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INTRODUCTION |
A large number of oncogenes encode altered forms of growth
factor receptor-tyrosine kinases (1, 2). Oncogenes derived from
cellular receptor-tyrosine kinases
(RTKs)1 are constitutively
activated by structural changes, such as point mutation, truncation,
and rearrangement (3-6). The activation of RTKs triggers
autophosphorylation of tyrosine residues, which then serve as specific
binding sites for cellular signaling proteins containing Src-homology 2 (SH2) or phosphotyrosine binding domains (7-9). Individual SH2 domains
recognize specific phosphotyrosine residues flanked by distinct
sequences (10). Signal transduction by RTKs involves activation of
multiple pathways following association with different cellular target proteins.
The env-sea oncogene is derived from a cellular
protein-tyrosine kinase, c-sea, the extracellular and transmembrane
domains of which have been replaced by viral envelope sequences (11). Due to the fusion with viral envelope domain, the env-sea protein has a
structure similar to RTKs (12). Previous studies showed that the
env-sea tyrosine kinase activity is necessary for transformation by the
env-sea protein (13) and that cell surface localization, oligomerization by viral envelope domains, and autophosphorylation are
all necessary for the transforming activity of the env-sea protein
(14-16). Using a temperature-sensitive mutant, previous work
demonstrated that the phosphorylation of the Shc proteins correlated
with the activation of the tyrosine kinase activity of the env-sea
protein (17). However, Shc did not form a stable complex with the
env-sea protein. In contrast, the Grb2 protein did associate with the
env-sea protein (17). To date, no other proteins have been demonstrated
to be involved in env-sea signaling.
The env-sea protein is a member of the hepatocyte growth factor
receptor subfamily. All members of this family have a bidentate motif,
comprising two tandemly arranged SH2 domain binding sites at the
carboxyl terminus (18-20), which functions as a multisubstrate interaction site (21). In the Met protein, it has been demonstrated that the tyrosine closest to the carboxyl terminus within this motif,
tyrosine 1356, plays a major role in signaling by this receptor (22,
23). Although this motif is conserved in Sea, the sequences surrounding
the tyrosine are quite different from those in Met. In particular,
env-sea has two potential Grb2 binding sites, whereas Met has only one,
and Grb2 binding has been shown to play a major role in Met signaling
(24, 25). However, there is no information on the role of this motif in
signaling by env-sea, nor is there any knowledge of substrates that can
bind to this motif. Hence, we have undertaken a structure-function
analysis to address the role of this motif in signaling by env-sea. We constructed mutants in which these tyrosines are changed to
phenylalanine residues. In this report, we show that the conserved
bidentate motif is essential for the transformation of Rat1 cells by
the env-sea protein. However, in contrast to Met, substitution of each
tyrosine alone either had no effect on transformation or actually
enhanced transformation. To study downstream signaling pathways
mediated by the tyrosine residues in the motif, we looked for
protein-protein interactions at this site. Our data indicate that Grb2,
phosphatidylinositol 3-kinase (PI3 kinase), and the tyrosine
phosphatase SHP-2 can all bind to this motif, but there was no simple
correlation between association of these proteins and transformation.
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EXPERIMENTAL PROCEDURES |
Reagents, Cells, and Antibodies--
All reagents, unless
specified, were purchased from Sigma. All cell lines were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum. S13 Rat1 cells are Rat1 fibroblasts expressing a
temperature-sensitive env-sea protein (11). S13 Rat1 cells were grown
at 35 and 39.5 °C with 7% CO2. Rat1 and COS1 cells were
grown at 37 °C with 7% CO2.
The sea-specific antibody was generated against the PstI to
ClaI fragment of the env-sea gene and was
characterized previously (26). Anti-Shc antibody directed against the
SH2 domain was provided by Dr. Pawson. Anti-SHP-2 antibody was a gift
from Dr. Kazlauskas. A monoclonal antibody that recognizes the M45
epitope tag was kindly provided by Dr. Hearing. Anti-phosphotyrosine
antibody (4G10) and anti-MAP kinase antibody were gifts from Dr.
Morrison and Dr. Davis, respectively. Monoclonal antibodies against
Grb2 and SHP-2 were purchased from Transduction Laboratories. The
polyclonal antibodies against Grb2 and glutathione
S-transferase (GST) were obtained from Santa Cruz
Biotechnology, and the antibody against PI3 kinase was from Upstate Biotechnology.
Site-directed Mutagenesis and Construction of the Expression
Vector--
Cloning of the full-length env-sea gene
(pS13-1) was reported previously (11). To generate tyrosine to
phenylalanine substitutions at the carboxyl-terminal end, the
site-directed mutagenesis method of overlap extension (27) was used.
SacI and ClaI fragment (amino acids 1161-2091)
containing the bidentate motif was subcloned into pBluescript
(Stratagene). For the Y557F mutant, the sense primer was
5'-ggtgagcacttcatcaacatggc-3', and the antisense primer was
5'-gccatgttgatgaagtgctcacc-3'. For the Y564F mutant, the sense primer
was 5'-gctgtcaccttcgtcaacctggag-3', and the antisense primer was
5'-ctccaggttgacgaaggtgacagc-3'. For the Y557F/Y564F mutant, the sense
primer was 5'-ggtgagcacttcatcaacatggctgtcaccttcgtcaacc-3', and the
antisense primer was the same as for the Y564F mutant. Primary
polymerase chain reaction was done with the sense and T7 primers and
also with the antisense and T3 primers. Primary polymerase chain
reaction products were used for overlap extension with T3 and T7
primers. Final products were purified, digested with SacI
and ClaI, and then subcloned into pS13-1. The entire env-sea
sequence containing the point mutations was digested with XbaI and KpnI and subcloned into an expression
vector, pMT2, at the EcoRI site. To generate the M45 epitope
tag inside the env-sea protein, the sense primer
5'-tcgaggatcggagtagggatcgcctacctccttttgagacagagacgcggatcc-3' and the
antisense primer
5'-tcgaggatccgcgtctctgtctcaaaaggaggtaggcgatccctactccgatcc-3' were
generated. Oligomers were annealed and ligated into pS13-1 at the
XhoI site in the amino-terminal envelope sequence. The correct orientation was confirmed by sequencing.
DNA Transfection--
To generate Rat1 cell lines overexpressing
the env-sea proteins, Rat1 cells were co-transfected with pMT2/M45
env-sea and pRSVneo by the calcium phosphate method as descried by Chen
and Okayama (28). Transfected cells were selected in Dulbecco's modified Eagle's medium containing G418 (400 µg ml, Life
Technologies, Inc.). For transient transfection into COS1 cells,
FuGENETM 6 reagent (Boehringer Mannheim) was used according
to manufacturer's protocol.
Cell Lysates, Immunoprecipitation, and Western
Blotting--
Cells were lysed in buffer containing 1% Triton, 10%
glycerol, 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, and protease inhibitors (1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml, and 10 µg of aprotinin/ml).
Lysates were precleared by centrifugation. Protein concentrations were determined using the BCA protein assay system (Pierce). 300 µg of
total cell lysate was incubated with antibodies for 2-3 h at 4 °C,
and then 20 µl of 50% protein A-Sepharose (Amersham Pharmacia Biotech) was added and incubated for an additional 2 h.
Immunoprecipitates were washed four times in the lysis buffer,
solubilized by boiling in Laemmli sample buffer, subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred
to nitrocellulose membrane (Schleicher & Schuell). For Western
blotting, membranes were blocked in 3% bovine serum albumin in
phosphate-buffered saline containing 0.1% Triton X-100 for at least
1 h at room temperature and then incubated with indicated
antibodies for at least 2 h. Immunoreactive bands were visualized
by enhanced chemiluminescence kit (Amersham Pharmacia Biotech)
Soft Agar Colony Forming Assay--
Clones of Rat1 cells
transfected with the different mutant and wild type forms of the
env-sea protein were screened for equivalent levels of expression of
the various env-sea proteins. Cells that were identified to express
equivalent amounts of env-sea protein were seeded into 0.3% agar
(Difco) supplemented with 10% fetal bovine serum at a density of
105 or 106 cells per 60-mm plate and then
incubated at 35 °C. The cells were fed with soft agar medium at day
5, and colonies were counted on day 10.
Purification of GST Fusion Proteins--
Bacteria
expressing GST-Shc (SH2) were provided by Dr. Pawson. Dr. Bar-Sagi and
Dr. Keegan provided bacteria expressing full-length GST-Grb2 and
full-length GST-SHP-2. The SH2 domain(s) of each protein was polymerase
chain reaction-amplified and subcloned into an expression plasmid,
pGEX-KG (29). Overexpressed GST fusion proteins were purified using
glutathione-Sepharose beads (Amersham Pharmacia Biotech) dialyzed and
concentrated (3 mg/ml) for microinjection. For an in vitro
binding experiment using GST-SHP-2 (SH2s), 600 µg of lysates was
incubated with 5 µg of GST fusion proteins on glutathione-Sepharose
for 2 h at 4 °C, and then bound proteins were detected by
Western blotting with anti-M45 antibody.
Microinjection--
S13 Rat1 cells were plated onto
acid-washed coverslips, grown to subconfluent density at 39 °C, and
then placed in Dulbecco's modified Eagle's medium with 0.5% fetal
bovine serum for 22-24 h prior to microinjection. GST fusion proteins
were microinjected into the cytoplasm. After injection, cells were
incubated at 35 or 39 °C for an additional 12-14 h, fixed in 3.7%
formaldehyde in phosphate-buffered saline for 1 h at room
temperature, and then permeabilized with 0.1% Triton X-100 in
phosphate-buffered saline for 3 min at room temperature. The coverslips
were incubated with rabbit anti-GST antibody (Santa Cruz) in
phosphate-buffered saline containing bovine serum albumin (2 mg/ml) at
37 °C for 1 h and then with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG. To visualize cells, the
actin cytoskeleton was stained with rhodamine-conjugated phalloidin
(Molecular Probes). Fluorescent microscopy was used to analyze the results.
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RESULTS |
Tyrosine 557 and 564 Mediate the Transforming Ability of the
env-sea Protein in Rat1 Cells--
The bidentate motif is made up of
two tandemly arranged potential SH2 domain binding sites
(Y557INMAVTY564VNL). This motif is shown
schematically in Fig. 1. To investigate whether tyrosine residues in this motif are essential for the transforming ability of env-sea, we generated point mutants that have
phenylalanine for tyrosine substitutions at each site individually (Y557F and Y564F) or at both residues (Y557F/Y564F) in Rat1 cells. In
addition, because the sequences carboxyl-terminal of the tyrosine can
influence SH2 domain binding, we converted the sequence around Tyr-564
to that of Tyr-557
(Y557/Y557).2 To monitor
expression, an epitope tag (M45) was generated at the amino-terminal
end of the envelope sequence. In Rat1 cells, the env-sea protein is
synthesized as a glycosylated protein of 155 kDa (gp155) that undergoes
proteolytic processing to generate the proteins gp85 and gp70; thus,
anti-M45 antibody can recognize gp155 but not gp70 (Fig. 1). In Fig.
2A, the upper bands
represent the mature, glycosylated form of env-sea proteins (gp155),
whereas the lower bands represent the immature,
unglycosylated precursor proteins. Expression levels for all mutant
env-sea proteins were comparable to wild type env-sea protein (Fig.
2A), and for the Y557F/Y564F mutant, they were generally
higher than wild type. To see whether any of these mutations affected
the autophosphorylation activity of gp70, we performed
anti-phosphotyrosine Western blotting of immunoprecipitates made with
anti-sea antibodies from equivalent amounts of cell lysates. As can be
seen, none of these mutations had any major effect on
autophosphorylation (Fig. 2B). The level of phosphorylation
of the Y557F/Y564F mutant was slightly lower than the other proteins,
especially if one considers the higher expression levels seen with this
mutant. However, this presumably reflects the loss of two
phosphorylation sites.

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Fig. 1.
Schematic representation of the env-sea
protein. This diagram illustrates the proteolytic processing of
the env-sea protein and also indicates the location of the bidentate
motif and the M45 epitope tag.
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Fig. 2.
Point mutations in the carboxyl-terminal
tyrosine residues do not affect the expression and the
autophosphorylation of the env-sea protein. Lysates from parental
Rat1 cells, stable cell lines expressing wild type or various mutant
env-sea proteins, were prepared. A, 30 µg of total cell
lysates was immunoblotted with anti-M45 antibody to detect M45-tagged
env-sea proteins. B, 300 µg of total cell lysates was used
to immunoprecipitate env-sea proteins with anti-sea antibody.
Immunoprecipitates were then immunoblotted with anti-phosphotyrosine
antibody. The positions of the env-sea precursor protein
(pr), gp155, and gp70 are indicated.
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To determine the effect of these mutations on transformation, the same
cell lines were used to measure colony-forming abilities in soft agar.
The results are shown in Fig. 3 and
quantified in Table I. Cells expressing
the Y557F mutant protein were able to transform cells to a level
comparable to cells expressing wild type env-sea protein. In contrast,
cells expressing the Y564F mutant protein formed even more colonies
than wild type cells. Cells expressing the Y557/Y5572
mutant protein produced fewer and smaller colonies than those induced
by the wild type env-sea protein. Cells expressing the env-sea protein
that have phenylalanine substitutions at both tyrosine residues
(Y557F/Y564F) completely lost their ability to grow in agar. These data
demonstrate that the tyrosines within this motif are essential for
transformation, and in contrast to the results using comparable mutants
in the Met protein, mutation of tyrosine 564 actually enhanced
transformation.

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Fig. 3.
Mutations at both Tyr-557 and Tyr-564 are
necessary to completely abrogate the transforming activity of the
env-sea protein. The transforming ability of wild type and mutant
env-sea proteins was compared in a colony forming assay in soft agar.
Rat1 cells expressing wild type or mutant env-sea proteins were seeded
into 0.3% agar, and colonies were counted after 10 days of
incubation.
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Grb2 Interacts with the env-sea Protein and Shc--
We reasoned
that the differences in transforming ability among mutant env-sea
proteins come from changes in association with one or more SH2
domain-containing cellular target proteins. To further study
protein-protein interactions mediated by these tyrosine residues, we
utilized transient expression in COS1 cells. In COS1 cells, wild type
and all mutant env-sea proteins were expressed at similar levels (Fig.
4A). Tyrosine phosphorylation
of the env-sea proteins was monitored by immunoprecipitation with an
anti-sea antibody followed by Western blotting with an
anti-phosphotyrosine antibody. Tyrosine phosphorylation levels remained
approximately the same for wild type, Y557F, Y557/Y557,2
and Y564F env-sea proteins but were lower for the Y557F/Y564F mutant
protein (Fig. 4B). This may reflect the fact that the
Y557F/Y564F has lost two phosphorylation sites and only keeps the basal
level of the autophosphorylation. Expression of a K318R env-sea
protein, which has a point mutation in the ATP binding pocket that
inactivates the kinase activity of Sea, lacked detectable tyrosine
phosphorylation. This demonstrates that the tyrosine phosphorylation
detected in the env-sea proteins is dependent on the kinase activity of
Sea.

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Fig. 4.
Expression of wild type and mutant env-sea
proteins in COS1 cells. COS1 cells in a 60-mm plate were
transiently transfected with 5 µg of the indicated constructs. Cell
lysates were made 22-24 h later. A, 30 µg of cell lysates
was immunoblotted with anti-M45 antibody to detect M45-tagged env-sea
proteins. B, 300 µg of cell lysates were
immunoprecipitated with anti-sea antibody and precipitated proteins
were resolved by 7.5% SDS-PAGE and detected with anti-phosphotyrosine
antibody. The positions of the env-sea precursor protein
(pr), gp155, and gp70 are indicated.
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The env-sea protein has previously been shown to be in a complex with
the Grb2 protein, but the binding site has never been identified. Both
tyrosines 557 and 564 are followed by the amino acid sequence
XNX (Fig. 1), which is a consensus Grb2 binding site (10). To investigate which tyrosine is the binding site for Grb2,
co-immunoprecipitation experiments were performed. COS1 cells were
transfected with the various constructs and the expression levels of
the env-sea protein determined to be equivalent using Western blotting
(Fig. 5A).Wild type or mutant
proteins were immunoprecipitated with an anti-sea antibody, and
co-immunoprecipitated Grb2 was detected by immunoblotting. Grb2 was
readily detected as being associated with the Y557F mutant protein as
well as with wild type env-sea protein (Fig. 5B). There were
only very weak Grb2 signals detected with Y557/Y5572 and
Y564F proteins. When Grb2 was immunoprecipitated and the immunoprecipitates were blotted with anti-M45 antibody, again, the
Y557F mutant and wild type env-sea proteins were detected as being
co-immunoprecipitated with Grb2, and the other three mutant proteins
were not detected (Fig. 5C). This indicates that although
both sites are consensus binding sites for Grb2, the env-sea protein
and Grb2 form a complex in cells primarily through tyrosine 564.

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Fig. 5.
Grb2 is associated with env-sea primarily
through tyrosine 564. A, 30 µg of total cell lysates
was immunoblotted with anti-M45 antibody to detect M45-tagged env-sea
proteins. B, wild type and various mutant env-sea proteins
from transiently transfected COS1 cells were immunoprecipitated with
anti-sea antibody and immunoblotted with anti-Grb2 antibody.
C, the same set of lysates was immunoprecipitated with
anti-Grb2 antibody. Immunoprecipitates were resolved and immunoblotted
with anti-M45 antibody. D, the same filter as in
B was stripped and reprobed with anti-phosphotyrosine
antibody. pr, precursor protein.
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To determine whether the mutations in the env-sea protein affect the
association of tyrosine-phosphorylated proteins with Grb2, we stripped
the filter used in Fig. 5C and reprobed it with an
anti-phosphotyrosine antibody. As shown in Fig. 5D, Grb2 was associated with several tyrosine-phosphorylated proteins in cells expressing the wild type env-sea protein. COS1 cells transfected with
vector alone or with the Y557F/Y564F mutant construct showed basal
levels of the tyrosine phosphorylation of Grb2-associated proteins
(Fig. 5D). This indicates that although the Y557F/Y564F mutant protein is an active kinase, it does not induce the
phosphorylation of Grb2-associated proteins or of any other cellular
proteins (data not shown). In addition to the env-sea proteins gp155
and gp70, there were three prominent bands in cells expressing wild type and the other three mutant proteins, indicated by the
arrows in Fig. 5D. The molecular weights of these
three proteins are similar to those of the Shc proteins. Previous
studies have demonstrated that Shc is tyrosine-phosphorylated following
activation of the env-sea protein and associates with Grb2 (17). To
confirm that the mutant env-sea proteins were able to induce tyrosine
phosphorylation of the Shc proteins, cells expressing equivalent
amounts of wild type and mutant proteins (Fig.
6A) were immunoprecipitated
with anti-Shc antibody, and then immunoprecipitates were blotted with anti-phosphotyrosine antibody (Fig. 6B). The levels of
tyrosine-phosphorylated Shc was increased to similar levels in cells
expressing the Y557/Y557,2 Y557F, or Y564F mutant proteins,
as well as in cells expressing the wild type protein. In contrast,
there was no increase in the level of tyrosine phosphorylation in cells
expressing either the dead kinase mutant, K318R, or in the Y557F/Y564F
mutant.

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Fig. 6.
Shc is tyrosine-phosphorylated and associates
with Grb2. A, 30 µg of total cell lysates was
immunoblotted with anti-M45 antibody to detect M45-tagged env-sea
proteins. B, cell lysates were prepared from transiently
transfected COS1 cells and used to immunoprecipitate Shc proteins.
Immunoprecipitates were resolved by 10% SDS-PAGE and immunoblotted
with anti-phosphotyrosine antibody. C, the same cell lysates
were resolved on a 10% low cross-linking gel and immunoblotted with
anti-MAP kinase antibody. MAPK* indicates the mobility of
the activated form of MAP kinase. pr, precursor
protein.
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Shc is phosphorylated in Y564F-expressing cells and associates with
Grb2. This interaction between Grb2 and Shc has been shown previously
to lead to activation of downstream effectors, such as MAP kinase.
Therefore, even though Grb2 is unable to associate directly with the
Y564F protein, the phosphorylation of Shc that is induced by the Y564F
protein could induce activation of MAP kinase. We decided to test this
possibility. MAP kinase has been shown to require both tyrosine and
threonine phosphorylation for activation (30). These phosphorylation
events result in a characteristic delayed mobility of MAP kinase by
SDS-PAGE (31). Therefore, we used this mobility shift assay to
determine whether the Shc-Grb2 complex formation correlates with the
level of MAP kinase activation. As shown in Fig. 6C,
activation of MAP kinase in Y564F mutant-expressing cells was equal to
that found in the wild type-, Y557/Y557-,2 and Y557F
mutant-expressing cells. In contrast, there was no increase in MAP
kinase activation in cells expressing the Y557F/Y564F or K318R mutant
proteins. Therefore it appears that all the mutant forms of env-sea
protein that cause transformation can induce Shc tyrosine
phosphorylation and subsequent MAP kinase activation.
Phosphatidylinositol 3-Kinase Associates with the env-sea Protein
Primarily through Tyrosine 557--
Examination of the amino acid
sequence of the bidentate motif reveals that the site surrounding
Tyr-557 is a perfect consensus binding site for PI3 kinase
(YXXM) (Fig. 1). To determine whether PI3 kinase is bound to
this site on the env-sea protein, we transfected the different forms of
env-sea into COS1 cells. Analysis of env-sea protein expression
revealed equivalent levels of expression (Fig. 7A), so we went ahead and
formed immunoprecipitates with anti-PI3 kinase antibodies and analyzed
the precipitates for the presence of the phosphorylated env-sea protein
by Western blot. As can be seen in Fig. 7B, the wild type
env-sea protein was co-immunoprecipitated by the PI3 kinase antibodies.
In addition, the tyrosine-phosphorylated p85 subunit of the PI3 kinase
enzyme was also readily detected in the immunoprecipitates. In
contrast, mutation of Tyr-557 to phenylalanine drastically reduced the
association of PI3 kinase with the env-sea protein, and
tyrosine-phosphorylated p85 was also barely detectable. (Reprobing of
the blot with antisera against PI3 kinase indicated equivalent levels
of p85 in all lanes (data not shown).) Mutation of Tyr-564 had little
effect on PI3 kinase association or p85 phosphorylation. Duplication of
the Tyr-557 site in mutant Y557/Y5572 appeared to increase
the association of the phosphorylated env-sea protein and PI3 kinase
(Fig. 7B). Because the Y557/Y5572 mutant has a
decreased transforming ability, there does not appear to be a simple
relationship between PI3 kinase association and transformation by these
env-sea mutants.

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Fig. 7.
PI3 kinase associates with the env-sea
protein primarily through tyrosine 557. COS1 cells in a 60-mm
plate were transiently transfected with 5 µg of indicated constructs.
Cell lysates were made 22-24 h later. A, 30 µg of cell
lysates was immunoblotted with anti-M45 antibody to detect M45-tagged
env-sea proteins. B, cell lysates were prepared from
transiently transfected COS1 cells and used to immunoprecipitate PI3
kinase proteins. Immunoprecipitates were resolved by 10% SDS-PAGE and
immunoblotted with anti-phosphotyrosine antibody. The mobility of the
p85 subunit of PI3 kinase, gp155, and gp70 are indicated.
pr, precursor protein.
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SHP-2 Interacts with the env-sea Protein and Plays a Positive Role
in MAP Kinase Activation--
The tyrosine phosphatase SHP-2 is
thought to be a positive regulator in RTK signaling (32-34).
Therefore, we investigated whether SHP-2 is involved in the env-sea
signaling. First, we investigated whether SHP-2 can serve as a
substrate of the env-sea protein by co-expressing the SHP-2 protein
with either a wild type env-sea protein or the catalytically dead
version of the env-sea protein, K318R (Fig.
8A, top panel). SHP-2 was not
tyrosine-phosphorylated when it was expressed alone (lane
2). When SHP-2 was expressed with the wild type env-sea protein,
SHP-2 became tyrosine-phosphorylated and physically interacted with the
env-sea protein (Fig. 8A, lane 3). However, when it was
expressed with the catalytically inactive K318R env-sea protein, there
was no phosphorylation of SHP-2 (lane 4). Those data
strongly suggest that SHP-2 is a substrate of the env-sea protein.
Interestingly, a tyrosine-phosphorylated protein with an apparent
molecular mass of 90 kDa (pp90) co-immunoprecipitated with SHP-2
(lane 3). We identified this protein as the SHP-2 substrate SHPS-1 (data not shown).

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Fig. 8.
SHP-2 physically interacts with the env-sea
and plays a positive role in MAP kinase activation. A,
COS1 cells were transfected with control vector (lane 1),
SHP-2 (lane 2), or SHP-2 with either wild type env-sea
(lane 3) or K318R env-sea (lane 4). SHP-2 was
immunoprecipitated with polyclonal anti-SHP-2 antibody.
Immunoprecipitates were resolved by 7.5% SDS-PAGE and then
immunoblotted with anti-phosphotyrosine antibody (top
panel). Whole cell lysates were immunoblotted with a mixture of
anti-M45 and anti-SHP-2 antibodies to show the expression levels
(bottom panel). The mobility of gp155, gp70, SHP2, and the
unknown protein pp90 are shown. B, 30 µg of total cell
lysates was immunoblotted with anti-M45 antibody to detect M45-tagged
env-sea proteins. C, lysates from the above cells expressing
wild type or mutant env-sea proteins were used to immunoprecipitate
with anti-SHP-2 antibody, and bound proteins were detected with
anti-M45 antibody. D, wild type or various mutant sea
proteins were immunoprecipitated with anti-sea antibody and resolved.
Bound proteins were detected by Western blotting with anti-SHP-2
antibody. E, 5 µg of GST-SHP-2 (SH2) proteins bound to
glutathione-Sepharose was incubated with 600 µg of cell lysates.
Complexes were washed and resolved by 7.5% SDS-PAGE and immunoblotted
with anti-M45 antibody. F, COS1 cells were transfected with
control vector (lane 1), env-sea (lane 2), or
env-sea with either wild type SHP-2 (lane 3) or C459S SHP-2
(lane 4). Lysates were resolved on a 10% SDS-PAGE gel and
immunoblotted with anti-MAP kinase antibody (top panel).
MAPK* indicates the mobility of the activated form of MAP
kinase. The same lysates were Western blotted with a mixture of M45 and
SHP2 antibodies to determine the expression levels (bottom
panel). pr, precursor protein.
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Next, we tried to map the binding site for SHP-2 on the env-sea
protein. Lysates containing equivalent amounts of env-sea protein (Fig.
8B) were immunoprecipitated with anti-SHP-2 antibody, and
the precipitates were immunoblotted with anti-M45 antibody to detect
associated various forms of the env-sea proteins (Fig. 8C).
There were a strong association for wild type env-sea protein, a weaker
association for Y557F, and an even weaker association for Y564F. The
reverse immunoprecipitation in which we immunoprecipitated Sea and
Western blotted with anti-SHP-2 gave similar results (Fig. 8D). No stable association could be detected for either the
Y557/Y5572 or Y557F/Y564F mutants. To extend this analysis,
we utilized an in vitro binding assay using a GST fusion
protein containing both of the SH2 domains of SHP-2. The in
vitro assay showed that only wild type and Y557F were able to
associate with SHP-2 (Fig. 8E), indicating that tyrosine 564 is the primary binding site for SHP-2. This GST pull-down assay also
suggests that the SH2 domains of SHP-2 are responsible for the
association with the env-sea protein. Next, we asked whether the
phosphatase domain has any role in env-sea signaling. Activation of
SHP-2 has been suggested to play a role in the subsequent activation of
MAP kinase. We cotransfected COS1 cells with either wild type SHP-2 or
mutant SHP-2 cDNA that has serine substituted for cysteine in the
phosphatase domain (C459S SHP-2) along with wild type env-sea
constructs. The C459S SHP-2 mutation has been demonstrated to be a
catalytically inactive phosphatase (35). Cell lysates were analyzed
using the activation-dependent mobility shift assay for MAP
kinase activation (Fig. 8F). The expression of the wild type
env-sea protein led to the activation of MAP kinase (lane
2). Overexpression of wild type SHP-2 together with env-sea had no
further effect on MAP kinase activation by the env-sea protein
(lane 3). However, when the env-sea protein was coexpressed
with the C459S SHP-2 mutant, MAP kinase activation was almost
completely blocked. The mechanism by which C459S SHP-2 mutant can
inhibit MAP kinase activation remains to be determined.
Microinjected SH2 Domains of Grb2 or SHP-2 Block the Transformation
of S13 Rat1 Cells by the env-sea Protein--
Grb2 and SHP-2 associate
with the env-sea protein through phosphorylated tyrosine residues using
their SH2 domains. To assess whether those associations are necessary
for the transformation by the env-sea protein, we microinjected GST
fusion proteins containing the SH2 domains of Grb2 or SHP-2 into
transformed cells and investigated whether microinjected SH2 domains
can interfere with endogenous Grb2 or SHP-2 to revert the
transformation. As a control, we injected either GST alone or a GST
fusion protein containing the SH2 domain of Shc, which does not bind to
the env-sea protein. For this experiment, we utilized S13 Rat1 cells
transformed by a temperature-sensitive env-sea protein so that we could
easily monitor the transformed phenotype of injected cells. As shown in
Fig. 9A, S13 Rat1 cells at the
nonpermissive temperature of 39 °C look flat and round (left
panel). When the cells were shifted to the permissive temperature of 35 °C for about 12 h, they restored their transformed
morphology, became refractile, and elongated with membrane ruffles
(right panel). These morphological differences were used as
an indicator of the transformed phenotype. The experimental strategy
was to inject cells at the nonpermissive temperature and then shift
them to the permissive temperature. If the injected protein interferes with env-sea signaling then it should be able to block the appearance of the transformed phenotype. S13 Rat1 cells, which were microinjected with GST or GST-Shc (SH2), looked flat and round at the nonpermissive temperature and were restored to a typical transformed morphology 12 h after growth at the permissive temperature. These results demonstrate that microinjection itself does not have any effect on the
morphological changes due to the temperature. In contrast, when cells
were injected with SH2 domains of either Grb2 or SHP-2, the cells were
unable to restore their transformed morphology following temperature
shift to the permissive temperature (Fig. 9B). These results
indicated that the tyrosine residues in the bidentate motif are
essential for the transforming potential of the env-sea protein and
recruit signaling proteins, such as Grb2 and SHP-2.

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Fig. 9.
Microinjected SH2 domains of Grb2 or SHP-2
block the transformation of S13 rat1 cells by the env-sea protein.
A, cells at the nonpermissive temperature (left
panel) and at the permissive temperature (right panel)
were visualized by staining actin cytoskeleton with
rhodamine-conjugated phalloidin. B, cells grown at the
nonpermissive temperature were microinjected with indicated proteins
and then incubated at the indicated temperature for additional 12 h. Injected cells were identified by staining with anti-GST
antibody.
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DISCUSSION |
Very little is known about pathways that mediate signaling by the
env-sea protein. In this paper, we have examined the role of the
tyrosine residues in the bidentate motif in signaling by the env-sea
protein. Substitution of both tyrosine residues to phenylalanine
completely abolished the ability to transform cells. Surprisingly, the
single mutation of tyrosine 564 to phenylalanine increased the
transforming ability of the env-sea protein. This implies that
phosphorylated tyrosine 564 may recruit one or more proteins, which
have negative regulatory roles; unfortunately, the identity of such
proteins is presently unknown. The Y557F mutant had 70% transforming
efficiency compared with wild type env-sea protein. This implies some
redundancy in the ability of the env-sea protein to activate pathways
that lead to the transformed phenotype. The overall transforming
activity of the wild type env-sea protein may result from cooperation
between signaling pathways mediated by both tyrosine residues.
It had been demonstrated previously that the Grb2 protein bound to the
activated env-sea protein, but the site of this interaction was unknown
(17). Here, we show that Grb2 associates with the env-sea protein
primarily through tyrosine 564. The association of the Grb2-Sos complex
with tyrosine-phosphorylated receptors has been directly implicated in
the activation of the Ras/MAP kinase signaling pathway. Interestingly,
the Y564F mutant, which could not associate efficiently with Grb2, had
better transforming activity than the Y557F mutant, which could
directly associate with Grb2. This apparent discrepancy could be
explained by the phosphorylation of the Shc proteins. Previous work
demonstrated that phosphorylation of the Shc proteins correlates with
the transforming activity of the env-sea protein, although the Shc
proteins do not form a stable complex with the env-sea protein (17).
Tyrosine phosphorylation of the Shc proteins was increased in cells
expressing Y564F mutant to the same level as in cells expressing wild
type and Y557F mutant, but not in cells expressing the nontransforming Y557F/Y564F mutant. The lack of an increase in Shc phosphorylation in
cells expressing the kinase active Y557F/Y564F mutant indicates that
although a stable complex between Shc and env-sea cannot be detected,
there may be some transient association because Shc phosphorylation is
not seen when both tyrosine are mutated. MAP kinase activation in cells
expressing the Y564F mutant was also equivalent to that in cells
expressing the wild type env-sea protein. This indicates that stable
complex formation between Grb2 and the env-sea protein is not necessary
for the activation of the Ras/MAP kinase pathway. In the case of
Tpr-Met, which is the oncogenic counterpart of hepatocyte growth factor
receptor (c-Met), the association between Shc and Grb2 was not
sufficient to give the full transforming activity (22, 36). However,
overexpression of Shc has been shown to result in the malignant
transformation of NIH3T3 cells, suggesting that the formation of a
trimeric complex of phosphorylated Shc, Grb2, and Sos can be a key
event in the activation of Ras (37, 38).
We were also able to demonstrate for the first time the association of
PI3 kinase with the env-sea protein and identify the site as Tyr-557.
However, there was no correlation of the association of PI3 kinase with
transformation by the mutants. This is reminiscent of the binding and
activation of PI3 kinase by hepatocyte growth factor receptor/Met. In
this case, PI3 kinase is not associated with transformation by Met but
rather with the ability of this receptor to induce cell motility. It
will be interesting to use these mutants of env-sea in future studies
to determine whether the env-sea protein can induce cell motility and
whether PI3 kinase activation plays a major role in this effect.
SHP-2 has been described as a positive regulator of multiple RTK
signaling pathways (32-34). Therefore, we investigated whether SHP-2
is involved in env-sea transformation. SHP-2 was shown to physically
interact with the env-sea protein and become phosphorylated. SHP-2 is
also tyrosine-phosphorylated following PDGF, epidermal growth factor,
and fibroblast growth factor stimulation of cells (39-41) and is
constitutively tyrosine-phosphorylated in v-Src- and p210
Bcr-Abl-transformed cells (39, 42). However, phosphorylation of SHP-2
was not detected in insulin signaling (43). SHP-2 is phosphorylated on
one or both of the tyrosine residues in its carboxyl terminus
(tyrosines 542 and 580), and Grb2 has been shown to bind to tyrosine
542 following PDGF stimulation (40). This observation led to a model in
which SHP-2 might act as an adaptor to couple Grb2 binding to the PDGF
receptor (40). However, direct association between Grb2 and the PDGF
receptor was demonstrated later (44), suggesting that the adaptor model
is not the only mechanism involving Grb2 in PDGF signaling. Grb7 has
also been demonstrated to associate with SHP-2 through tyrosine 580 by
the yeast two-hybrid method (45). Thus, the physiological importance of
tyrosine phosphorylation of SHP-2 is unclear. However, it is interesting to note that SHP-2 exists in more than four different forms
of tyrosine-phosphorylated protein in COS1 cells when co-expressed with
the env-sea protein (data not shown). Therefore, it is possible that
tyrosine-phosphorylated SHP-2 molecules may recruit multiple signaling
proteins due to their phosphorylation status.
We mapped the binding sites for SHP-2 on the env-sea protein using
in vivo co-immunoprecipitation and in vitro GST
pull-down experiments. Both individual tyrosine residues are able to
bind SHP-2, but the presence of both tyrosine residues was necessary to
bring about the full affinity of SHP-2 binding. This fits well to
recent biochemical studies concerning SHP-2, in which, using biphosphorylated tyrosine-based activation motif peptides, it was
demonstrated that the tandem SH2 domains of SHP-2 confer 20-50-fold higher specificity than the individual SH2 domain (46).
It has been shown that the phosphatase activity of SHP-2 is essential
for insulin signaling (47, 48) and epidermal growth factor signaling
(49, 50) in tissue culture cells and for fibroblast growth factor
signaling in Xenopus early development (51, 52). Therefore,
we investigated the role of the phosphatase activity of SHP-2 in the
env-sea signaling. Overexpression of the catalytically inactive SHP-2
significantly inhibited the MAP kinase activation induced by the
env-sea protein, whereas overexpression of wild type SHP-2 had no
effect on the MAP kinase activation. This indicates that the catalytic
activity of SHP-2 is necessary for MAP kinase activation by env-sea.
However, it is unclear how the phosphatase activity of SHP-2 actually
regulates MAP kinase activation.
Here, we demonstrate that both Grb2 and SHP-2 associate with the
activated env-sea protein through tyrosine residues in the bidentate
motif. To assess the importance of these interactions, we microinjected
S13 Rat1 cells with SH2 domains from either Grb2 or SHP-2 to interfere
with association by endogenous proteins. Microinjected SH2 domains from
both blocked transformation. In contrast, microinjection of the SH2
domain from Shc had no effect on transformation. This indicates that
Grb2 and SHP-2 may be critical positive regulators in transformation by
the env-sea protein. However, it is also possible that the binding of
the SH2 domains could also interfere with the association of other
signaling molecules. The microinjection data demonstrate that
interactions between the bidentate motif and signaling molecules are
key for transformation by the env-sea protein and confirm the data
acquired from the mutagenesis experiments.
The env-sea protein is a member of the hepatocyte growth factor family
of growth factor receptors. Like other members of this family, the
env-sea protein seems to engage critical signaling pathways through its
bidentate motif. However, the mutational analysis reported here
demonstrates that the consequences of mutation of the individual
tyrosines are different between the family members. This presumably
reflects the sequence differences surrounding the tyrosines that are
seen between the different family members, and this may also explain
the different biological activities seen between these receptors (53,
54). These differences would be predicted to lead to the activation of
a different constellation of signaling molecules. Further studies will
be necessary to identify additional molecules that are activated by the
env-sea protein.