligomers, which have been reported to occur with ErbB
family receptors upon EGF
stimulation
(6, 45, 57) . It is also known that
dimerization and clustering of receptors are reversible
processes
(6) . ErbB2/c-Neu may first heterodimerize with the
human EGF-R and become enzymatically activated by a mechanism
independent of phosphorylation; the activated ErbB2/c-Neu could then be
released and dimerize with another ErbB2/c-Neu molecule, which would
precipitate transphosphorylations. Distinguishing between these
possibilities will require further analysis of the physical and
functional interactions between ErbB family receptors.
Surprisingly,
we observed that the stimulation of ErbB2/c-Neu in vitro autokinase activity and SHC binding by kinase-defective EGF-Rs was
actually greater than that seen with wild-type receptors
(Fig. 8C and 9). This enhanced ErbB2/c-Neu signaling
activity in the cells expressing kinase-defective receptors might
reflect the failure of kinase-defective receptors to induce a
desensitizing feedback loop and/or the ability of
tyrosine-phosphorylated wild-type EGF-Rs to compete with ErbB2/c-Neu
for binding to SHC. Whatever the mechanism, the fact that the
endogenous ErbB2/c-Neu apparently compensates for the lack of EGF-R
kinase activity could explain the surprisingly large activation of the
MAP kinase pathway in these cells.
Substantial evidence is
accumulating that receptors can form functional hetero-oligomeric
complexes and that the specificity of signaling can depend on the
formation of these hetero-oligomers. For example, the activated EGF
receptor binds to and phosphorylates ErbB3, which apparently lacks
intrinsic kinase activity. This transphosphorylation is hypothesized to
be largely responsible for the activation of phosphatidylinositol
3`-kinase by ErbB3 upon EGF stimulation
(58, 59) . We
have observed that GAP, the GAP-associated protein p62, the nonreceptor
tyrosine kinase JAK1, and p91STAT1 do not become
tyrosine-phosphorylated following EGF stimulation of kinase-defective
EGF-Rs in B82L cells, although all these phosphorylations are
substantially stimulated by the wild-type receptor. If, as we suggest,
signaling by the kinase-defective receptor is mediated by ErbB2/c-Neu
and perhaps other related EGF receptor family members, these ErbB
receptors may diverge from the EGF receptor (ErbB1) in the signaling
pathways they activate. Perhaps ErbB2/c-Neu specifically signals to SHC
and the MAP kinase pathway but not to other pathways which include GAP,
JAK1, or p91STAT1. The different ErbB family members and their
heterodimerization with the EGF receptor could be a route to provide
different specificities for EGF in different cell types.
The ability
of EGF receptors to heterodimerize with and to activate related but
nonidentical endogenous receptors explains two otherwise peculiar
attributes of mutant EGF receptors: (i) it has been reported that EGF
receptor mutants devoid of autophosphorylation sites signal effectively
to a subset of EGF-inducible
pathways
(45, 60, 61) . These signaling events
could occur through heterologous receptors, thus bypassing the need for
autophosphorylation sites on the EGF-R. (ii) Whereas most reports
indicate that kinase-defective EGF-Rs do not induce
c-fos(32, 34) , one recent publication reports
the contrary
(62) . Since the c-fos promoter contains
binding sites for p91STAT1 as well as for the SRF (which can be
regulated by MAP kinase), it is possible that the earlier results used
conditions in which c-fos expression was dependent on p91STAT1
(which is not phosphorylated in response to kinase-defective EGF-Rs),
whereas the more recent work utilized cells and conditions in which the
SRF was limiting for gene expression. Thus, these findings support the
interesting suggestion of Selva et al.(34) that
selective, partial signaling by the kinase-defective EGF receptors may
be useful in investigating the role of MAP kinase in signal
transduction.
Ectopic expression of receptor mutants has been a
powerful tool for understanding the physical and functional
interactions of receptors with cellular signaling molecules. The
results reported here, showing that kinase-defective EGF receptors can
induce the phosphorylation and activation of the related ErbB2/c-Neu
receptor, raise the caution that interactions between mutant receptors
and endogenous receptors of the same family can confound the
interpretation of such signaling studies but can also provide the
opportunity to study the effects of lateral communication between
regulatory receptors at the surface of cells.
We are grateful to G. N. Gill for providing the B82L
cell lines and to S. Parsons, C. M. Silva, D. McCarley, A. Larner, and
H. S. Earp for providing both antibodies and helpful technical
advice/suggestions. We also thank J. T. Parsons for insightful
discussions.
(
)
is a 170-kDa transmembrane receptor tyrosine
kinase
(1, 2) . EGF-R ligands, such as EGF or
transforming growth factor-
, bind to the extracellular domain of
the receptor and induce receptor dimerization and
clustering
(3, 4, 5, 6) ; this in turn
results in activation of the receptor's intracellular tyrosine
kinase activity
(7) . The activated, clustered receptors
transphosphorylate other co-clustered receptors, creating binding sites
for cellular proteins containing SH2 domains, and become activated to
continue the signal transduction cascade. Well characterized substrates
for the receptor include phospholipase C-
(8, 9) ,
the GTPase-activating protein of p21
(GAP)
(10) , and the SH2 containing adapter protein
SHC
(11, 12) . The nonreceptor tyrosine kinase JAK1 and
transcription factors of the STAT family also become
tyrosine-phosphorylated following EGF stimulation
(13) .
(16, 17, 18, 19) .
The activated p21
-GTP is then able to bind and
facilitate activation either of Raf or other serine/threonine protein
kinases, which in turn stimulate the activity of MEK (MAPK or Erk
kinase) (20-26). MEK acts as a dual specificity kinase which
phosphorylates MAP kinase on both a threonine and a tyrosine, thereby
activating MAP kinase
(27, 28, 29, 30) .
phosphorylation or activation, fos or myc gene
expression, cell surface transport processes, or increases in
intracellular calcium
concentration
(31, 32, 33, 34) . More
recently, however, Campos-Gonzalez and Glenney
(35) , Selva
et al.(34) , and Coker et al.(36) have
reported that EGF stimulation of kinase-defective EGF-R mutants can
still induce activation of MAP kinase.
Cell Culture
The B82L parental cells and the
B82L cells expressing the human wild-type (Lys) EGF
receptor and the mutated, kinase-defective (Met
) EGF
receptor were a generous gift from Dr. G. N. Gill (University of
California, San Diego, CA) and have been described
previously
(32) . A mutant dihydrofolate reductase gene provides
a dominant selectable marker for the B82L cells overexpressing the EGF
receptor
(37) . These cells were maintained in
-minimum
essential medium (Life Technologies, Inc.) containing 10% dialyzed
fetal bovine serum and 2 µM methotrexate. Before
experiments, the cells were kept in the absence of methotrexate (in
Dulbecco's modified Eagle's medium (DMEM) containing 10%
fetal bovine serum) for at least 24 h. The B82L parental cells were
maintained in DMEM containing 10% fetal bovine serum. Dishes of the
confluent cells (1-2
10
cells/100-mm dish)
were then incubated overnight (8-12 h) in serum-free DMEM before
stimulation with 100 ng/ml EGF (Upstate Biotechnology, Inc. Catalog
number 01-107).
Immunoprecipitation and Immunoblotting
Cells were
left untreated(-) or exposed to EGF (+) for 3 min. The cells
were washed twice with ice-cold phosphate-buffered saline (PBS) and
lysed in lysis buffer containing 50 mM Tris, pH 7.4, 150
mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1
mM EDTA, 1 mM NaVO
, 10
µg/ml pepstatin A, 50 µg/ml leupeptin, 0.02 TIU/ml of
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 40 mM
4-nitrophenyl phosphate Na
salt. Insoluble material was
removed by centrifugation at 4 °C for 15 min at 14,000 rpm.
Supernatants containing equal amounts of protein (500 µg) were
incubated for 2 h at 4 °C with either: 1) 4 mg of washed protein
A-Sepharose (Pharmacia Biotech Inc.) and a specific rabbit polyclonal
antibody or rabbit nonimmune serum (as a control, data not shown) or 2)
100 µl of washed protein G-agarose (Boehringer Mannheim) and a
specific mouse monoclonal antibody. After washing the
immunoprecipitates three times with lysis buffer (or two washes with
lysis buffer and 2 washes with Tris-buffered saline), Laemmli sample
buffer was added, and the samples were boiled for 2 min. The samples
were analyzed by SDS-PAGE and transferred to nitrocellulose filters for
Western blot analysis.
MAP Kinase and MEK Assays
The B82L parental cells,
as well as the B82L cells overexpressing the kinase-defective
(K-) and wild-type (K+) EGF receptors were left
untreated(-) or exposed to EGF (+) for 5 min. The activities
of ERK2 (p42) and ERK1 (p44) MAP kinases were determined by
immunoprecipitation using TR2 and R1798 rabbit polyclonal antibodies
and performing in vitro kinase reactions using myelin basic
protein as a substrate, as described
(38) . The activities of
MEK1 and MEK2 were determined by immunoprecipitation using rabbit
polyclonal antibodies (raised against either a COOH-terminal peptide
from MEK1 or a NH-terminal peptide from MEK2) and
performing in vitro kinase reactions using recombinant
kinase-defective MAP kinase as a substrate, as described (38).
Nonimmune serum controls were done (data not shown) to ensure that the
kinase activities were specific. Western blot analysis using anti-MAP
kinase and anti-MEK antibodies were also done to ensure that equal
amounts of MAP kinase or MEK proteins were present in the
immunoprecipitations (data not shown).
Immunoprecipitation and Immunoblotting of Endogenous
Murine EGF-Rs
Cells were grown in large scale in DMEM containing
10% fetal bovine serum (without methotrexate) for at least 24 h before
cell lysis. For each immunoprecipitation, 4-10 150-mm confluent
dishes were washed twice with ice-cold PBS. The plates were then
incubated with 2 ml of ice-cold PBS containing 1 mM EDTA, and
the cells were scraped into 15-ml conical tubes (8 ml/tube). The cells
were pelleted by centrifugation and the PBS-EDTA was decanted. The
pelleted cells were lysed in 1 ml radioimmune precipitation lysis
buffer (containing 50 mM Tris, pH 7.4, 150 mM NaCl,
1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA,
1 mM NaVO
, 10 µg/ml pepstatin A,
50 µg/ml leupeptin, 0.02 TIU/ml of aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 40 mM 4-nitrophenyl phosphate
Na
salt) and thoroughly vortexed. The lysed cells were
incubated on ice for 10 min, and insoluble material was removed by
centrifugation at 4 °C for 15 min (twice) at 14,000 rpm.
Supernatants containing equal amounts of protein (1-24 mg) were
incubated for 2 h at 4 °C with 4 mg of washed protein A-Sepharose
(Pharmacia) and a rabbit nonimmune serum. The samples were pelleted by
centrifugation and the precleared supernatants were incubated with
either: 1) 4 mg of washed protein A-Sepharose and a specific anti-EGF-R
rabbit polyclonal antibody 1382 (which recognizes murine or human
EGF-Rs) or 2) 4 mg of washed protein A-Sepharose and a rabbit nonimmune
serum. After washing the immunoprecipitates three times with lysis
buffer, Laemmli sample buffer was added, and the samples were boiled
for 2 min. The samples were analyzed by SDS-PAGE and transferred to
nitrocellulose filters. Western blot analysis was done using either an
anti-EGF-R-specific rabbit polyclonal antibody 22 (which recognizes
murine or human EGF-Rs) or RC20H, a horseradish peroxidase linked
anti-phosphotyrosine antibody (data not shown). Antibodies 1382 and 22
were generously provided by Dr. H. S. Earp, University of North
Carolina, Chapel Hill, NC.
RESULTS
In Vivo Tyrosine Phosphorylation and In Vitro Kinase
Activity of Wild-type and Kinase-defective EGF-Rs
We have
examined EGF-R signaling by using a mouse cell line (B82L) transfected
with and overexpressing either the wild-type human EGF-R (K+) or a
kinase-defective human EGF-R (K-) that contains a point mutation
in which lysine 721 is changed to methionine. Expression and activity
of the kinase-negative and wild-type receptors isolated from K+,
K-, and B82L parental cells were analyzed by Western blot
analysis and by measuring in vitro autokinase activity. The
B82L parental cells contained no detectable EGF-Rs
(Fig. 1A), and therefore no tyrosine phosphorylation or
autokinase activity was detected in the EGF-R immunoprecipitates from
these cells (Fig. 1, B and C). The
kinase-defective EGF-R showed very little induced in vivo tyrosine phosphorylation following EGF stimulation. However, after
prolonged overexposure of the phosphotyrosine immunoblot
(Fig. 1B), EGF-induced in vivo phosphorylation
of kinase-defective EGF-Rs was detectable, at a level less than 10%
that of the wild-type EGF-Rs. This small amount of in vivo phosphorylation occurred in spite of the fact that the receptors
were almost devoid of intrinsic kinase activity, as assayed by an
in vitro autokinase reaction (Fig. 1C). We
suspect that an endogenous (murine) tyrosine kinase is responsible for
the low level in vivo tyrosine phosphorylation taking place on
the kinase-defective EGF-Rs, in agreement with the suggestion of Selva
et al.(34) .
Figure 1:
In
vivo tyrosine phosphorylation and in vitro kinase
activity of wild-type and kinase-defective EGF-Rs. Confluent B82L mouse
parental cells or B82L cells overexpressing either the kinase-defective
(B82L K-) or wild-type (B82L K+) human EGF
receptor were serum-starved overnight and treated with (+) or
without (-) 100 ng/ml of EGF for 2 min before being lysed. EGF
receptors were immunoprecipitated from the cell lysates using an EGF
receptor-specific monoclonal antibody (108) and separated on a 7.5%
polyacrylamide gel. The gel was transferred to nitrocellulose, and
Western blot analysis was done using either EGF receptor specific
monoclonal antibodies (3A and 4A) (A) or a
phosphotyrosine-specific antibody (RC20H) (B). The
immunoprecipitated EGF receptors were also used to perform in vitro kinase assays, which were analyzed by SDS-PAGE and exposed to film
for 17 h (C). We found that immunoprecipitated wild-type
EGF-Rs were constitutively active; there was no difference in the
in vitro autokinase enzymatic activity in the presence or
absence of in vivo EGF stimulation. The reduction of
P incorporation into EGF-Rs from B82L K+ cells
(+EGF) compared with B82L K+ (-EGF) is likely due to
in vivo phosphorylation on the receptor (before the cells are
lysed), which reduces the number of sites available for
autophosphorylation by [
-
P]ATP in the
in vitro kinase assay.
Activation of the MAP Kinase Pathway following EGF
Stimulation of Wild-type or Kinase-defective EGF-Rs
The
activities of p42 and p44 MAP kinases (ERK2 and ERK1) following EGF
stimulation were determined using antibodies specific for p42 and p44
MAP kinases (Fig. 2, A and B). As expected,
there was no significant EGF-inducible p42 or p44 MAP kinase activation
in the B82L parental cells, which lack EGF-Rs. In the B82L cells which
overexpress wild-type EGF-Rs, there was a clear activation of both p42
and p44 MAP kinase activity after EGF stimulation. Moreover, there was
also a significant activation (25-50% of what is seen in the
K+ cells) of both p42 and p44 MAP kinase upon EGF stimulation of
kinase-defective EGF-Rs. Since kinase-defective EGF-Rs can signal to
MAP kinase family members, we examined whether the normal upstream
activators of MAP kinase, MEK1 and MEK2, were also stimulated by EGF
treatment of kinase-defective EGF-Rs. EGF-induced activation of MEK1 or
MEK2 occurred in a manner which was very similar to p42 and p44 MAP
kinase. MEK1 and MEK2 were clearly activated following EGF stimulation
of either the wild-type or (to a lesser degree) the kinase-defective
receptors (Fig. 2, C and D). Therefore, ERK1,
ERK2, MEK1, and MEK2 are all activated by kinase-defective EGF-Rs in
similar relative proportions (e.g. 25-50% of the level
induced by wild-type receptors), and thus, kinase-defective EGF-Rs do
not selectively activate a subset of MEK or MAP kinase isoforms. As
discussed below, this suggests that similar pathways are used by
wild-type and kinase-defective EGF-Rs in activating MAP kinase.
Figure 2:
Activation of ERK1 and ERK2 MAP kinases,
MEK1, and MEK2 following EGF stimulation of wild-type or
kinase-defective EGF-Rs. Confluent B82L mouse parental cells or B82L
cells overexpressing either the wild-type (B82L K+) or
kinase-defective (B82L K-) human EGF receptor were
serum-starved overnight and treated with (+) or without (-)
100 ng/ml of EGF for 5 min before being lysed. Either ERK2 (A)
or ERK1 MAP kinase (B) or MEK1 (C) or MEK2
(D) was immunoprecipitated and assayed for enzyme activity as
described under ``Experimental
Procedures.''
Tyrosine Phosphorylations Induced by EGF Stimulation of
Kinase-defective EGF-Rs
Since the data in Fig. 1suggested
stimulation of kinase-defective EGF-Rs can activate an endogenous
tyrosine kinase, we determined whether other proteins (besides MAP
kinase) might become tyrosine-phosphorylated following addition of EGF
to cells expressing kinase-defective receptors (Fig. 3). In
addition to MAP kinase, EGF stimulation of kinase-defective receptors
induced the phosphorylation of proteins in the 40-60 kDa range,
as well as at 90 kDa, and (faintly) at 120 kDa.
Figure 3:
Tyrosine phosphorylation events induced by
EGF stimulation of wild-type or kinase-defective EGF-Rs. B82L mouse
parental cells or B82L cells overexpressing either the kinase-defective
(B82L K-) or wild-type (B82L K+) human EGF
receptor were grown to confluence. The cells were then serum-starved
overnight and treated with (+) or without (-) 100 ng/ml of
EGF for 3 min before being lysed. Equivalent amounts of protein were
used to make whole cell lysates. These whole cell lysates were then
electrophoresed on a 7.5% polyacrylamide gel and transferred to
nitrocellulose. Phosphotyrosine levels were measured by doing Western
blot analysis using an anti-phosphotyrosine rabbit polyclonal antibody.
The molecular weights of prestained protein markers are indicated on
the left.
Two of the
phosphorylated bands seen in Fig. 3(p46 and p52) correspond in
molecular weight to isoforms of SHC, an SH2-containing adapter protein
which is a well characterized substrate for the EGF-R. In order to
determine whether SHC becomes tyrosine-phosphorylated following EGF
stimulation of kinase-defective EGF-Rs, SHC was immunoprecipitated from
the same cell lysates used in Fig. 3. The immunoprecipitates were
separated by SDS-PAGE and blotted with anti-phosphotyrosine antibodies.
Fig. 4A demonstrates that three isoforms of SHC clearly
became tyrosine phosphorylated after EGF stimulation of the
kinase-defective EGF-R.
Figure 4:
SHC is tyrosine-phosphorylated and
associates with the EGF-R following EGF stimulation of wild-type or
kinase-defective EGF-Rs. B82L mouse parental cells or B82L cells
overexpressing either the kinase-defective (B82L K-) or
wild-type (B82L K+) human EGF receptor were grown to
confluence. The cells were then serum starved overnight and treated
with (+) or without (-) 100 ng/ml of EGF for 3 min before
being lysed. Equivalent amounts of protein were used to make whole cell
lysates. SHC was immunoprecipitated from these cell lysates using a
SHC-specific polyclonal antibody (or nonimmune rabbit serum as a
negative control, data not shown), and the immunoprecipitations were
electrophoresed on 7.5% polyacrylamide gels. Two identical gels were
transferred to nitrocellulose for anti-phosphotyrosine (RC20H) or
anti-EGF-R Western blot analysis (A). Anti-SHC Western blot
analysis was also done (data not shown) to guarantee that equivalent
amounts of SHC were present in all samples. The anti-EGF receptor
Western blot was done using human EGF receptor-specific monoclonal
antibodies (3A and 4A) to detect whether either kinase-defective
(K-) or wild-type (K+) EGF receptors can associate with SHC
in an EGF-dependent manner (B).
Since SHC is known to bind to the wild-type
EGF-R upon EGF stimulation (12), we investigated whether SHC could also
bind to the kinase-defective receptor. Anti-EGF-R Western blot analysis
of the SHC immunoprecipitates (Fig. 4B) showed that SHC
did bind to both the wild-type and kinase-defective EGF-Rs in an
EGF-dependent manner.
Figure 5:
SHC associates with Grb2 following EGF
stimulation of wild-type or kinase-defective EGF-Rs. B82L mouse
parental cells or B82L cells overexpressing either the wild-type
(B82L K+) or kinase-defective (B82L K-)
human EGF receptor were grown to confluence. The cells were then
serum-starved overnight and treated with (+) or without (-)
100 ng/ml of EGF for 3 min before being lysed. SHC was
immunoprecipitated from these cell lysates using a SHC-specific
polyclonal antibody, and the immunoprecipitations were electrophoresed
on a 15% polyacrylamide gel. The gel was transferred to nitrocellulose
and Western blot analysis was done using a Grb2-specific monoclonal
antibody. Whole cell lysates were separated on the same gel to show
that equivalent amounts of Grb2 were present in the cell lysates.
Anti-SHC Western blot analysis was also done (data not shown) to
guarantee that equivalent amounts of SHC were present in all samples.
IP, immunoprecipitation.
Tyrosine Phosphorylations Which Fail to be Induced by EGF
Stimulation of Kinase-defective EGF-Rs
Besides SHC, other
cellular proteins which have been reported to become
tyrosine-phosphorylated following stimulation of wild-type EGF
receptors include GAP
(10) , the tyrosine kinase JAK1 and the
tran-scription factor p91STAT1
(13) . Protein tyrosine
phosphorylations with M values consistent with
possible phosphorylation of these proteins were observed following
stimulation of both wild-type and (to a lesser extent) kinase defective
EGF-Rs ( Fig. 3and Fig. 4). To determine whether
kinase-defective receptors can cause these phosphorylations, the
proteins were immunoprecipitated, separated by PAGE, and their tyrosine
phosphorylation assessed by Western blotting with an
anti-phosphotyrosine antibody.
Figure 6:
GAP, GAP-associated protein p62, JAK1, and
p91STAT1 are tyrosine-phosphorylated following EGF stimulation of
wild-type, but not kinase-defective, EGF-Rs. B82L mouse parental cells
(data not shown) or B82L cells overexpressing either the
kinase-defective (B82L K-) or wild-type (B82L
K+) human EGF receptor were grown to confluence. The cells
were then serum-starved overnight and treated with (+) or without
(-) 100 ng/ml of EGF for 3 min before being lysed. A,
GAP was immunoprecipitated from these cell lysates using GAP-specific
monoclonal or polyclonal antibodies and the immunoprecipitates were
electrophoresed on a 7.5% polyacrylamide gel. The gel was transferred
to nitrocellulose and Western blot analysis was done using either a
GAP-specific polyclonal antibody or a phosphotyrosine specific
horseradish peroxidase-linked antibody (RC20H). Either JAK1
(B) or JAK2 (C) or p91STAT1 (D) was
immunoprecipitated (using specific polyclonal antibodies) from these
cell lysates, and the immunoprecipitations were electrophoresed on 7.5%
polyacrylamide gels. The gel was transferred to nitrocellulose, and
Western blot analysis was done using either JAK1 or JAK2 or
p91STAT1-specific polyclonal antibodies or a phosphotyrosine-specific
horseradish peroxidase-linked antibody (RC20H). IP,
immunoprecipitation.
Similar
experiments were also done with p91STAT1
(13, 39) , since
a 90-kDa protein was tyrosine-phosphorylated following EGF
stimulation of kinase-defective EGF-Rs. As was found with GAP and p62,
phosphorylation of p91STAT1 occurred through the wild-type receptors,
but not kinase-defective EGF-Rs (Fig. 6D).
No Endogenous Murine EGF-Rs Are Detected in the B82L
Parental Cells
It has previously been observed that B82L cells
do not contain detectable endogenous EGF-R mRNA or protein, as assessed
by RNA blot analysis and ligand binding studies
(37) . However,
Hack et al.(40) have suggested that a very low level
of endogenous murine EGF-Rs was detectable by anti-EGF-R
immunoprecipitation from B82L cells and phosphotyrosine Western blot
analysis. These authors proposed that endogenous wild-type EGF-Rs might
somehow be responsible for EGF-induced signaling by the overexpressed
human kinase-defective EGF-Rs. We performed similar tests for
endogenous murine EGF-Rs in B82L cells, by doing immunoprecipitations
and Western analysis using rabbit polyclonal antibodies that recognize
both murine and human EGF-Rs. We also included positive controls (using
mouse Swiss 3T3 fibroblast cells and B82L cells overexpressing human
kinase-defective EGF-Rs) and negative controls (by using a nonimmune
rabbit serum for immunoprecipitations).
Figure 7:
No endogenous murine EGF-Rs are detected
in the B82L parental cells. B82L mouse parental and B82L cells
overexpressing human kinase-defective EGF receptors (B82L
K-) and mouse Swiss 3T3 cells were grown to confluence in
large quantities. A, endogenous murine EGF receptors were
immunoprecipitated from B82L and B82L K- (11 mg of
protein/immunoprecipitation (IP) and Swiss 3T3 (2.5 mg of
protein/immunoprecipitation) cell lysates using either a rabbit
polyclonal antibody 1382 (which recognizes murine and human EGF-Rs) or
a rabbit nonimmune serum as a negative control. B, endogenous
murine EGF receptors were immunoprecipitated from B82L (24 mg of
protein/immunoprecipitation) and Swiss 3T3 (8 mg of protein/
immunoprecipitation) cell lysates using either a rabbit polyclonal
antibody 1382 (which recognizes mouse and human EGF-Rs) or a rabbit
nonimmune serum as a negative control. The immunoprecipitates were
separated on 7.5% polyacrylamide gels and transferred to
nitrocellulose. Western blot analysis was then done using the rabbit
polyclonal antibody 22 which recognizes mouse and human EGF
receptors.
Enhanced Stimulation of ErbB2/c-Neu Enzymatic Activity
and Binding to SHC following EGF Stimulation of Kinase-defective
EGF-Rs
In addition to testing for endogenous mouse EGF-Rs
(ErbB1), we also investigated whether other EGF-R/ErbB family members,
particularly ErbB2 (or c-Neu), might participate in signaling by
kinase-defective EGF-Rs. Therefore, we immunoprecipitated ErbB2/c-Neu
from the B82L cell lines using a monoclonal antibody that recognizes
murine ErbB2/c-Neu. Endogenous ErbB2/c-Neu was clearly present in all
three cell lines (Fig. 8A) and became
tyrosine-phosphorylated (Fig. 8B) and activated
(Fig. 8C) upon EGF stimulation of either the wild-type
or kinase-defective EGF-Rs. There was no EGF-inducible phosphorylation
or activation of ErbB2/c-Neu in the B82L parental cell line and this is
expected for two reasons: there are no detectable mouse ErbB1/EGF-Rs in
the B82L cells (as seen in Fig. 7), and EGF does not detectably
bind ErbB2/c-Neu or any other known ErbB family members, besides
ErbB1
(41, 42, 43) . Therefore, we hypothesize
that the kinase-defective EGF-R (as well as the wild-type receptor) is
able to heterodimerize with and activate endogenous mouse ErbB2/c-Neu
which is present in B82L cells.
Figure 8:
Endogenous murine ErbB2/c-Neu is
tyrosine-phosphorylated and activated following EGF stimulation of
wild-type or kinase-defective EGF-Rs. B82L mouse parental cells or B82L
cells overexpressing either the wild-type (B82L K+) or
kinase-defective (B82L K-) human EGF receptor were grown
to confluence. The cells were then serum-starved overnight and treated
with (+) or without (-) 100 ng/ml of EGF for 3 min before
being lysed. c-Neu was immunoprecipitated from these cell lysates using
a c-Neu-specific monoclonal antibody and the immunoprecipitates were
electrophoresed on a 7.5% polyacrylamide gel. The gel was transferred
to nitrocellulose, and Western blot analysis was done using either the
c-Neu-specific monoclonal antibody (A) or a horseradish
peroxidase-linked anti-phosphotyrosine antibody, RC20H (B).
In vitro autokinase assays with the c-Neu immunoprecipitations
were also done (Fig. 7C). IP,
immunoprecipitation.
It has been reported that SHC is a
substrate for ErbB2/c-Neu and that phosphorylation of carboxyl-terminal
sites located on ErbB/c-Neu are required for both the phosphorylation
of SHC by ErbB/c-Neu and complex formation between the SHC and
ErbB/c-Neu proteins
(44) . Since the EGF-R and ErbB2/c-Neu can
form heterodimers
(45) , we investigated whether SHC binds to
ErbB2/c-Neu following EGF treatment of wild-type or kinase-defective
EGF-Rs. We found that ErbB2/c-Neu binds to SHC in an EGF-dependent
manner as assessed by co-immunoprecipitation (Fig. 9).
Interestingly, both the in vitro autokinase reaction
(Fig. 8C) and the binding experiments (Fig. 9)
indicate that the activation and binding of ErbB2/c-Neu to SHC is
greatly enhanced upon EGF stimulation of kinase-defective EGF-Rs,
compared with that seen with wild-type receptors. Perhaps the kinase
activity of ErbB2/c-Neu can compensate for the lack of tyrosine kinase
activity of the kinase-defective EGF-Rs. Therefore, activation of the
MAP kinase pathway by kinase-defective EGF-Rs may occur via the
activation of ErbB2/c-Neu.
Figure 9:
ErbB2/c-Neu binds to SHC following EGF
stimulation of wild-type or kinase-defective EGF-Rs. B82L mouse
parental cells or B82L cells overexpressing either the wild-type
(B82L K+) or kinase-defective (B82L K-)
human EGF receptor were grown to confluence. The cells were then
serum-starved overnight and treated with (+) or without (-)
100 ng/ml of EGF for 3 min before being lysed. SHC was
immunoprecipitated from these cell lysates using a SHC-specific
polyclonal antibody (or nonimmune rabbit serum as a negative control,
data not shown), and the immunoprecipitates were electrophoresed on a
7.5% polyacrylamide gel. The gel was transferred to nitrocellulose, and
Western blot analysis was done using a c-Neu-specific monoclonal
antibody. Anti-SHC Western blot analysis was also done (data not shown)
to guarantee that equivalent amounts of SHC were present in all
samples. IP, immunoprecipitation.
DISCUSSION
Even though it is generally believed that EGF receptor
signaling depends on its tyrosine kinase activity, it is clear that
mutant, kinase-defective EGF-Rs are capable of robustly activating the
MAP kinase pathway. Although one might argue that these results could
be explained by residual kinase activity retained by these mutant
receptors, substantial evidence indicates that this is not the case.
First, virtually no in vitro kinase activity associated with
the immunopurified, kinase-defective EGF-R could be detected, unless
the immunopurification was performed under nonstringent conditions
(which presumably allows co-isolation of a contaminating tyrosine
kinase), in agreement with previously published results
(34) .
Second, two different kinase-defective EGF-receptor mutants have been
shown to activate MAP kinase: the Lys mutant (in the ATP
binding site), which was used in the experiments reported here, and an
Asp
mutant (in the catalytic
loop)
(34, 35, 36) . Whereas the first mutation
likely renders the receptor unable to properly bind ATP, the second
mutation eliminates the receptor's ability to catalyze phosphate
transfer. Thus, although it is difficult to prove unequivocally that
the mutant receptors are totally devoid of kinase activity, no evidence
supports the suggestion that the partial signaling activity of
kinase-defective receptors is due to residual receptor kinase activity.
endogenous wild-type
receptors per cell can dramatically stimulate phosphorylation of SHC.
Moreover, if this hypothesis were correct, the endogenous receptors
would have to be activated by a receptor mutant which has been
reported to function as a ``dominant-negative'' with respect
to endogenous EGF-R signaling
(47, 48) . Finally,
amplified signaling by low levels of endogenous receptors cannot easily
be reconciled with the strikingly dichotomous signaling activated by
the kinase-defective EGF-R, in which the SHC/MAP kinase pathway is
substantially activated but other tyrosine phosphorylations (e.g. GAP/p62 or JAK/STAT) which are equally prominent in response to
wild-type receptors are not detectably activated by the
kinase-defective receptors. Thus, although it is not possible to prove
that there are no endogenous EGF receptors in the B82L cells,
or that very low levels of receptors do not contribute to the
signaling, the weight of the evidence argues strongly against this
possibility.
in rat hepatocytes is reportedly inhibited by pertussis
toxin
(49, 50) , implying that the EGF-R is able,
directly or indirectly, to activate G
in this cell
type. This suggestion is further strengthened by reports that
G
directly associates with the EGF receptor in an
EGF-dependent manner and that pertussis toxin pretreatment of
hepatocytes inhibits this association (50). It has also been
demonstrated that thrombin treatment of CCL39 hamster fibroblasts
activates a MAP kinase pathway by stimulating a pertussis
toxin-sensitive G-protein
(51) . Therefore, it is possible that
kinase-defective EGF-Rs might signal to MAP kinase through an
EGF-inducible, pertussis toxin-sensitive G-protein, such as
G
. However, we have found that pertussis toxin treatment of
B82L cells causes only a marginal inhibition of EGF-induced MAP kinase
activation, by either wild-type or kinase-defective EGF-Rs (data not
shown). Thus, pertussis toxin-sensitive G-proteins appear not to be
significantly involved in signaling to MAP kinase in this system.
does become
tyrosine-phosphorylated in cells expressing kinase-defective EGF-Rs,
and thus it is possible that another kinase of this family participates
in the signaling to MAP kinase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.