The ErbB family of receptors, which include the
epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4
mediate the actions of a family of bioactive polypeptides. EGF signals
through EGFR, whereas heregulin (HRG) signaling is initiated through
binding to either ErbB3 or ErbB4. In this report we studied the role of protein-tyrosine phosphatase SHP-2 in ErbB-mediated activation of
mitogen-activated protein kinase (MAPK) by overexpressing SHP-2 mutants
in COS-7 cells. We demonstrate that enzymatic activity and both
NH2- and COOH-terminal SH2 domains of SHP-2 are
required for EGF-induced MAPK activation, but not for c-Jun
amino-terminal kinase stimulation or MAPK activation which occurred in
response to myristoylated son of sevenless, activated Ras, or phorbol
ester. Dominant-negative forms of SHP-2 had no effect on EGF-stimulated interaction of GRB2 with EGFR or SHC, nor did they influence
phosphorylation of SHC and SHC/EGFR association. The same mutant SHP-2
structures that inhibited EGF-mediated stimulation of MAPK also blocked
HRG
/
-induced MAPK activation. EGF or HRG
caused SHP-2 SH2
domains to engage multiple phosphotyrosine proteins, and mutation of
either domain disrupted these associations. These results demonstrate that SHP-2 performs a common and essential function(s) in
ligand-stimulated MAPK activation by the ErbB family of receptors.
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INTRODUCTION |
The ErbB family of receptors, which include epidermal growth
factor receptor (EGFR1;
ErbB1), ErbB2, ErbB3, and ErbB4, mediate the biological actions of a
family of growth factors which are structurally related to EGF (1).
This family of bioactive peptides, which includes EGF, transforming
growth factor
, amphiregulin, heparin-binding EGF-like growth
factor, betacellulin, epiregulin, and heregulin
/
(HRG; neu
differentiation factor, neuregulin, acetylcholine receptor-inducing
activity, glial growth factor) elicits numerous cellular responses such
as mitogenesis, differentiation, trophism, and motility (1). Signaling
from ErbBs involves a process of receptor homo- and heterodimerization,
which is initiated by engagement of ligand with a specific ErbB
receptor (1). EGF and amphiregulin require the presence of EGFR for
signaling (2), whereas HRG-induced signal transduction occurs after
binding of ligand to either ErbB3 or ErbB4 on cells that co-express
ErbB2 (3, 4).
The protein-tyrosine phosphatase SHP-2 (PTP1D, SHPTP2, PTP2C, SHPTP3,
or Syp) contains two Src homology 2 (SH2) domains (5) and appears to
play a critical role in mitogenic responses to EGF and insulin, but not
to platelet-derived growth factor (6-10). Although it is not clear how
SHP-2 functions as a positive mediator of EGF signaling, stimulation of
cells with EGF has been shown to drive the association of SHP-2 with a
number of proteins including a 115-kDa phosphotyrosine
(Tyr(P))-containing protein (11), GRB2-associated binder-1 (Gab1) (12),
SHP substrate 1 (SHPS-1)/signal-regulatory protein
(SIRP
) (13,
14), and GRB2 via the COOH-terminal SH3 domain of GRB2 (15). In
Drosophila, membrane targeting of the SHP-2 homologue,
corkscrew, is sufficient for R7 photoreceptor development in
the absence of receptor tyrosine kinase activity (16), and a downstream
target called daughter of sevenless (Dos) has been identified (17, 18).
In the work reported here, we show that SHP-2 function appears to
represent a common point(s) of convergence in signaling downstream of
activated EGFR, ErbB2, ErbB3, and ErbB4. Furthermore, enzymatic
activity and both NH2- and COOH-terminal SH2 domains of
SHP-2 are required for EGF-induced mitogen-activated protein kinase
(MAPK) stimulation.
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MATERIALS AND METHODS |
Antibodies, Reagents, and cDNAs--
Monoclonal antibodies
(mAb) against influenza hemagglutinin protein epitope (HA) and c-Jun
amino-terminal kinase (JNK) were obtained from Boehringer Mannheim and
Pharmingen, respectively. mAbs against MAPK, SHC, GRB2, SHP-1, and
SHP-2 were obtained from Transduction Labs. EGF was obtained from
PeproTech, Inc. and HRG
177-228 and HRG
1177-228 were provided by Berlex Biosciences. The
expression vectors for SHP-1 and SHP-2 structures have been described
previously (19). Epitope-tagged SHP-2 cDNAs were generated by
subcloning into pcDNA3.1 myc/his (Invitrogen), and DNA sequence was
confirmed. Expression vectors for HA-MAPK, HA-JNK, HA-SHC, and
myristoylated cdc25 domain of Sos (myr-Sos) have been described by Coso
et al. (20). ErbB3 and ErbB4 cDNAs were generously
provided by Jacalyn Pierce (NCI, National Institutes of Health) and
Greg Plowman (Bristol-Myers Squibb), respectively, and were subcloned
into pCMV5. The myc-GRB2 cDNA was generously provided by Robert
Weinberg (Whitehead Institute).
Transient Transfections--
Subconfluent COS-7 cells were
transfected with cDNAs as described in the figure legends using the
DEAE-dextran/chloroquine technique (21). Cells were allowed to recover
for 24 h, serum-starved overnight, and stimulated with 10 nM growth factor or phorbol ester, and cell lysates were
generated as described previously (15). Lysate protein concentrations
were determined using the Bio-Rad detergent compatible protein assay.
In all instances, consistent expression of transfected cDNAs was
confirmed by Western blotting analysis of 10 µg of lysate as
described previously using enhanced chemiluminescence detection
(15).
Immunoprecipitations (IP) and Kinase Assays--
IPs were
performed as described previously (15). For HA-MAPK kinase assays the
immune complex was washed once with 20 mM Hepes (pH 7.4)
containing 2 mM EGTA and 10 mM
MgCl2 (kinase buffer) prior to assay. Kinase buffer (20 µl) containing 20 µM ATP, 2 µCi of
[32P]ATP and 20 µg of myelin basic protein (MBP) was
added to the immune complex and incubated for 30 min at room
temperature. Reaction was terminated by adding 10 µl of 4×
SDS-polyacrylamide gel electrophoresis sample buffer and boiling for 4 min. Reaction mixture was resolved in an 8-16% SDS-polyacrylamide gel
electrophoresis gel, and proteins were transferred to a polyvinylidene
difluoride membrane. Autoradiography was performed followed by Western
blotting to confirm consistent IP of kinases. JNK activity was
determined using 100 µM SKAIPS peptide substrate (22)
exactly as described above, and reaction was terminated by the addition
of 10 µl of 8.5% H3PO4. Reaction mixture was
spotted onto P81 phosphocellulose paper and washed, and radioactivity
was quantitated in a scintillation counter.
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RESULTS AND DISCUSSION |
COS-7 Cells as a Model System to Study the Functional Role of SHP-2
in EGF-induced MAPK Activation--
Because COS-7 cells can be easily
transfected and used to transiently express proteins, we tested the
feasibility of using COS-7 cells to study the role of SHP-2 in
activation of MAPK by EGF. cDNAs encoding wild type (WT) or
catalytically inactive SHP-2 with a Cys to Ser mutation at residue 459 (C459S) were co-transfected into cells along with an HA epitope-tagged
MAPK (extracellular signal-regulated kinase 2). Cells were stimulated
with 10 nM EGF for 5 min and lysed, HA-MAPK was
immunoprecipitated using anti-HA mAb, and enzymatic activity in the
immunoprecipitates was determined using MBP as substrate. As shown in
Fig. 1, expression of catalytically inactive SHP-2 in COS-7 cells significantly inhibited EGF-induced MAPK
activation (lane 4), consistent with previous
findings in 293 cells (9, 10). SHP-1, like SHP-2, is a protein-tyrosine phosphatase that contains two SH2 domains in tandem (23).
Overexpression of catalytically inactive SHP-1 (C455S) did not
significantly inhibit EGF-stimulated MAPK activation (lane
8) confirming the specificity of inhibition by C459S SHP-2.
Further, stimulation of MAPK by expression of an activated form of Ras
(Q61L) was unaffected by active or inactive forms of either SHP-2 or
SHP-1 (lanes 9-12). Thus, COS-7 cells
appear to be an excellent model to study the role of SHP-2 in
EGF-induced activation of MAPK.

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Fig. 1.
Effect of catalytically inactive SHP-1 and
SHP-2 on activation of MAPK by EGF and activated Ras in COS-7
cells. COS-7 cells were transiently transfected with 0.2 µg of
HA-MAPK cDNA and 0.8 µg of pCMV5 expression vector encoding
either wild type SHP-2 (WT; lanes 1 and 2), catalytically inactive SHP-2 (C459S;
lanes 3 and 4), wild type SHP-1
(lanes 5 and 6), or catalytically
inactive SHP-1 (C455S; lanes 7 and
8) as described under "Materials and Methods."
Serum-starved cells were stimulated with 10 nM EGF for 5 min (lanes 2, 4, 6, and
8) and lysed, and IPs were performed with anti-HA mAb.
Immune complex kinase assays were performed using
[32P]ATP and MBP, and incorporation of 32P
into MBP was analyzed by autoradiography (upper
panel), IP of HA-MAPK (middle panel),
and consistent expression of SHP-2 and SHP-1 (bottom
panel) were confirmed by Western blotting. In
lanes 9-12, 0.2 µg of HA-MAPK cDNA, 0.5 µg of activated Ras cDNA (Q61L), and 0.8 µg of either WT SHP-2,
C459S SHP-2, WT SHP-1, or C455S SHP-1 cDNA were transfected into
cells, serum-starved, and lysed, and HA-MAPK activity was
determined.
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Catalytic Activity and Both SH2 Domains of SHP-2 Are Required for
EGF-stimulated MAPK Activation, but Not for JNK Activation--
To
gain additional insight into which SHP-2 elements are required for
EGFR-mediated MAPK activation, COS-7 cells were transfected with a
truncated form of SHP-2 that contains both NH2- and
COOH-terminal SH2 domains (residues 1-244), but lacks the
COOH-terminal phosphatase domain. This structure was a potent inhibitor
of EGF-regulated activation of MAPK (Fig.
2A, lane
6). In contrast, MAPK activation by phorbol 12-myristate
13-acetate (PMA), myristoylated cdc25 domain of son of sevenless
(myr-Sos), or Ras Q61L was not influenced by expression of SHP-2 SH2
domains (Fig. 2A, lane 21; Fig.
2B, lanes 3 and 8)
demonstrating that attenuation of EGF-stimulated MAPK activity by this
structure was highly selective. However, EGF-induced MAPK activation
was rescued by mutations that render the SH2 domains incapable of
binding Tyr(P) residues (R32E in the NH2-terminal SH2
domain or R138E in the COOH-terminal SH2 domain) (Fig. 2A,
lanes 8 and 10). These results
indicate that both SH2 domains are essential for stimulation of MAPK by
EGFR. Expression of C459S SHP-2 had no significant effect on activation of MAPK by PMA (Fig. 2A, lane 19),
myr-Sos, or Ras Q61L (Fig. 2B, lanes 2 and 7).

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Fig. 2.
Evaluation of role of SHP-2 structures on
MAPK and JNK activation by EGF. In A, COS-7 cells were
transfected with 0.2 µg of HA-MAPK cDNA along with 0.8 µg of
cDNA encoding either WT SHP-2, C459S SHP-2, residues 1-244 of
SHP-2 (SH2s), residues 1-244 with R32E mutation
(SH2s:R32E), or residues 1-244 with R138E
mutation (SH2s:R138E). Cells were stimulated with
10 nM EGF for 5 min (lanes 2,
4, 6, 8, and 10) or with 10 nM PMA for 10 min (lanes 17,
19, 21, 23, and 25) and
lysed, and HA-MAPK activity was determined. To confirm expression of
SHP-2s pooled aliquots of lysates derived from identically transfected
dishes were analyzed by Western blotting (lanes
11-15). In B, cells were transfected
exactly as in A, except that 0.5 µg of cDNA encoding
either myr-Sos or Ras Q61L were co-transfected with HA-MAPK and SHP-2
cDNAs. In C, cells were transfected exactly as in
A, except 0.2 µg of cDNA encoding HA-JNK was
transfected instead of HA-MAPK, and cells were treated with EGF for 15 min. Immune complex kinase assays were performed using
[32P]ATP and SKAIPS peptide (22). The results are
expressed as fold induction relative to unstimulated cells and
represent means ± S.E. of duplicate experiments.
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To further investigate the role of SHP-2 in mediating downstream
signaling events, we tested the ability of C459S SHP-2 and the SH2
domains to interfere with EGF-stimulated activation of JNK
(stress-activated protein kinase). The SHP-2 constructs were transfected into COS-7 cells along with HA epitope-tagged JNK (HA-JNK),
and JNK enzymatic activity was determined in anti-HA immunoprecipitates
using SKAIPS peptide as substrate (22). As shown in Fig. 2C,
none of the SHP-2 structures had any significant effect on EGF-induced
JNK activity, and similar results were obtained when JNK activity was
monitored using glutathione S-transferase-ATF2 fusion
protein as substrate (data not shown). These results demonstrate that
the requirement for SHP-2 function is highly specific to MAPK
activation by EGF.
Dominant-negative SHP-2 Mutants Do Not Interfere with EGF-induced
GRB2- and SHC-mediated Associations--
To further confirm that
expression of C459S or SHP-2 SH2s inhibit MAPK activation in a specific
manner and do not nonspecifically bind Tyr(P) proteins essential to
EGFR signaling we studied the effect these mutants had on Tyr(P)/SH2
domain interactions believed to be critical to EGFR function. A myc
epitope-tagged GRB2 was expressed in cells along with the various
SHP-2s, cells were stimulated with EGF, and myc-GRB2 was
immunoprecipitated from lysates (Fig. 3A). These data demonstrated
that none of the SHP-2 structures had any effect on association of GRB2
with EGFR or with SHC. In a complimentary experiment, cells were
transfected with an HA epitope-tagged SHC (p52) and HA-SHC was
immunoprecipitated (Fig. 3B). Again, none of the SHP-2
mutants influenced tyrosine phosphorylation of SHC or interaction of
SHC with EGFR. These results present a strong argument that
dominant-negative SHP-2s act to block MAPK stimulation specifically and
do not function via the nonspecific sequestration of Tyr(P)
proteins.

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Fig. 3.
Dominant-negative SHP-2 mutants do not
interfere with GRB2- and SHC-mediated interactions. In
A, COS-7 cells were transfected with 0.2 µg of myc-GRB2
cDNA along with 0.8 µg of cDNA encoding either WT, C459S,
SH2s, SH2s:R32E, or SH2s:R138E SHP-2s. Cells were stimulated with 10 nM EGF for 5 min (lanes 2,
4, 6, 8, and 10) and lysed,
and IPs were performed with anti-myc 9E10 mAb. Immunoprecipitates were
analyzed for the presence of Tyr(P), SHC, and GRB2 by Western blotting.
To confirm expression of SHP-2s pooled aliquots of lysates derived from
identically transfected dishes were analyzed by Western blotting
(lanes 11-15). In B, cells
were transfected exactly as in A, except 0.2 µg of
cDNA encoding HA-SHC was transfected instead of myc-GRB2, and
HA-SHC was immunoprecipitated.
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Catalytic Activity and Both SH2 Domains of SHP-2 Are Required for
HRG
- and
-Stimulated MAPK Activation--
We next addressed the
question of whether SHP-2 plays a role in MAPK activation by HRGs.
Western blotting analysis of lysates demonstrated that COS-7 cells do
not possess detectable amounts of ErbB3 or ErbB4 and HRGs did not
activate MAPK (data not shown). However, ectopic expression of either
ErbB3 or ErbB4 in COS-7 cells reconstituted both HRG
- and
-stimulated MAPK activation (Fig.
4A and B,
lanes 2 and 3). Expression of C459S
SHP-2 or SHP-2 SH2s together with either ErbB3 or ErbB4 abrogated HRG
- or
-induced MAPK activation (Fig. 4, A and
B, lanes 6, 7,
10, and 11). Further, ligand-dependent MAPK activation was unaffected by mutated
SHP-2 SH2 domains (lanes 14, 15,
18, and 19). HRG
- and
-stimulated MAPK
activation in these cells was not affected by expression of C455S SHP-1
(data not shown). Thus, SHP-2 function is essential to HRG-stimulated
MAPK activation, and the required SHP-2 moieties are identical with
those observed for EGF signaling. In addition, cells were
co-transfected with ErbB3 and myc epitope-tagged SHP-2 cDNAs and
stimulated with EGF or HRG
, and anti-myc immunoprecipitates were
evaluated for the presence of Tyr(P) proteins (Fig. 4C). These results demonstrated that C459S and SHP-2 SH2s engage several common Tyr(P) proteins in response to EGF and HRG
and bound these
proteins to a greater extent than WT SHP-2 (lanes
2, 3, 5, 6, 8,
and 9). Mutation of either SH2 domain inhibited these associations (lanes 11, 12,
14, and 15) thereby providing a potential molecular basis for the ability of C459S and SHP-2 SH2s to block ErbB-mediated MAPK activation.

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Fig. 4.
Evaluation of role of SHP-2 structures on
HRG-induced MAPK activation. In A, COS-7 cells were
transfected with 0.2 µg of HA-MAPK cDNA, 0.25 µg of ErbB3
cDNA along with 0.8 µg of cDNA encoding either WT, C459S,
SH2s, SH2s:R32E or SH2s:R138E SHP-2s. Cells were stimulated for 5 min
with EGF (E), HRG ( ), or HRG ( ) and lysed, and
HA-MAPK activity was determined. B was performed exactly as
in A except that ErbB4 cDNA was transfected into cells
instead of ErbB3 cDNA. C was performed exactly as in
A except that 0.2 µg of myc epitope-tagged SHP-2 cDNAs
were transfected instead of HA-MAPK. IPs were performed with anti-myc
mAb, and immunoprecipitates were analyzed for Tyr(P) and SHP-2 by
Western blotting. A control IP was performed on lysates derived from
cells transfected with WT by replacing anti-myc with MOPC.21 control
mAb (lanes 16 and 17).
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A Common Requirement for SHP-2 in MAPK Activation by the ErbB
Family of Receptors--
Our findings indicate that SHP-2 represents a
common and essential point(s) of convergence in signaling downstream of
ErbB receptors, regardless of which receptor combinations are
activated. The dominant-negative effects observed by C459S and SHP-2
SH2s were found to be highly specific in that (i) these mutants did not
inhibit MAPK stimulation by myr-Sos, Ras Q61L, or activated protein
kinase C (PMA), (ii) these mutants did not interfere with GRB2
interaction with SHC and EGFR and SHC/EGFR association, (iii) overexpression of C455S SHP-1 did not block MAPK stimulation, and (iv)
these mutants did not inhibit JNK activation. Because both MAPK and JNK
stimulation is at least partially mediated by Ras (20), our findings
suggest that SHP-2 functions in a Ras-independent pathway which leads
to or allows for MAPK activation. The observation that mutation in
either SHP-2 SH2 domain rescues ErbB-induced MAPK activation reveals
that both domains are required for this response. This finding is
important because deletion or simultaneous binding of both SH2 domains
by Tyr(P) stimulates SHP-2 enzymatic activity (24, 25). These results
suggest that SHP-2 SH2 domains need to simultaneously engage a Tyr(P)
protein(s) in order for SHP-2 to act as a positive mediator of ErbB
receptor-induced MAPK stimulation.