(Received for publication, July 6, 1995; and in revised form, January 23, 1996)
From the
To investigate the mechanisms underlying the enhanced mitogenic
activity of the truncated epidermal growth factor receptor (EGFR)
lacking the C-terminal autophosphorylation sites (973-EGFR), we
studied the intracellular signaling pathways in NR6 cells expressing
human wild type EGFR and
973-EGFR. Microinjection of
dominant/negative p21
(N17) completely inhibited
EGF-induced DNA synthesis in both cell types. EGF stimulated Shc
phosphorylation as well as the formation of wild type EGFR
Shc
complexes. In contrast, EGF stimulated Shc phosphorylation without
formation of
973-EGFR
Shc complexes. Tyrosine-phosphorylated
Shc formed complexes with Grb2
Sos, and microinjection of anti-Shc
antibody and Shc-SH2 GST fusion protein inhibited EGF stimulation of
DNA synthesis in both cell lines. EGF markedly increased ErbB2 tyrosine
phosphorylation in wild type EGFR cells. In
973-EGFR cells, ErbB2
was tyrosine phosphorylated in the basal state and EGFR stimulated
further phosphorylation of ErbB2. In addition to ErbB2, additional
proteins were tyrosine phosphorylated in
973-EGFR cells, mostly in
the molecular mass range of 120-170 kDa. Taken together with our
findings indicating coupling of ErbB2 to Shc, these data suggest the
importance of an alternative signaling pathway in
973-EGFR cells
mediated by the formation of heterodimeric structures between the
truncated EGFR and ErbB2, followed by coupling through Shc to
Grb2
Sos and the p21
pathway, ultimately
leading to mitogenesis.
Epidermal growth factor (EGF) ()stimulates the
intrinsic tyrosine kinase activity of the epidermal growth factor
receptor (EGFR)(1) . The activated EGFR phosphorylates its own
tyrosine residues as well as intracellular substrate proteins. For
EGFR, five autophosphorylation sites (tyrosine 992, 1068, 1086, 1148,
and 1173) located at the C terminus are identified as the major binding
region for Src homology (SH) 2 domain containing
proteins(2, 3) . SH2 and/or phosphotyrosine binding
domain containing proteins such as phospholipase
C-
(4) , GTPase-activating protein of
p21
(5) , Grb2(6) , and Shc (7, 8) are direct candidates to signal downstream.
p21
has been shown to play an important role in
EGF-induced mitogenic signaling(9, 10) .
p21
is active in its GTP-bound form (11) and this activation is mainly controlled by the
translocation of the p21
guanine nucleotide
exchange factor (GEF), the Son of Sevenless (Sos) protein, to the cell
membrane, where it is in proximity to p21
(12, 13, 14, 15, 16, 17) .
The proline-rich region of Sos binds to the SH3 domain of Grb2 and
preformed Grb2
Sos complexes exist within unstimulated cells (17, 18, 19, 20, 21) .
Through the Grb2-SH2 domain, Grb2
Sos complexes can bind to
tyrosine phosphorylated
EGFRs(17, 18, 19, 20, 21, 22, 23) .
Alternatively, the SH2 domain of Grb2 can also bind to phosphorylated
Shc, which itself can recognize EGFR autophosphorylation motifs via its
SH2 and N-terminal phosphotyrosine binding
domain(18, 19, 20, 21, 22, 24, 25) .
Thus, EGFR
Grb2
Sos and/or EGFR
Shc
Grb2
Sos
provide a mechanism whereby EGF can stimulate p21
GTP formation. Recently, we reported the importance of Shc
as an adaptor protein transducing mitogenic signaling from activated
EGFR to p21
activation (26, 27) . Although the association of phosphorylated
EGFRs with these adaptor proteins provides the mechanism to trigger
downstream signals, mutant EGFRs in which the tyrosine phosphorylation
sites were replaced with phenylalanine signal to the same extent as
wild type EGFRs do (28) . In addition, mutant EGFRs truncated
at amino acid residue 973 (
973-EGFR) which lack the C-terminal
five autophosphorylation sites mediate enhanced cell cycle
progression(2, 29, 30) . Since neither Shc
nor Grb2, which are important for p21
activation, directly binds to
973-EGFR following EGF
stimulation, an alternative pathway leading to cell cycle progression
bypassing p21
may reside in cells expressing
973-EGFRs. Alternatively, since Shc was still tyrosine
phosphorylated without association with the EGFR mutant lacking the
autophosphorylation sites(3) , the formation of
Shc
Grb2
Sos complexes may lead to p21
activation initiated even by the truncated EGFR.
In this
report, we directly evaluated the importance of p21in
973-EGFR mediated mitogenic signaling using a single cell
microinjection assay, and identified an alternative mechanism leading
to the p21
activation. Here, we show that ErbB2
plays an important role in NR6 cells expressing
973-EGFRs.
Figure 1:
Inhibition of DNA synthesis by
microinjection of dominant/negative p21(N17).
Serum-starved wild type EGFR (panel A) and
973-EGFR (panel B) cells were microinjected with 2 mg/ml N17-Ras
containing 5 mg/ml rabbit IgG. BrdU incorporation stimulated by 160
nM EGF, 10% serum or neither in the injected cells (open
bars) and uninjected cells (solid bars) on the same
coverslip was determined as described under ``Experimental
Procedures.'' Cumulative data are shown and results are the means
± S.E. of three experiments.
Figure 2: EGF-stimulated tyrosine phosphorylation of Shc. A, time course of Shc phosphorylation. Serum-starved cells were treated with 160 nM EGF for the indicated times. B, dose-response of Shc phosphorylation. Serum-starved cells were treated with the indicated concentrations of EGF for 5 min. After treatment, cell lysates were immunoprecipitated with anti-Shc antibody. The immunoprecipitates were separated by SDS-PAGE and transferred to Immobilon-P. Immunoblots were performed with anti-phosphotyrosine antibody. Molecular masses of predominant Shc isoform (52 kDa), EGFR (170 kDa), and Shc-associated proteins pp145 (145 kDa) are shown by arrows. Results are representative of three separate experiments.
Figure 3:
Inhibition of DNA synthesis by
microinjection of anti-Shc antibody. Serum-starved wild type EGFR (panel A) and 973-EGFR (panel B) cells were
microinjected with 5 mg/ml anti-Shc antibody. EGF (160 nM) or
serum (10%) were then added, and BrdU incorporation in the injected
cells (open bars) and uninjected cells (solid bars)
on the same coverslip was determined as described under
``Experimental Procedures.'' Cumulative data are shown and
results are the means ± S.E. of four
experiments.
Figure 4:
Inhibition of DNA synthesis by
microinjection of Shc-SH2 GST fusion protein. Serum-starved wild type
EGFR (panel A) and 973-EGFR (panel B) cells were
microinjected with 5 mg/ml Shc-SH2 GST fusion protein containing 5
mg/ml rabbit IgG. Cells were treated with EGF (160 nM) or
serum (10%), and BrdU incorporation in the injected cells (open
bars) and uninjected cells (solid bars) on the same
coverslip was determined as described under ``Experimental
Procedures.'' Cumulative data are shown and results are the means
± S.E. of three experiments.
Figure 5:
EGF stimulated complex formation.
Serum-starved wild type EGFR cells (panels A, B, and C) and 973-EGFR cells (panels D, E, and F) were treated without or with 160 nM EGF for 2 min
at 37 °C. Cell lysates were immunoprecipitated with control
preimmune IgG, anti-Shc, anti-EGFR, or anti-Grb2 antibody. The
immunoprecipitates were separated by SDS-PAGE and transferred to
Immobilon-P. Immunoblots were performed with anti-phosphotyrosine (panels A and D), anti-Shc (panels B and E), or anti-Grb2 (panels C and F) antibody.
Results are representative of three separate
experiments.
Figure 6:
EGF-stimulated complex formation with
Shc-SH2 GST fusion protein. Serum-starved wild type EGFR cells (lanes 1 and 2) and 973-EGFR cells (lanes 3 and 4) were stimulated without (lanes 1 and 3) or with (lanes 2 and 4) 160 nM EGF for 2 min at 37 °C. After treatment, cell lysates were
affinity-precipitated with Shc-SH2 GST fusion protein. The precipitates
were separated by SDS-PAGE and transferred to Immobilon-P, and
immunoblots were performed with anti-phosphotyrosine antibody.
Molecular masses of EGFR (170 kDa), and ErbB2 (185 kDa), and
Shc-associated protein pp145 (145 kDa) are shown by arrows.
Results are representative of two separate
experiments.
Figure 7:
EGF stimulation of GEF activity in
membrane fraction. Serum-starved cells were stimulated without or with
160 nM EGF for 2 min at 37 °C. A membrane fraction was
then prepared and GEF activity was determined by measuring the
dissociation of protein-bound [H] GDP
radioactivity. Results in wild type EGFR (closed bars) cells
and
973-EGFR cells (open bars) are expressed as the
percentage of [
H]GDP released in 15 min, and are
shown as the mean ± S.E. of three separate
experiments.
Figure 8: EGF stimulation of ErbB2 phosphorylation. Serum-starved cells were stimulated without or with 160 nM EGF for 2 min at 37 °C. After treatment, cell lysates were immunoprecipitated with anti-ErbB2 specific antibody. The immunoprecipitates were separated by SDS-PAGE and transferred to Immobilon-P. Immunoblots were performed with anti-phosphotyrosine antibody. Molecular mass of ErbB2 (185 kDa) is shown by an arrow. Results are representative of three separate experiments.
EGF stimulates mitogenesis by activating the tyrosine kinase
activity of the EGFR which then sets in motion a signaling cascade
eventually leading to cell cycle progression(1) . Although not
completely understood, many elements of this signaling pathway have
been defined. One of the initial steps in EGF action involves binding
of various SH2 and phosphotyrosine binding domain-containing molecules
to the phosphotyrosine motifs within the autophosphorylated EGFR. These
proteins, such as Grb2, Shc, and phospholipase C-, then further
propagate the biologic signals initiated by autophosphorylation of the
EGFR(4, 5, 6, 7) . The five EGFR
autophosphorylation sites are contained within the receptor C terminus,
and it has recently been shown that cells expressing mutant EGFRs in
which the five autophosphorylation sites have been deleted by
C-terminal truncation are normally responsive to EGF, with respect to
stimulation of DNA synthesis(2, 29, 30) .
This is surprising, given the presumed importance of EGFR
phosphotyrosine motifs in docking SH2 containing signaling proteins to
the EGFR. This suggested the presence of an alternate pathway for EGF
action in cells expressing these mutant receptors. To evaluate this
possibility, we have studied the intracellular signaling mechanisms in
transfected cells overexpressing a truncated EGFR (
973-EGFR) from
which all five tyrosine autophosphorylation sites are deleted.
An
important step in normal EGF action is formation of
ShcGrb2
Sos complexes which lead to the generation of
p21
GTP and subsequent activation of the MAP kinase
pathway(26, 27) . To determine whether p21
GTP was critical to EGF action in
973-EGFR cells, we
conducted single cell microinjection studies using dominant negative
Ras(N17) protein. As expected, inhibition of p21
completely prevented the ability of EGF to stimulate DNA
synthesis in wild type EGFR cells. Interestingly, the same result was
observed in
973-EGFR cells. Thus, despite the fact that the
973-EGFR contains no autophosphorylation sites, and, therefore,
cannot bind to Grb2 or Shc directly(2, 3) ,
p21
is still necessary for mitogenic signaling in these
cells.
Given these results, we next explored potential upstream
elements connecting the 973-EGFR to p21
. We have
previously shown that EGF mediated formation of Shc
Grb2
Sos
complexes is the predominant mechanism coupling wild type EGFRs to the
Ras pathway(27) . The current studies confirm these results,
but also show that EGF stimulation of
973-EGFR cells leads to
comparable dose and time dependent tyrosine phosphorylation of Shc, as
compared to wild type EGFR cells. Interestingly, when Shc
immunoprecipitates from wild type and
973-EGFR cells were probed
with anti-phosphotyrosine antibody, the predominant phosphoprotein
co-precipitating with Shc in wild type EGFR cells was the EGFR,
whereas, in
973-EGFR cells, a pp145 and pp185 protein were the
only ones visualized. pp185 was found to be ErbB2. The identity of
pp145 remains unknown, although it is not the truncated EGFR.
Interestingly, when lysates from EGF stimulated
973-EGFR cells
were precipitated with a Shc-SH2 GST fusion protein, pp145 and pp185
were readily precipitated and identified (Fig. 6). Thus, despite
the absence of any autophosphorylation sites, the
973-EGFR was
normally capable of mediating ligand induced Shc tyrosine
phosphorylation. Since this truncated receptor did not coprecipitate
with either intact Shc protein or the Shc-SH2 GST fusion protein, these
results suggest an alternate pathway coupling the truncated EGFR to
Shc.
To determine whether Shc was a functionally significant
molecule conveying signals between 973-EGFR and p21
,
we conducted microinjection studies using anti-Shc antibodies and a
Shc-SH2 GST fusion protein. Microinjection of either reagent inhibited
EGF-induced DNA synthesis by 80-90% in wild type EGFR cells.
While inhibition was clearly demonstrated in
973-EGFR cells, the
magnitude of this inhibition was less than in wild type EGFR cells.
Thus, although Shc is clearly an important signaling molecule mediating
EGF stimulated DNA synthesis in
973-EGFR cells, the partial
inhibition by the microinjected reagents suggests that alternative
pathways are also operative. The failure of either anti-Shc antibody or
Shc-SH2 GST fusion protein to block serum-stimulated DNA synthesis
confirms that additional mechanisms to activate p21
exist. In all cases the amount of anti-Shc antibody or Shc-SH2
GST fusion protein microinjected into the cells was optimal, because
when higher concentrations of these reagents were microinjected, no
additional inhibition was observed (data not shown). Some component of
mitogenic signaling could partially bypass Shc to activate p21
in
973-EGFR cells. In this regard, a predominant pp145 kDa
protein was phosphorylated in response to EGF in these cells, whereas,
this protein was only minimally phosphorylated in wild type EGFR cells.
The identity and function of this protein are unknown, but it is
possible that pp145 represents a new signaling molecule which provides
an input to p21
, independent of the
Shc
Grb2
Sos pathway. Furthermore, EGF stimulation led to a
much broader spectrum of tyrosine phosphorylation of substrate proteins
in
973-EGFR cells compared to wild type EGFR cells, confirming a
broader array of substrate specificities in this cell
line(36) .
Our studies indicate that one mechanism
explaining the alternative signaling pathway in cells expressing
truncated EGFRs involves activation of ErbB2. Four members of the EGFR
related family of receptor tyrosine kinases have been identified
including ErbB1/EGFR (1) , ErbB2(41) ,
ErbB3(38, 39) , and ErbB4 (40) and it has been
demonstrated that activated ErbB2 or ErbB3 can lead to tyrosine
phosphorylation of Shc and stimulation of p21 GTP
formation (32, 43, 44, 45) .
Expression of ErbB3 and ErbB4 is limited to certain epithelial cell
types, and they are not expressed in fibroblasts, whereas, ErbB2 has
been identified in fibroblast cell lines(46) . Taken together
with the fact that the NR6 cells used in the current studies are
derived from fibroblasts, ErbB2 is a logical candidate signaling
molecule in these cells. Furthermore, we demonstrated that no
significant tyrosine phosphorylation of ErbB3 was observed in either
wild type EGFR or
973-EGFR cells (data not shown). It has been
demonstrated that ErbB2 can complement with the EGFR in mediating EGF
induced signals(47, 48) . Although the ligand for
ErbB2 has not been identified, the ErbB2 protein can be phosphorylated
on tyrosine residues by the activated EGFR(49, 50) .
This may be due to intermolecular cross phosphorylation of ErbB2 by the
EGFR(49) , or intramolecular transphosphorylation within
heterodimers formed between ErbB2 and EGFR
molecules(42, 50, 51) . Since a kinase
deficient mutant ErbB2 can suppress normal EGFR signaling in a
dominant/negative fashion (52) , heterodimer formation between
EGFR and ErbB2 appears to be the more important mechanism. In the
current studies, ErbB2 was heavily tyrosine phosphorylated in basal
973-EGFR cells, whereas basal ErbB2 phosphorylation was undetected
in wild type EGFR cells. Following EGF stimulation, an increase in
ErbB2 phosphorylation was seen in both cell types. Importantly,
co-precipitation studies demonstrated that ErbB2 was associated with
Sos, Shc, and Grb2 in both basal and EGF stimulated
973-EGFR cells
(data not shown). Taken together with the fact that the
973-EGFR
can not interact directly with SH2-containing adaptor proteins such as
Shc and Grb2, it would appear that ErbB2 plays a major role in EGF
induced mitogenic signaling in
973-EGFR cells. This would occur by
transphosphorylation of ErbB2 by the
973-EGFR within heterodimer
complexes. Tyrosine phosphorylated ErbB2 may also lead to activation of
signaling pathways by other mechanisms. For example, a broad array of
tyrosine phosphorylated proteins was detected in ErbB2 precipitates
from
973-EGFR cells, and, in particular, a predominant 145 kDa
tyrosine phosphorylated protein, whose phosphorylation state was EGF
dependent in
973-EGFR cells, was readily identified. Whether pp145
or some other signaling molecule participates in an alternate signaling
pathway coupling
973-EGFRs to p21
, independent of
Shc
Grb2
Sos, remains to be elucidated.
In summary, our
results demonstrate the importance of p21 in the
mitogenic signal transduction pathway mediated by
973-EGFR.
Although the molecular coupling of EGFR
Shc
Grb2
Sos is
important as a common mechanism to activate p21
, an
alternative pathway resides in
973-EGFR cells. Our evidence
suggests that ErbB2 plays an important role in coupling the truncated
EGFR to p21
activation. These results indicate a novel
signal transduction mechanism involving ErbB2 in
973-EGFR cells.