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INTRODUCTION |
Tyrosine phosphorylation of proteins is central to receptor
tyrosine kinase (RTK)1 signal
transduction (1-5). Some of the most extensively studied RTKs include
the epidermal growth factor receptor (6), the platelet-derived growth
factor receptor (7), the fibroblast growth factor receptor (8), the
insulin receptor (9), and the Met receptor. The signaling activity of
RTKs is controlled by a distinct class of enzymes known as
phosphotyrosyl phosphatases (PTP) (9-17). Recent advances in the area
of PTPs indicate that tyrosine phosphorylation and dephosphorylation of
signaling proteins must be integrated for normal cell growth,
differentiation, migration, and development (18). Uncontrolled tyrosine
phosphorylation of RTKs and/or their downstream targets results in
abnormal cell growth and differentiation that lead to diseases such as
cancer and developmental problems (6, 19, 20). Thus, the PTPs play
major role in maintaining proper cellular signaling.
PTPs encompass a large family of transmembrane and non-transmembrane
enzymes (14, 15, 18). Among the non-transmembrane mammalian PTPs, only
two are Src homology 2 (9) domain-containing, namely SHP1 (also called
PTP1B, SH-PTP1, PTP1C, HCP, or SHP) and SHP2 (also known as SH-PTP2,
SH-PTP3, PTP2C, PTP1D, or SYP). SHP2 is a ubiquitously expressed
mammalian PTP, whereas its close relative, SHP1, is primarily
restricted to hematopoietic cells (11, 12, 14, 21). SHP1 and SHP2 show
significant structural homology but play opposite physiological roles,
the former being a negative and the latter being primarily a positive
regulator of tyrosine kinase signaling (14, 16, 18). Both PTPs possess
two tandemly arranged SH2 domains in their N-terminal region, a
phosphatase domain in their C-terminal regions and tyrosine
phosphorylation sites in their extreme C-terminal regions (7, 11, 22). A Drosophila PTP, Corkscrew, also has similar structure but
shows functional similarity only to SHP2 (23-25).
SHP2 directly binds to some autophosphorylated RTKs such as the
epidermal growth factor receptor (6), the platelet derived growth
factor receptor (7), and the Met receptor (7, 14, 23, 26) or to
tyrosine phosphorylated adaptor proteins such as insulin receptor
substrate 1 and 2 (27, 28), Shc, fibroblast growth factor receptor
(FGFR) substrate 1 and 2 (FRS1 and 2) (8, 29), and Grb2 adaptor binder
1 and 2 (30-34). These interactions are essential for growth factor-
or cytokine-induced activation of the Ras-extracellular
signal-regulated kinase (ERK) pathway, but the molecular mechanism by
which SHP2 effects these functions is unknown. There has been a great
deal of effort to identify SHP2 substrates. For instance, it was shown
that SHP2 dephosphorylates SHP2 substrate 1 (SHPS1) under
in vitro conditions (35-37), but the in vivo
relevance of these findings is not clear. A stumbling block in defining
the molecular mechanism of SHP2 could be that the physiological
substrates (9) of SHP2 have not yet been identified. Thus,
identification of such substrates would be a stepping stone toward
elucidating the mechanism of action of SHP2 in RTK signaling.
Substrate-trapping mutants of PTPs have been shown to be ideal reagents
for substrate identification. It was demonstrated that such mutants of
PTPs can be produced by mutation of Asp to Ala in the conserved WPD
loop (38). For instance, Asp to Ala mutants of PTP1B, TC-PTP, and
PTP-PEST helped identify EGFR, p52Shc, and
p130Cas as candidate substrates, respectively (38-40). It
was also demonstrated that a Cys to Ser mutation in the signature motif
(41) makes PTPs become substrate trapping (42-44), but with less
efficiency. Here, we report the development of a new trapping mutant
for SHP2 by introducing Cys to Ser and Asp to Ala double mutations that were superior to the single Cys to Ser mutant. Surprisingly, and in
contrast to widely held views, an Asp to Ala mutant of SHP2 was
non-trapping. To our knowledge, this is the first report on the
development of an efficient trapping mutant of SHP2 and the first
demonstration in intact cells that EGFR and Gab1 are target substrates.
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MATERIALS AND METHODS |
Cells, Cell Culture, Antibodies, et Cetera--
The cell types
used in this study were COS-1, human embryonic kidney 293T, and
NIH-3T3. All were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum and maintained at 37 °C with
7% CO2. The epidermal growth factor was purchased from
Invitrogen. Anti-PTP1D, anti-RasGAP, anti-p130Cas, anti-FYB, and
anti-p190 were purchased from BD Transduction Laboratories. Anti-phosphotyrosine monoclonal antibody (4G10) was a gift from Dr.
Morrison, and anti-SOS1, anti-Gab1, and anti-PDGFR antibodies were
purchased from Upstate Biotechnology. Polyclonal antibodies against
SHP2 and EGFR were raised by immunizing rabbits with glutathione S-transferase (GST) fusion proteins of the amino-terminal
region and the kinase domain, respectively. Horseradish
peroxidase-conjugated secondary antibodies were purchased from Amersham
Biosciences. For fluorescent quantitation of band intensities, alkaline
phosphatase-conjugated anti-rabbit or anti-mouse secondary antibodies
were purchased from Zymed Laboratories Inc., whereas
the AttoPhos fluorescent substrate system was from Promega.
Plasmid Construction and Site-directed
Mutagenesis--
Construction of WT-SHP2 or C459S-SHP2 mutant into the
pMT2 vector was described previously (45). The D425A-SHP2 or
D425A/C459S-SHP2 mutants were obtained by introducing the indicated
point mutation to the WT or the C459S mutants using complementary
primers that span the WPD loop region. The Stratagene site-directed
mutagenesis kit and protocol were used to produce the mutants.
Introduction of the point mutation was confirmed by sequencing the
relevant region.
For purification of His-tagged WT or C459S proteins,
corresponding cDNA was cut out from the pMT2 vector with
EcoRI and then inserted in-frame into pET-28(a+) vector
(Novagen) using the same restriction site. Orientation of inserts was
analyzed by restriction digestion followed by protein expression.
Cell Transfection, Preparation of Lysates, Immunoprecipitation,
and Immunoblotting--
Plasmid constructs were transfected into
COS-1, 293T, or NIH-3T3 cells grown to ~60-70% confluency in 6-cm
dishes using the FuGENE transfection regent as recommended by the
manufacturer (Invitrogen). After transfection, cells were incubated for
~30 h, serum-starved for ~12 h, and then stimulated with 100 ng/ml EGF or 10% fetal calf serum. All immunoprecipitation and
immunoblotting experiments were performed as described previously
(46).
Fluorescent Quantitation of Protein Band
Intensities--
Separated proteins on nitrocellulose membranes were
stained with a primary antibody for 2 h at room temperature or
overnight at 4 °C, washed three times, and then stained with
alkaline phosphatase-conjugated secondary antibody for 1 h at room
temperature. After washing three times, membranes were exposed to
AttoPhos fluorescent substrate for 1 min, and band intensities
were measured on a PhosphorImager using ImageQuant software (Storm 860 PhosphorImager, Amersham Biosciences).
In Vitro Phosphatase Assay--
Dephosphorylation of substrate
phosphotyrosyl proteins was performed as described previously (47).
Briefly, EGFR or Gab1 was immunoprecipitated from COS-1 or 3T3 cells
with anti-EGFR or anti-Gab1 antibody, respectively, washed 2 times with
lysis buffer and once with phosphatase assay buffer (50 mM
sodium acetate, pH 5.5, 50 mM NaCl, 10 mM
dithiothreitol, and 2 mM EDTA). Beads were resuspended in
the phosphatase buffer, varying concentrations of WT-PTP were added,
and then the beads were incubated for 30 min at 30 °C with shaking.
The double mutant (DM) protein was used as a negative control in the
phosphatase assay. Reactions were performed in a total volume of 100 µl and stopped by adding Laemmli sample loading buffer and boiling.
The level of tyrosine phosphorylation of substrate proteins was
analyzed by immunoblot with an anti-Tyr(P) antibody.
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RESULTS |
Despite intensive and extensive research to identify physiological
substrates and define the site of action of SHP2, the problem has
remained virtually unresolved. We reasoned that identification of the
physiological substrates of SHP2 would be a logical step forward. For
this, we produced an efficient trapping mutant of SHP2 that would be
instrumental in identifying target phosphotyrosyl substrate proteins.
Production of a Trapping Mutant of SHP2--
Previous reports
showed that trapping mutants of PTPs can be produced by mutation of Asp
to Ala in the conserved WPD loop (38). We utilized the same principle
and introduced an Asp to Ala point mutation into WT-SHP2. WT-SHP2,
D425A-SHP2, or C459S-SHP2, herein after referred to as WT, D425A, and
C459S, respectively, were transfected into COS-1 cells, and EGF-induced
coprecipitation of phosphotyrosyl proteins was analyzed by immunoblot.
In cells transfected with the C459S construct, three phosphotyrosyl
protein bands identified as p70, p120, and p170 were observed. In cells transfected with the WT or D425A, relatively weaker bands of p70 and
p170 were observed. Reblotting with anti-SHP2 and anti-EGFR antibodies
showed that the p70 and the p170 were SHP2 and the EGFR, respectively
(data not shown). These results showed that the D425A protein was
non-trapping.
We hypothesized that the trapping ability of the C459S mutant might be
improved by introducing an additional mutation. For this, we introduced
a D425A mutation into the C459S mutant and produced a double mutant
SHP2 (D425A/C459S-SHP2), herein after referred to as the DM protein.
The DM protein, together with the above mentioned constructs, were
transiently overexpressed in COS-1 cells, and expression was monitored
by immunoblot; all were expressed at approximately five times that of
the endogenous SHP2 (Fig. 1A,
bottom). The pattern of tyrosine-phosphorylated proteins was
similar in all cases except p70, which was significantly intense in the
C459S and the DM cells (Fig. 1A, top). Several
phosphotyrosyl proteins coimmunoprecipitated with the DM protein, which
were identified as p170, p150, p120 and p90 (Fig. 1B,
top). Lower amounts of p170 and p120 also coprecipitated
with the C459S protein as well. In the case of the WT or the D425A
protein, a significantly lower amount of p170 only was observed.
Reblotting with anti-EGFR and ant-SHP2 antibodies reconfirmed that p170
and p70 were EGFR and SHP2, respectively (Fig. 1B,
middle and bottom). Interestingly, the tyrosine
phosphorylation level of the D425A protein was similar to the WT,
suggesting that it could be enzymatically active. By visually comparing
band intensities (both anti-Tyr(P) and anti-EGFR blots), it was
apparent that significantly higher amounts of phosphotyrosyl proteins
coprecipitated with the DM protein, suggesting that the DM protein was
the most efficient substrate-trapping mutant.

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Fig. 1.
The DM protein trapped a 170-kDa and several
other phosphotyrosyl proteins in EGF-stimulated COS-1 cells.
A, anti-Tyr(P) (pY) immunoblot (IB) of
total cell lysates (TCL) show the presence of similar amount
and pattern of tyrosine-phosphorylated proteins in all experimental
groups and also to show expression of the different SHP2 proteins.
B, immunoprecipitation (IP) of COS-1 cell lysates
with anti-SHP2 and immunoblot with the indicated antibodies,
i.e. anti-Tyr(P) (pY, top), anti-EGFR
(EGFR, middle), and anti-SHP2 (SHP2,
bottom). C, total cell lysates of 293T cells
blotted with anti-Tyr(P) (top) and anti-SHP2
(bottom). D, immunoprecipitation of 293T cell
lysates with anti-SHP2 and immunoblot with the indicated antibodies,
i.e. anti-Tyr(P) (top), anti-EGFR
(middle) and anti-SHP2 (bottom). Vec,
vector; D/A, D425A-SHP2;
C/S, C495S-SHP2.
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Because COS-1 cells express higher amounts of the EGFR, which was the
most tyrosine-phosphorylated protein upon EGF stimulation, it is
possible that this might have influenced the interaction. To test this
possibility, we conducted the same experiment in 293T cells, which
express significantly lower amounts of the EGFR (Fig. 1C,
top). As in COS-1 cells, four phosphotyrosyl proteins that
correspond in molecular weight to p170, p150, p120, and p90 coimmunoprecipitated with the DM protein, and lower amounts of p170 and
p120 coimmunoprecipitated with the C459S protein (Fig. 1D,
top). Again, significantly lower amounts of p170 were
detected with the vector, the WT, or the D425A proteins. Reprobing the membrane with anti-EGFR and anti-SHP2 antibodies confirmed that p170
and p70 were EGFR and SHP2, respectively (Fig. 1D,
middle and bottom). Therefore, the trapping of
EGFR by the DM protein was an inherent property of this mutant but not
a concentration artifact. Furthermore, it is also important to note
that the DM protein had no obvious effect on the overall tyrosine
phosphorylation of the cellular proteins. The
tyrosine-phosphorylated proteins trapped by the DM protein were a
very specific subset of the overall tyrosine-phosphorylated proteins
(compare Fig. 1, A with B and C with
D). This indicated that the DM protein was reacting in a
very specific manner with the cellular proteins.
Time Course Studies on the Trapping of EGFR by Mutant SHP2
Proteins--
We examined the dynamics of substrate trapping by the DM
protein at various time points of stimulation. A higher amount of EGFR
coprecipitated with the DM protein at all time points tested and seemed
to accumulate over time (Fig. 2,
top panel). Despite the presence of a high amount
of tyrosine-phosphorylated EGFR and a comparable expression level of
SHP2 constructs (Fig. 2, middle and bottom), a
lower amount of EGFR coprecipitated with the C459S protein and did not
show any increase over time. Similarly, a significantly lower amount of
EGFR coprecipitated with the WT and D425A proteins. These findings
suggested that the DM protein formed a stable complex with its target
substrate EGFR, which would be a desirable characteristic to isolate
and identify other SHP2 substrates.

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Fig. 2.
EGFR trapped by the DM protein accumulated
over time. Lysates from COS-1 cells transfected with the indicated
plasmid constructs were immunoprecipitated (IP) with
anti-SHP2 and immunoblotted (IB) with anti-EGFR
(top). The same membrane was stripped and reblotted with
anti-SHP2 to show immunoprecipitation of an equal amount of SHP2 in all
lanes (bottom). Total cell lysate (TCL) was
immunoblotted with anti-Tyr(P) (pY) to show the presence of
an equal amount of tyrosine-phosphorylated EGFR in all stimulated cells
(middle). Vec, vector; D/A,
D425A-SHP2; C/S, C495S-SHP2.
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SHP2 Partially Dephosphorylates the EGFR--
The trapping of the
EGFR by the DM protein indicated that it could be a target substrate of
SHP2. We thus determined the level of EGFR tyrosine phosphorylation in
COS-1 cells transfected with the SHP2 constructs used in Fig. 1.
Although the expression of all SHP2 proteins was comparable (Fig.
3A, bottom), the
tyrosine phosphorylation level of the EGFR was moderately higher only
in the DM cells (Fig. 3A, top). Interestingly,
overexpression of the WT protein did not result in global
dephosphorylation of the EGFR (compare vector (V)
versus WT, Fig. 3A, top), suggesting that only a few phosphotyrosine (pY, Fig. 3A,
top) residues were targeted. To better compare these
observations, band intensities were quantified. In the vector, the WT
and the D425A transfected cells, the level of EGFR tyrosine
phosphorylation was virtually identical, ~50-fold over basal.
However, in the C459S and the DM cells it was 60- and 90-fold over
basal, respectively (Fig. 3B). When comparison was made
between the vector, the WT, or the D425A and the C459S or the DM, the
level of EGFR tyrosine phosphorylation in the C459S and the DM cells
was 1.2 and 1.8-fold higher, respectively.

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Fig. 3.
SHP2 partially dephosphorylated EGFR.
A, immunoprecipitation (IP) with anti-EGFR and
immunoblot (IB) with anti-Tyr(P) (pY,
top) of COS-1 cells transfected with the indicated SHP2
constructs. The same membrane was stripped and blotted with anti-EGFR
(middle) to show the presence of an equal amount of EGFR in
all lanes and stripped with anti-SHP2 (bottom) to
demonstrate expression of the different SHP2 proteins. B,
quantitation of the level of EGFR tyrosine phosphorylation.
C, immunoblot analysis of the level of EGFR tyrosine
phosphorylation following expression of increasing amounts of the WT
protein. The DM protein was used as a control. TCL, total
cell lysate; V, vector plasmid without SHP2 insert;
D/A, D425A-SHP2; C/S,
C495S-SHP2
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The dephosphorylation of EGFR by SHP2 was also tested by expression of
an increasing amount of the WT protein. Maximum dephosphorylation was
achieved at 5 µg of DNA per 6-cm plate of confluent COS-1 cells, and
no further dephosphorylation was observed by raising the DNA
concentration to 10 µg (Fig. 3C), indicating that SHP2 is
not a global phosphatase. As expected, cells transfected with 10 µg
of the DM construct did not show any dephosphorylation of the EGFR.
These results were consistent with specific and partial dephosphorylation and suggested that the EGFR might be an in
vivo substrate of SHP2.
SHP2 Dephosphorylates EGFR and Gab1 in Vitro--
To further
confirm that phosphotyrosyl proteins trapped by SHP2 are target
substrates, we carried out an immunocomplex in vitro
phosphatase assay. We concentrated only on the EGFR and Gab1, because
these are the only identified substrates (Figs. 1 and 6) against which
specific antibodies are available. EGFR from COS-1 cells stimulated
with EGF or Gab1 from 3T3 cells stimulated with serum was
immunoprecipitated with a specific antibody and then subjected to a PTP
assay using varying concentrations of purified WT protein (see
"Materials and Methods" for details). The WT protein partially
dephosphorylated both the EGFR and Gab1, whereas the DM protein did not
(Fig. 4, A and B,
top). In both cases, the addition of five times more WT
protein did not result in global dephosphorylation, further confirming
that whether it is inside or outside the cell, SHP2 is very specific.
Reblotting with anti-EGFR (Fig. 4A, bottom) or
anti-Gab1 (Fig. 4B, bottom) showed that a
comparable amount of protein was present in all lanes.

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Fig. 4.
SHP2 dephosphorylates EGFR and Gab1 in
vitro. COS-1 or NIH-3T3 cells were grown to confluency,
serum starved for 6 h, treated with 1 mM vanadate for
30 min, and then stimulated with EGF or serum for 10 min, respectively.
Lysates prepared from these cells were subjected to immunoprecipitation
(IP) with anti-EGFR or anti-Gab1 antibody and then to an
immunocomplex phosphatase assay as described under "Materials and
Methods." Note that the WT protein partially dephosphorylated EGFR
(A) and Gab1 (B) but not the DM protein. The
immunoblots (IB) presented were representatives of at least
three independent experiments. pY, Tyr(P).
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Subset of SHP2 Substrates Vary Depending on Stimulus and Cell
Type--
In EGF-stimulated COS-1 cells, the EGFR and three
unidentified phosphotyrosyl proteins were shown to be SHP2 substrates.
We treated SHP2-transfected COS-1 cells with another stimulus, namely 10% fetal calf serum. Anti-Tyr(P) blot showed trapping by the DM
protein of four phosphotyrosyl proteins that were identified as p190,
p150, p120, and p90 (Fig. 5A,
top). What was different from the case with EGF-stimulated
cells was the presence of p190 and the absence of p170 (EGFR),
suggesting that SHP2 substrates vary depending on the stimulus. Despite
comparable expression levels (Fig. 5A, bottom), a
relatively lower amount of p190 and p90 only were trapped by the C459S
protein. Furthermore, a significantly lower amount of p190
coprecipitated with the endogenous or overexpressed WT and the D425A
proteins. For the purpose of comparing the COS-1 cell results with
other cell type, we conducted a similar experiment in NIH-3T3 cells
using only three of the SHP2 proteins. Again, the DM protein trapped
four phosphotyrosyl proteins identified as p190, p150, p120, and p90
(Fig. 5B, top). In case of the C459S protein,
p190 and a significantly lower amount of p150 and p90 were trapped.
Some amount of p190 also coprecipitated with the WT protein, which may
represent an SH2 domain-mediated interaction. These results indicated
that the subset of SHP2 target substrates varies depending on the
stimulus and/or cell type.

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Fig. 5.
The DM protein identified four phosphotyrosyl
proteins as its target substrates in serum-stimulated cells and only
p190 in PDGF-stimulated cells. COS-1 (A and
C) or NIH-3T3 (B and D) cells were
transfected with an expression plasmid containing the indicated SHP2
constructs and stimulated either with serum or PDGF. Lysates prepared
from these cells were subjected to immunoprecipitation (IP)
with anti-SHP2 and immunoblot (IB) with anti-Tyr(P)
(pY) antibodies (top panel, in all
cases). Corresponding membranes were stripped and reblotted with
anti-SHP2 antibody to show expression of SHP2 proteins
(bottom panel, in all cases). Time course study
with serum stimulation in COS-1 cells is shown in C. Vec, vector; D/A, D425A-SHP2;
C/S, C495S-SHP2.
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Time course studies in COS-1 cells with serum stimulation revealed that
only the DM protein was able to trap significantly higher amounts of
target substrates at all time points tested, and the amounts seemed to
slightly increase over time, at least in case of p190 and p90 (Fig.
5C). Both in COS-1 and NIH-3T3 cells, the trapping of p90
and p150 was serum stimulation-independent, although stimulation
enhanced the amount. In contrast, the amount of target substrates
trapped by the C459S protein was low and largely serum
stimulation-dependent.
Previous studies demonstrated that SHP2 dephosphorylates the PDGFR
under in vitro conditions (47). Because serum contains PDGF
and the molecular weight of p190 was close to that of the PDGFR, we
assumed that p190 could be the PDGFR. For this, we overexpressed the DM
protein in NIH-3T3 cells and then stimulated them with PDGF for 10 min.
Lysates prepared from these cells were subjected to immunoprecipitation
with anti-SHP2 and immunoblot analysis first with anti-Tyr(P) and later
with anti-PDGFR antibodies. An anti-Tyr(P) blot detected p190 in
PDGF-stimulated cells (Fig. 5D, top).
Interestingly, only p190 was trapped by the DM protein, although there
were several phosphotyrosyl protein bands in the corresponding lysate
(compare +PDGF IP with +PDGF TCL in Fig. 5D), suggesting that the interaction was highly specific.
However, reprobing the membrane with several anti-PDGFR antibodies
could not positively identify p190 as the PDGFR (data not shown). Thus, the identity of p190, p150, and p90 remain unknown. Further reprobing of the membrane with anti-SHP2 antibody showed the presence of equivalent amount of the DM protein in all lanes (Fig. 5D,
bottom).
Identification of the Other Phosphotyrosyl Protein
Bands--
We made efforts to identify the other phosphotyrosyl
proteins that were trapped by the DM protein using a variety of
antibodies against known proteins in the indicated molecular weight
range. Analysis with anti-p130Cas, anti-FYB, and anti-SHP2
substrate 1 antibodies gave negative results (data not shown). Another
potential candidate substrate is Gab1, which was shown to interact with
SHP2 (33). Immunoblotting anti-SHP2 immunoprecipitates with anti-Gab1
showed the presence of a higher amount of Gab1 in the DM cells as
compared with the WT or the C459S cells (Fig.
6A, top), although
the expression level of all SHP2 proteins was similar (Fig.
6A, bottom). These experiments were repeated in a
reciprocal way, i.e. immunoprecipitation with anti-Gab1 and
immunoblotting with anti-SHP2. Significantly higher amount of the DM
protein coprecipitated with Gab1 as compared with the WT or the C459S
proteins (Fig. 6B, top panel).
Reprobing with anti-Gab1 antibody showed that there was an
equivalent amount of Gab1 in all lanes (Fig. 6B,
middle). Immunoblot with anti-Tyr(P) of Gab1
immunoprecipitates showed that the tyrosine phosphorylation level of
Gab1 was higher in the DM cells as compared with the WT or the C459S
cells (Fig. 6B, bottom). Similar results were obtained in NIH-3T3 cells (data not shown). Thus, Gab1 could be a
substrate of SHP2.

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Fig. 6.
The DM protein identified Gab1 as its target
substrate. COS-1 cells were transfected with the indicated SHP2
constructs, and lysates prepared from these cells were subjected to
immunoprecipitation (IP) and immunoblot (IB)
analysis. A, immunoprecipitation with anti-SHP2, and
immunoblot with anti-Gab1 (top) and anti-SHP2
(bottom). B, immunoprecipitation with anti-Gab1
and immunoblot with anti-SHP2 (top), anti-Gab1
(middle), and anti-Tyr(P) (pY,
bottom). C/S, C495S-SHP2.
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DISCUSSION |
Genetic and biochemical studies have demonstrated that SHP2 is
primarily a positive effector of RTK signaling. Deletion of the N-SH2
or the phosphatase domain or mutation of the conserved Cys residue to
Ser (C459S) abrogated the biological activity of SHP2 (48, 49). Mice
homozygous for the N-SH2 deletion mutant die in the uterus before day
E10.5 from multiple defects in mesoderm development (19). Also,
microinjection of SH2 or phosphatase deletion mutant mRNAs into
Xenopus eggs caused abnormal embryonic development
(49, 50). However, the mechanism(s) by which SHP2 effects its
biological activity is unknown. We reasoned that identification of
physiological substrates might be a stepping stone in elucidating the
molecular mechanism of SHP2 function. Here, we report the development
of an efficient substrate-trapping mutant of SHP2 and also
demonstrate for the first time in intact cells that EGFR, Gab1, and
three other as yet unidentified phosphotyrosyl proteins are
candidate physiological substrates.
Trapping mutants of PTPs can be produced by the mutation of Asp to Ala
in the conserved WPD loop (38, 40, 51). We utilized the same principle
and produced an Asp to Ala mutant of SHP2, but found that it was not
trapping. In line with our findings, it was recently reported that the
Asp to Ala mutant of RPTP
was also non-trapping (52). These findings
are in contrast with previous reports wherein the introduction of an
Asp to Ala mutation to PTP1B and TC-PTP resulted in substrate-trapping
mutants (38-40). Thus, this principle might not apply to all PTPs.
Structural studies on the PTP domain of SHP1 bound to a phosphopeptide
derived from SHPS-1 showed that the WPD loop did not close as expected
(53, 54). Because SHP1 is a close structural relative of SHP2, we speculate that the Asp in the WPD loop of SHP2 may not serve as an
efficient proton donor/acceptor for the hydrolysis of the thiol-ester intermediate. This observation is further supported by the finding that
the D425A protein autodephosphorylated itself comparable with the WT
(Fig. 1A, top panel), suggesting that
the mutation did not cause loss of enzymatic activity. As demonstrated
for VHR (41), it could be that the hydroxy amino acid Thr, immediately C-terminal to the Arg in the signature motif of SHP2, might be responsible for the hydrolysis of the thiol-ester intermediate.
The above findings led us to resort to improving the trapping ability
of the C459S mutant. We reasoned that, although the D425A mutant of
SHP2 per se is non-trapping, it might reinforce the ability
of the C459S mutant. Indeed, the DM protein showed a superior trapping
ability. Thus, although the Asp in the WPD loop of SHP2 does not seem
to have a role in the catalytic process, it could be close enough to
cause electrostatic repulsion on the phosphate moiety of the substrate.
Our results are consistent with this hypothesis, because the DM protein
efficiently trapped the EGFR, Gab1, and three other, as yet
unidentified, phosphotyrosyl proteins (Figs. 1, 5, and 6). Xie
et al. recently reported that the Asp to Ala and
Cys to Ser double mutant PTP1b was non-trapping (55). Although it was
not clear from the presented data or explanations given, it may simply
reflect the structural and functional differences between the two proteins.
SHP2 interacts with autophosphorylated RTKs or tyrosine phosphorylated
adaptor proteins (7, 8, 23, 26, 29, 32, 56, 57). Because the SH2
domains in all constructs used were intact, it is possible that some
amount of EGFR bound to the DM protein was SH2-mediated. Indeed, a low
amount of EGFR was detected in the WT and D425A mutants. However,
compared with the amount of EGFR that coprecipitated with the DM
protein, the SH2 domain-mediated interaction was extremely low and thus
cannot account for the observed difference.
It could also be argued that the interaction of the DM protein with
EGFR was indirect via an unknown target substrate of SHP2 that binds to
EGFR. However, the partial dephosphorylation of EGFR in the control and
the WT cells as compared with the C459S and the DM suggested otherwise.
If the interaction was indirect or if EGFR was not a substrate of SHP2,
we would expect similar levels of EGFR tyrosine phosphorylation in all
experimental groups. Thus, the most likely explanation is a direct
trapping of the EGFR by the PTP domain of the DM protein. The
time-dependent nature of the accumulation of
enzyme-substrate complexes following stimulation (Figs. 2 and
5C) suggested that the complex, once formed, is stable. Given that the interaction of the DM protein with candidate substrates occurred in intact cells following ligand stimulation and that the
trapping was very specific (Figs. 1, 5, and 6), it is highly likely
that EGFR and Gab1 represent physiological SHP2 substrates. Thus, the
DM protein is an ideal tool for the isolation and identification of
physiological substrates of SHP2 and as well for identification and
characterization of specific tyrosine phosphorylation sites on substrates.
Another point worth mentioning is that the double mutation may cause
structural changes that affect the specificity of the DM protein. The
specificity of the SH2 domain-containing PTPs is regulated at two
levels, i.e. proper cellular translocation mediated by the
SH2 domains and inherent substrate discrimination mediated by the PTP
domain. Because both SH2 domains are intact in all of the SHP2 proteins
used, including the DM protein, proper cellular translocation to a
substrate microdomain has not been compromised by the double mutation.
The crystal structure of SHP2 showed that its catalytic activity is
controlled by an intramolecular interaction that involves the N-SH2 and
the PTP domains (58). Critical residues that mediate this interaction
are Asp-61 and Glu-76 on N-SH2, and Arg465 and main-chain amine groups
of the "signature motif" on the PTP domain (58, 59).
Furthermore, Arg-465 and main-chain amide groups of the signature
motif on the PTP domain are critical for coordinating the
phosphate moiety of substrate proteins upon binding to the active site.
Again, these residues are intact in the DM protein, and, thus, the
double mutation is unlikely to cause either disregulation or loss of substrate specificity. The demonstration that the DM protein
selectively trapped only p190 in PDGF-stimulated samples (Fig.
5D) is an excellent example for this conclusion.
In EGF-stimulated cells, the level of tyrosine phosphorylation of the
EGFR was moderately higher in DM-transfected cells. Furthermore,
expression of increasing amounts of the WT protein did not result in
global dephosphorylation of the EGFR. Interestingly, an in
vitro immunocomplex phosphatase assay on EGFR and Gab1 (Fig. 4)
also provided results that mirrored those in intact cells (Figs. 3 and
6). These findings were consistent with partial dephosphorylation of
the EGFR and Gab1 by SHP2. Therefore, SHP2 is a very specific PTP that
only targets certain phosphorylation sites on substrate proteins.
Recent reports on the DEP-1 receptor-like PTP also show that it
dephosphorylates the Met receptor partially (60). However, previous
work that compared the chimeras of the SH2 and PTP domains of SHP1 and
SHP2 toward EGFR as a substrate concluded that SHP2 does not
dephosphorylate the EGFR (61). The basis for this conclusion could be
that no comparison between the WT and the phosphatase-defective mutant
of SHP2 was made and that complete but not partial dephosphorylation was probably the expectation.
Important questions that remain to be answered are which tyrosine
residue(s) on the EGFR are targets of SHP2 PTP activity and why?
Previous work has shown that SHP2 preferentially dephosphorylated a
phosphopeptide derived from Tyr-992 of the EGFR under in
vitro conditions (62). Similarly, Corkscrew, the
Drosophila homologue of SHP2, dephosphorylated a single
autophosphorylation site on the Torso RTK that serve as a binding site
for RasGAP, the down-regulator of GTP-Ras (25). Thus, it could be that
SHP2 dephosphorylates a few phosphotyrosine residues on the EGFR that
may serve as binding sites for negative regulatory proteins.
We also found that Gab1 is a substrate of SHP2. Gab1 is an adaptor
protein with several tyrosine residues that become phosphorylated upon
induction by growth factors or cytokines (30, 32-34, 56). These
phosphotyrosines serve as binding sites for SH2 domain-containing signaling proteins, the most noted being SHP2 and
phosphatidylinositol-3 kinase (PI3K) (30, 34, 56). The same question as
for the EGFR could be asked, i.e. why does SHP2
dephosphorylate Gab1, whose tyrosine phosphorylation is essential for
signal transduction? Possible explanations would again be similar,
i.e. that SHP2 might be dephosphorylating negative
regulatory sites on Gab1. Again, a direct answer to this question must
await mutational analysis of phosphorylation sites on Gab1.
The identities of p90, p150, and p190 are currently unknown. Although
the PDGFR is a plausible candidate for p190, we were unable to
positively identify this protein to be the PDGFR, given the limitations
of the currently available antibodies. Thus, identification of p90,
p150, and p190 must be addressed in future studies that utilize protein
isolation and peptide sequencing and, if necessary, cDNA cloning,
nucleotide sequencing, and characterization.
The findings that the subset of SHP2 substrates varied depending on the
stimulus suggested that target proteins of SHP2 PTP activity vary,
depending on the pathways that become activated and/or the cell type.
The ability of SHP2 to act on a different set of substrates in
different signaling pathways might determine its functional specificity
in mediating cell growth, differentiation, or motility.