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INTRODUCTION |
Migration of cells is an integral part of many different
physiological and pathological processes. Embryonic development, angiogenesis, wound healing, and tumor spreading require cell motility.
Signal transduction through growth factor receptors with intrinsic
protein-tyrosine kinase activity leads to induction of directed
cellular migration, chemotaxis. Activation of the tyrosine kinase, upon
growth factor binding and dimerization of receptors, results in
receptor autophosphorylation (1). Phosphorylated tyrosine residues
present binding sites for signal transduction molecules containing one
or two copies of so called Src homology 2 domains, a conserved stretch
of about 100 amino acid residues, which mediates the interaction with
the phosphotyrosine and the following 3-6 C-terminal amino acid
residues (2). Binding of signal transduction molecules to the receptor
leads to initiation of signaling cascades, through activation of
enzymatic activities intrinsic to or associated with the signal
transduction molecules. The signaling cascade is eventually established
as a cellular response, such as migration, proliferation, and differentiation.
Platelet-derived growth factor
(PDGF)1 acts on a wide
spectrum of cells, including mesenchymal cells (3). There are two receptors for PDGF, denoted the PDGF
- and
-receptors (4, 5). The
receptors are similar in structure, with an extracellular part
organized in five immunoglobulin-like domains, a single transmembrane stretch and an intracellular part containing the kinase domain. The
kinase domain is interrupted by the insertion of a noncatalytic kinase
insert. More than 10 autophosphorylation sites have so far been
identified in the PDGF
-receptor (6). Several of these have been
shown to interact with Src homology 2 domain-containing proteins, which
have been implicated in transduction of signaling cascades leading to
cellular migration. Thus, two autophosphorylation sites
(Tyr740 and Tyr751) in the kinase insert of the
receptor bind the regulatory subunit (p85) of phosphatidylinositol (PI)
3-kinase. PI 3-kinase activity has been shown to be a prerequisite for
PDGF-induced cellular migration (7, 8). The two autophosphorylation
sites in the carboxyl-terminal tail (Tyr1009 and
Tyr1021) mediate binding of phospholipase C-
(PLC-
),
which is implicated in regulation of chemotaxis (8, 9). In addition,
phosphorylated Tyr1009 binds the phosphotyrosine
phosphatase SHP-2 (also denoted PTP1D, Syp, and SH-PTP2) (10, 11).
SHP-2 activity has been implicated in cytoskeletal organization (12),
which may indicate a function for SHP-2 in cellular migration.
Numerous studies have shown that SHP-2 becomes tyrosine-phosphorylated
in growth factor-stimulated cells and stably associated with activated
growth factor receptors (13, 14). Tyrosine phosphorylation of SHP-2 has
been reported to induce its catalytic activity (15), although mutation
of the phosphorylatable tyrosine residues in SHP-2 does not
preclude its activation (16). SHP-2 may act as a negative regulator of
PDGF receptor function, by dephosphorylating the receptor or its
cognate substrates (17). On the other hand, SHP-2 is also implicated in
positive regulation of PDGF function, since tyrosine phosphorylation of
SHP-2 presents a binding site for the adaptor protein Grb2, which is
known to mediate Ras activation through complex formation with the
nucleotide exchange factor Sos (16, 18). However, the role of Grb2/Sos binding to SHP-2 is not clear, and mutation of the Grb-2-binding site
on SHP-2 does not interfere with PDGF-induced Erk-2 activation (16).
The recent report on targeted inactivation of the SHP-2 gene showed
that loss of SHP-2 was embryonally lethal and led to failure of
development of the vascular system (19). The reason for vascular
failure in these animals is not clear, but it is noteworthy that
migration is critical for the development of the vascular system
(vasculogenesis) as well as for formation of new vessels from
pre-existing ones (angiogenesis) (20, 21).
In this work, we have used PDGF-stimulated porcine aortic endothelial
(PAE) cells expressing the PDGF
-receptor or receptor mutants Y1009F
and Y1021F, to examine the potential role for SHP-2 in migration. PAE
cells expressing a Y1009F mutant PDGF
-receptor, which lacks the
SHP-2 binding site, failed to migrate toward PDGF. Moreover, treatment
with the tyrosine phosphatase inhibitors PAO and vanadate led to
attenuation of migration of PDGF-BB-stimulated PAE cells expressing the
wild type PDGFR-
. Our data indicate a role for SHP-2 in migration
via a pathway that acts parallel to the PI 3-kinase pathway and
involves regulation of focal adhesion kinase activity.
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MATERIALS AND METHODS |
Cell Culture--
PAE cells expressing the wild type PDGFR-
or mutant PDGFR-
Y1009F and Y1021F have been described previously
(22). Cells were cultured in Ham's F-12 medium (Life Technologies,
Inc.) supplemented with 10% fetal calf serum (Sigma) and antibiotics
at 37 °C and 5% CO2. For starvation, cells were kept in
serum-free medium supplemented with 0.1% serum bovine albumin.
Antibodies and Other Reagents--
The rabbit antiserum PDGFR-3,
specifically recognizing the PDGFR-
, has been described earlier
(23). Anti-SHP-2 monoclonal antibody and anti-Grb2 rabbit antiserum
were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The
monoclonal anti-phosphotyrosine antibody PY20 and the monoclonal
anti-FAK (2A7) antibody were from Transduction Laboratories. The rabbit
antiserum against PLC-
, raised against a synthetic peptide
corresponding to a part of human PLC-
was kindly provided by Dr.
Lars Rönnstrand (Ludwig Institute for Cancer Research, Uppsala,
Sweden). Peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse
immunoglobulins were from Amersham Pharmacia Biotech.
Phosphatidylinositol was from Lipid Products (Redhill, Surrey, United
Kingdom). Wortmannin, LY294002, phenylarsine oxide, and sodium
orthovanadate were purchased from Sigma. PDGF-BB was purchased from
PeproTech Inc.
Immunoprecipitation and Immunoblotting--
Serum-starved cells
were treated with 50 ng/ml PDGF-BB at 37 °C for 8 min, rinsed with
ice-cold phosphate-buffered saline (PBS) containing 0.1 mM
Na3VO4 and 1 mM dithiothreitol and
lysed in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 10% glycerol, 1%
TX-100, 0.5 mM Na3VO4, 1%
aprotinin (Trasylol, Bayer), 10 µg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride (Sigma). Cell lysates were
incubated with rabbit antiserum or monoclonal antibodies for 1 h
at 4 °C, followed by incubation with protein A-Sepharose CL-4B
(Pharmacia & Upjohn, Stockholm, Sweden) for 30 min at 4 °C. The
samples were eluted by boiling in SDS-sample buffer (4% SDS, 0.2 M Tris-HCl, pH 8.8, 0.5 M sucrose, 5 mM EDTA, 0.01% bromphenol blue, 2%
-mercaptoethanol)
and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Samples were then electrophoretically transferred onto nitrocellulose membrane (Hybond-C Extra; Amersham Pharmacia Biotech); the filter was
blocked in 3% bovine serum albumin, 0.2% Tween 20 in PBS at 4 °C
overnight and probed with specific antibodies for 1 h at room
temperature. After washing, the filter was incubated with horseradish
peroxidase-linked anti-rabbit or anti-mouse IgG (sheep, Amersham
Pharmacia Biotech; 1:1000); immune complexes were visualized through
enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). The blots
were reprobed after removal of the first probe by incubation in 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM
-mercaptoethanol for 30 min at 55 °C.
Immune Complex Kinase Assay--
After PDGF-BB stimulation, the
cells were lysed, and immunoprecipitation was performed with PDGFR-3
antiserum or anti-SHP-2 antibody. The immunoprecipitates were subjected
to kinase assays by incubation in 10 mM Tris-HCl, pH 7.5, 10 mM MnCl2, and 2 mM MgCl2, in the presence of 0.5 µCi of
[
32-P]ATP for 10 min at room temperature. Samples were
analyzed by SDS-PAGE. After electrophoresis the gel was treated in 2%
glutaraldehyde to fix the proteins to the gel and then with 0.5 M KOH at 55 °C for 45 min, to hydrolyze phosphorylation
on serine. The gel was dried and analyzed by autoradiography.
Chemotaxis Assay--
The assay was performed in a mini-Boyden
chamber as described (24) using micropore nitrocellulose filters (8 µm thick, 8-µm pore) coated with type-1 collagen solution at 100 µg/ml (Vitrogen 100; Collagen Corp.). Cells were trypsinized and
resuspended at a concentration of 5 × 105 cells/ml in
serum-free medium containing 0.25% bovine serum albumin. The cell
suspension was placed in the upper chamber and 0.25% bovine serum
albumin in serum-free medium without or with various concentrations of
PDGF-BB placed below the filter in the lower chamber. For each set of
experiments, random migration in the presence of serum-free medium
containing 0.25% bovine serum albumin served as control and is
referred to as 100% migration. All experiments were performed in
triplicates for every concentration of PDGF-BB. The assays were read in blind.
Determination of PTP Activity of SHP-2--
Anti-PDGFR-
immunoprecipitates immobilized on protein A-Sepharose beads were washed
twice with lysis buffer (see "Immunoprecipitation") without
vanadate and twice in the assay buffer containing 25 mM imidazole (pH 7.5), 0.1 mg/ml bovine serum albumin, 10 mM
dithiothreitol, 0.05% Triton X-100. The reaction mixture contained the
immobilized immune complexes (60 µl), assay buffer without Triton
X-100 (55 µl), and 32P-labeled Src optimal peptide (5 µl), the sequence of which is AEEEIYGEFEAKKKK (kindly provided by
Arne Östman (Ludwig Institute for Cancer Research)). After
incubation for 10 min at 30 °C, the reaction was terminated by the
addition of 290 µl of an acidic charcoal mixture containing 0.9 M HCl, 90 mM sodium pyrophosphate, 2 mM NaH2PO4, and 4% (w/v) Norit A. After centrifugation at 12.000 × g for 10 min,
released Pi present in the supernatant was determined by
scintillation counting. The assay did not work well when using anti-SHP-2 immunoprecipitated material or when many samples were processed simultaneously.
Inositol Phosphate Assay--
Cells were grown in 24-well plates
to subconfluency. They were washed in inositol-free Ham's F-12 medium
and then incubated with 0.5 ml of inositol-free Ham's F-12 medium
containing 0.25% fetal calf serum and 2 µCi/ml of
[2-3H]myoinositol (Amersham Pharmacia Biotech) for
24 h. Following labeling, cells were washed in Hanks'/Hepes
buffer at 37 °C for 20 min. Cells were then preincubated in 0.25 ml
of Hanks'/Hepes buffer containing 20 mM LiCl for 15 min at
37 °C before stimulation with PDGF-BB at the indicated
concentrations for 30 min. Stimulations were terminated by the addition
of 0.5 ml of acidified ice-cold methanol (methanol/HCl, 99:1 (v/v)),
and samples were extracted on ice. Formation of total inositol
phosphates was determined by ion exchange chromatography on Dowex
AG1-X8 (formate form, 100-200 mesh, Bio-Rad) as described previously
(25).
Actin Reorganization--
Actin reorganization and membrane
ruffling were analyzed essentially as described by Mellström
et al. (26). Cells were cultured on glass coverslips in
six-well plates. Quiescent subconfluent cells were stimulated for 15 min at 37 °C with 50 ng/ml PDGF-BB. The cells were fixed with 3.7%
paraformaldehyde (Merck) in PBS for 30 min at 4 °C and permeabilized
with 0.2% Triton X-100 for 20 min at room temperature. The coverslips
were rinsed three times with PBS and incubated with fluorescein
isothiocyanate-labeled phalloidin in PBS (0.66 µg/ml; Sigma) for 20 min at room temperature and then washed three times with PBS. For
examination by fluorescent microscopy, coverslips were covered with
antifade reagent (Molecular Probes, Inc., Eugene, OR).
PI 3-Kinase Assay--
PI 3-kinase activity was measured
in vitro, using anti-receptor antiserum for
immunoprecipitation from cell lines pretreated or not with phosphatase
inhibitors and stimulated with or without PDGF-BB. The immobilized
immunoprecipitates were used for lipid kinase reactions using
phosphatidylinositol as an exogenous substrate, and the reactions were
analyzed as described by Wennström et al. (7).
Assay for FAK Activity--
Serum-starved cells pretreated or
not with phosphatase inhibitors were stimulated with the indicated
concentrations of PDGF-BB in the absence or presence of the drugs or
vehicle (Me2SO) for the indicated time periods at 37 °C,
rinsed with ice-cold PBS, and lysed in 1 ml of lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet
P-40, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na3VO4, and 0.2 mM phenylmethylsulfonyl fluoride). The lysates were
incubated for 1 h at 4 °C with the anti-FAK monoclonal antibody
2A7, followed by incubation with rabbit anti-mouse Ig antiserum for an
additional 45 min at 4 °C. Immune complexes were collected using
protein A-Sepharose. The beads were washed twice with lysis buffer and
twice with FAK reaction buffer (10 mM Tris-HCl, 10 mM MnCl2, 2 mM MgCl2,
and 0.05% Triton X-100). In vitro phosphorylation was
performed for 15 min at room temperature in 25 µl of the reaction buffer containing 5 µCi of [
-32P]ATP, and the
reactions were stopped by the addition of 25 µl of 2× SDS sample
buffer. The samples were heated at 95 °C for 5 min and separated by
SDS-PAGE in 7% linear polyacrylamide gels. After fixation in
methanol/acetic acid, the gel was treated with 1 M KOH for
1 h at 55 °C, fixed again, dried, and exposed on RX films (Fuji).
 |
RESULTS |
Characterization of PAE Cell Lines Expressing Wild Type and Mutant
PDGFR-
--
It has previously been shown that phosphorylated
Tyr1009 and surrounding amino acid residues in the
PDGFR-
present a binding site for SHP-2 (10, 11). In order to
characterize the role of SHP-2 in PDGF-induced cellular responses, we
examined the properties of PAE cells expressing the wild type
PDGFR-
, the mutant Y1009F (lacking the binding site for SHP-2), and,
for comparison, the mutant Y1021F (lacking the binding site for
PLC-
). These cell lines have previously been reported to express
similar numbers of receptors and to transduce mitogenic signals with
similar efficiencies (22). Fig.
1A shows that PDGF-BB
stimulation of the different cell lines led to similar -fold induction
of receptor kinase activity. An immune complex kinase assay on SHP-2
immunoprecipitations showed that SHP-2 was tyrosine-phosphorylated and
in complex with the PDGFR-
after PDGF-BB stimulation of PAE cells
expressing the wild type receptor or the Y1021F mutant but not in cells
expressing the Y1009F mutant PDGFR-
(Fig. 1B).
Immunoprecipitation of SHP-2, followed by immunoblotting with
anti-phosphotyrosine antibodies (Fig. 1C), showed that the
extent of SHP-2 tyrosine phosphorylation in the Y1009F cells was
considerably reduced, as compared with the wild type PDGFR-
and
Y1021F cells. The fact that some level of tyrosine phosphorylation of
SHP-2 remained in the Y1009F cells could indicate that there are
additional binding sites for SHP-2 on the PDGFR-
. Although the
sensitive immune complex kinase assay (Fig. 1B) showed
complex formation between the wild type PDGFR-
and SHP-2, we were
not able to demonstrate such complexes in PDGF-treated, intact cells
(Fig. 1D).

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Fig. 1.
PDGF-BB-stimulated receptor tyrosine kinase
activity, tyrosine phosphorylation of SHP-2, and inositol phosphate
accumulation in cells expressing the wild type or mutant
PDGFR- . A and B,
PAE cells expressing the wild type, Y1009F, or Y1021F PDGF -receptor
( -R) were incubated in the absence ( ) or presence (+)
of 50 ng/ml PDGF-BB for 8 min at 37 °C. Cells were lysed and
immunoprecipitated with anti-receptor antiserum PDGFR-3 (A)
or anti-SHP-2 antibody (B), and samples were subjected to
kinase assays as described under "Materials and Methods," followed
by SDS-PAGE and autoradiography. C and D, the
different PAE cells lines expressing wild type or mutant receptors were
stimulated as above and processed for immunoprecipitation with the
anti-SHP-2 antibody or anti-PDGFR- antiserum as indicated. The
samples were separated by SDS-PAGE and transferred to nitrocellulose
and blotted with anti-SHP-2 antibody or anti-phosphotyrosine antibody
as indicated. Immunoreactive proteins were detected by enhanced
chemiluminescence. Ip, immunoprecipitation; Ib,
immunoblotting. E, accumulation of inositol phosphates in
the different cell lines was estimated by labeling cells with
[3H]myoinositol, followed by ion exchange chromatography
on Dowex AG-1-X8 resin. The data show means ± S.E. of at least
three experiments. *, significantly different from unstimulated at
p < 0.05 (Student's t test).
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It has been shown previously that PLC-
associates with
Tyr(P)1021 and, to a lesser extent, with
Tyr(P)1009 (22). PDGF-BB stimulation of the different cell
lines led to a 1.5-fold increase in formation of inositol phosphate
(Fig. 1E) in cells expressing the wild type PDGFR-
. In
the Y1009F cells, inositol phosphate formation was significantly
increased in response to PDGF stimulation, although not to the levels
seen in the wild type receptor cells. In contrast, no inositol
phosphate formation in response to PDGF stimulation could be detected
in cells expressing the Y1021F mutant receptor. These data show that
the Y1009F mutant receptor fails to associate with SHP-2 and mediate
its tyrosine phosphorylation but that it is still able to mediate
activation of PLC-
.
Phosphotyrosine Phosphatase Activity in PDGF-BB-stimulated PAE
Cells Expressing the Wild Type and Mutant PDGFR-
and Effects of
Phosphatase Inhibitors--
We examined the effect of SHP-2 tyrosine
phosphorylation and its catalytic activity. Phosphatase (PTP) activity
was measured using anti-receptor immunoprecipitates from the different
PAE cell lines treated or not with PDGF-BB. The receptor precipitates were incubated in the presence of a 32P-labeled synthetic
phosphopeptide, which served as a substrate for the PTP activity. The
PTP activity in unstimulated cells was used as a control and was set to
100%. As shown in Fig. 2A,
PDGF-BB-dependent increases in receptor-associated PTP
activities in PAE cells expressing the wild type and Y1021F PDGFR-
were 322 ± 48% and 471 ± 71%, respectively. In contrast,
the PDGF-BB-dependent increase in receptor-associated PTP
activity in PAE cells expressing the Y1009F mutant was 120 ± 15%. These data indicate that SHP-2 is the dominating PDGF
-receptor-associated phosphotyrosine phosphatase.

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Fig. 2.
PDGF-BB-dependent increase in PTP
activity in different cell types and effect of PTP inhibitors on PTP
activity and the receptor kinase activity. Serum-starved PAE cells
expressing the wild-type, Y1009F, or Y1021F PGDFR- and wild type
PGDFR- cells pretreated with PAO (0.6 µM) and vanadate
(Van; 60 µM) for 4 h were incubated with
or without 50 ng/ml PDGF-BB for 8 min at 37 °C. Cells were lysed and
immunoprecipitated with anti-receptor antibody. A,
immunoprecipitates were assayed for phosphotyrosine phosphatase
activity using synthetic 32P-labeled phosphopeptide as a
substrate. PTP activity in unstimulated cells served as control and was
set to 100% as described under "Materials and Methods." Each value
is the mean of duplicate determinations, and the results are
representative of three separate experiments. B,
anti-PDGFR- immunoprecipitates prepared from the cells treated with
PAO and vanadate and stimulated or not with PDGF-BB (50 ng/ml for 8 min
at 37 °C) were subjected to kinase assay as described under
"Materials and Methods," followed by SDS-PAGE and autoradiography.
DMSO, Me2SO.
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We next analyzed the effects of the phosphatase inhibitors PAO and
orthovanadate on PDGFR-
-coupled PTP activity (Fig. 2A). PAO has previously been shown to inhibit SHP-2 catalytic activity (27).
The PDGF-BB-induced increase in PTP activity in PDGFR-
immunoprecipitates was abolished (76 ± 7%) in PAO-treated PAE cells expressing the wild type receptor. In vanadate-treated PAE cells
expressing the wild type receptor, PTP activity was 94 ± 9%. PAO
and orthovanadate treatment for 4 h did not affect PDGF-BB-induced PDGFR-
kinase activity (Fig. 2B). PAO and orthovanadate
did also not affect tyrosine phosphorylation of SHP-2 or its expression levels (Fig. 3, A and
B). Furthermore, it is well established that
tyrosine-phosphorylated SHP-2 presents a binding site for Grb2, which
still occurred in cells treated with the phosphatase inhibitors (Fig.
3C). These data show that receptor association and tyrosine
phosphorylation, and thereby Grb2-binding, of SHP-2 remains after
treatment of cells with PAO or orthovanadate. In contrast, PDGF
-receptor-associated phosphatase activity is reduced to that in
unstimulated cells, indicating that the catalytic activity of SHP-2 is
inhibited by PAO and vanadate.

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Fig. 3.
Effects of PAO and vanadate on
PDGF-BB-stimulated tyrosine phosphorylation of SHP-2 and its
association with Grb2. Serum-starved PAE cells expressing
PDGFR- were incubated in the absence or presence of 0.1% dimethyl
sulfoxide (DMSO), 0.6 µM PAO, or 60 µM orthovanadate for 4 h at 37 °C and then
stimulated with 50 ng/ml PDGF for 8 min. Cell lysates were
immunoprecipitated with the anti-SHP-2 antibody. The samples were
separated by SDS-PAGE and immunoblotted with antibody to
phosphotyrosine (PY-20), the anti-SHP-2 antibody, or the anti-Grb2
antibody, respectively. Immunoreactive proteins were detected by
enhanced chemiluminescence. Ip, immunoprecipitation;
Ib, immunoblotting.
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Migration of PDGFR-
Wild Type and Mutant PAE Cells toward
PDGF-BB and Effects of Phosphatase Inhibitors--
To examine the
potential role of SHP-2 in PDGF-BB-stimulated chemotaxis, PAE cells
expressing wild type, Y1009F, or Y1021F PDGFR-
were examined in a
mini-Boyden chamber. Cells were loaded on one side of an 8-µm
micropore nitrocellulose filter, and PDGF-BB at different
concentrations was loaded on the other side. The ability of cells to
move through the filter, toward the chemoattractant, was measured by
Giemsa staining of the filter and counting of the cells. As
shown in Fig. 4A, PDGF-BB
stimulated a dose-dependent increase in cell migration,
with a maximal migration occurring at 100 ng/ml PDGF-BB. PAE cells
expressing the Y1021F mutant receptor also migrated efficiently but
with a different dose optimum, 50 ng/ml PDGF-BB, as compared with the
wild type receptor cells. In contrast, PAE cells expressing the Y1009F
mutant PDGFR-
migrated only very inefficiently at high doses of
PDGF-BB (Fig. 4A). These results indicate that receptor
binding of SHP-2 is a requirement for PDGF-BB-stimulated migration. We
further examined the effects of PAO and orthovanadate on
PDGF-BB-stimulated migration of PAE cells expressing the wild type
PDGFR-
. As seen in Fig. 4B, inclusion of PAO during the
migration assay led to a dose-dependent decrease of
PDGF-BB-stimulated (10 ng/ml) migration. Orthovanadate treatment also
led to a decrease in PDGF-BB-stimulated migration (Fig. 4C). 1 µM PAO and 300 µM orthovanadate
efficiently suppressed PDGF-BB-stimulated and basal cellular migration
(Fig. 4, B and C). The results are in accordance
with a role for SHP-2 in PDGF-BB-stimulated migration.

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Fig. 4.
Chemotaxis of different cell types and
effects of PTP inhibitors and PI 3-kinase inhibitors on PDGF-BB-induced
chemotaxis. A, PAE cells expressing the wild-type
(open circles), Y1021F (closed
circles), and Y1009F (open squares)
PDGFR- were analyzed for their migration toward different
concentrations of PDGF-BB. B-E, the effects of phosphatase
inhibitors and PI 3-kinase inhibitors on migration of PAE cells
expressing PDGF -receptor were measured by preincubation with
increasing concentrations of PAO (B), orthovanadate
(C), wortmannin (D), and LY294002 (E)
for 30 min. Migration toward 10 ng/ml PDGF-BB was measured in a
mini-Boyden chamber for 4 h in the presence of the drugs. Random
migration (PDGF-BB on both sides of the filter) is referred to as 100%
migration. Migrating cells were estimated as described under
"Materials and Methods." The data show means ± S.E. of at
least three experiments. *, significantly different from control at
p < 0.05 (Student's t test).
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Cellular migration involves reorganization of the actin cytoskeleton,
and veil-like actin-dense structures at the cell margin, denoted
membrane edge ruffles, have previously been shown to be an integral
part of cellular motility (20). We examined PAE cells expressing the
wild type PDGFR-
or the Y1009F and Y1021F mutants, respectively, for
their ability to respond to PDGF-BB stimulation with formation of
membrane edge ruffles. Fig. 5 shows that
edge ruffles were formed both on cells expressing wild type PDGFR-
and on the Y1021F cells but that no ruffles were detected on the
PDGF-BB-stimulated Y1009F cells. About 90% of the PDGF-treated wild
type receptor and Y1021F receptor cells exhibited membrane edge
ruffles, whereas 4% of the Y1009F cells showed these structures. These
data agree with the notion that cellular migration is dependent on the
formation of these membrane extensions.

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Fig. 5.
PDGF-BB-stimulated actin reorganization in
cells expressing wild type and mutant
PDGFR- . Cells expressing wild type,
Y1009F, or Y1021F PDGFR- were stimulated or not stimulated for 8 min
at 37 °C with 50 ng/ml PDGF-BB. Cells were permeabilized and stained
with fluorescein isothiocyanate-phalloidin, as described under
"Materials and Methods." The arrows indicate membrane
edge ruffling in wild type and Y1021F PDGFR- cells. Magnification, × 1000.
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Synergistic Effects of SHP-2 and PI 3-Kinase Inhibitors--
It
has been shown previously that activation of PI 3-kinase is a
prerequisite for PDGF-induced migration of cells (7, 8). In agreement,
Fig. 4, D and E, show a
dose-dependent decrease in PDGFR-
-mediated migration of
cells toward PDGF-BB when treated with the PI 3-kinase inhibitors
wortmannin and LY294002. The efficiency of inhibition of migration of
cells treated with LY294002 was less than for wortmannin-treated cells,
possibly since LY294002 appears to be a more specific PI 3-kinase
inhibitor. We tested whether activation of PI 3-kinase was disturbed in
cells expressing the SHP-2 binding site mutant receptor and in cells
treated with the phosphatase inhibitors. PAE cells expressing the
Y1009F receptor, as well as wild type PDGFR-
cells treated or not
with PAO, were stimulated with PDGF-BB and tested for PI 3-kinase
activation in vitro. This was performed by
immunoprecipitation with anti-receptor antiserum and incubation of the
precipitate in the presence of PI and [
-32P]ATP. Fig.
6, A and B, shows
that PDGF treatment induced phosphorylation of PI to similar extents in
cells expressing the wild type and mutant Y1009F PDGFR-
and that the
presence of PAO had no effect. These data indicate that PI 3-kinase and
SHP-2 regulates migration through distinct signal transduction
pathways.

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Fig. 6.
PI 3-kinase activity in different cell types
and effect of phosphatase inhibitors on PI 3-kinase activity.
Serum-starved PAE cells expressing wild-type, Y1009F, and Y1021F
PDGFR- (A) and PAE cells expressing the wild-type
PDGFR- pretreated with PAO (0.6 µM) for 4 h
(B) were incubated with or without 50 ng/ml PDGF-BB for 8 min at 37 °C. PI 3-kinase activity in anti-receptor antiserum
immunoprecipitates was measured by phosphorylation of
phosphatidylinositol (PIP), which was included as an exogenous
substrate in in vitro kinase assays, as described under
"Materials and Methods." Reactions were separated by thin layer
chromatography and analyzed by autoradiography. PI 3-kinase activity
was also not affected by treatment of cells with orthovanadate (60 µM; data not shown). DMSO, Me2SO;
ORI, origin.
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To substantiate this assumption, PAE cells expressing the wild type
PDGFR-
were treated with suboptimal concentrations of inhibitors for
PI 3-kinase (wortmannin or LY294002) or SHP-2 (PAO or orthovanadate),
individually or in combination. As seen in Table
I, low doses of either of these drugs had
no inhibitory effect or a weak inhibitory effect on PDGF-induced
migration. In contrast, treatment with combinations of the drugs at
these low concentrations led to suppression of migration. The
combination of PAO and wortmannin, as well as the combination of
orthovanadate and LY294002, led to a specific reduction in stimulated
migration, indicating that PI 3-kinase and SHP-2 act through
independent and synergistic molecular mechanisms.
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Table I
Effects of combination of tyrosine phosphatase inhibitors and PI-3
kinase inhibitors on PDGF-induced migration of PAE cells
PAE cells expressing PDGF -receptor were preincubated with PAO,
orthovanadate, wortmannin (Wort), and LY294002 (LY) alone or
combination for 30 min. Migration of the cells towards 10 ng/ml PDGF-BB
was measured in a mini-Boyden chamber for 4 h in the presence of
the drugs. Random migration in the presence of serum-free medium
containing 0.25% bovine serum albumin is referred to as 100%
migration. Migrating cells were estimated as described under
"Materials and Methods." The data show means ± S.E. of at
least three experiments.
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Regulation of Focal Adhesion Kinase Activity in PDGF-BB-stimulated
PDGFR-
Wild Type and Mutant PAE Cells--
PDGF is known to induce
activation of p125 FAK (28). Targeted inactivation of the FAK gene
leads to loss of cellular migration (29). To examine whether SHP-2
participates in regulation of PDGF-induced FAK activation, lysates of
PDGF-stimulated PAE cells expressing the wild type or Y1009F or Y1021F
mutant PDGFR-
were immunoprecipitated with a monoclonal antibody
reactive with FAK. The immunoprecipitates were incubated in kinase
buffer and [
-32P]ATP. Fig.
7 shows that FAK kinase activity was
induced in the wild type receptor cells in a dose-dependent
manner, with a maximal response seen at 5 ng/ml PDGF (Fig.
7D). At higher concentrations, FAK activity declined, in
agreement with data in previous reports showing
dose-dependent fluctuations in PDGF-induced tyrosine
phosphorylation of p125 FAK (28). In cells expressing the Y1021F
mutant, a similar concentration-dependent induction of FAK
kinase activity was seen (Fig. 7B). The -fold induction of
FAK kinase activity was consistently slightly lower in the Y1021F
cells, possibly implicating PLC-
in FAK activation. PDGF treatment
largely failed to affect FAK kinase activity in the Y1009F mutant
receptor cells (Fig. 7C). The expression levels of FAK were
unaffected by the PDGF treatment in all cell lines (lower
parts of Fig. 7, A, B, and
C).

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Fig. 7.
Dose-dependent PDGF-BB-stimulated
FAK kinase activity. Serum-starved PAE cells expressing the
wild-type (A), Y1021F (B), and Y1021F
(C) PDGF -receptors were incubated for 8 min at 37 °C
with the indicated concentrations of PDGF-BB. Cells were lysed and
immunoprecipitated with an anti-FAK antibody (2A7). The samples were
subjected to an in vitro kinase assay as described under
"Materials and Methods" followed by SDS-PAGE and autoradiography.
Lower parts of A, B, and
C show quantification of p125 FAK protein levels by
immunoprecipitation and immunoblotting with anti-FAK antibody.
D, p125 FAK kinase activity was quantitated by use of a
BioImaging Analyzer and expressed as -fold induction.
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We tested the kinetics of FAK activation in the wild type and mutant
Y1009F cell lines treated with 10 ng/ml PDGF. As shown in Fig.
8A, FAK activity was maximally
induced by 15-min stimulation in the wild type receptor cells, whereas
in the Y1009F cells, there was no time-dependent induction
of FAK activity over the 30-min stimulation period.

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Fig. 8.
Time-dependent PDGF-BB-stimulated
FAK kinase activity in the presence and absence of phosphatase
inhibitors. Serum-starved PAE cells expressing wild type and
Y1009F PDGFR- (A, top) as well as PAE cells
expressing PDGF -receptor preincubated for 4 h at 37 °C with
0.6 µM PAO and 60 µM vanadate
(B, top) were stimulated with 5 ng/ml PDGF-BB for
the indicated time periods. Cells were lysed and immunoprecipitated
with anti-FAK antibody. The samples were subjected to an in
vitro kinase assay. Below each autoradiogram
is shown quantification, expressed as -fold induction, by use of a
BioImaging Analyzer. DMSO, Me2SO.
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We went on to examine the effect of PDGF on FAK activation in
phosphatase inhibitor-treated PAE cells expressing the wild type
PDGFR-
, which were treated with 10 ng/ml PDGF-BB for different time
periods. As seen in Fig. 8B, PDGF-stimulated FAK activation seen in the absence of drugs (see Fig. 8A) was attenuated by
the drug treatment, and instead, FAK activity decreased with time in a
pattern very similar to that seen in cells expressing the Y1009F mutant
receptor. These data indicate that SHP-2 catalytic activity is critical
for PDGF-stimulated changes in FAK kinase activity.
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DISCUSSION |
In this paper, we show that PDGF-induced migration of cells
expressing PDGFR-
is dependent on the association of SHP-2 with the
PDGF receptor. Cells expressing a mutant Y1009F PDGFR-
, in which the
binding site for SHP-2 is removed, failed to migrate toward PDGF. PDGF
receptor immunoprecipitates from these cells did not contain any
phosphatase activity, but we did detect increased accumulation of
inositol phosphate in the PDGF-stimulated Y1009F cells, although not to
the levels seen in wild type receptor cells. For comparison, we
examined cells expressing another PDGFR-
mutant, Y1021F, which does
not bind PLC-
. These cells migrated efficiently toward PDGF.
Immunoprecipitated Y1021F receptors contained phosphatase activity, but
accumulation of inositol phosphates was not detected. Our data show
that PDGF receptor-associated phosphatase activity is important for
migration toward PDGF but that inositol phosphate accumulation is not
an absolute requirement for motility.
How is SHP-2 involved in PDGF-induced cellular migration?
PDGF-dependent changes in FAK activity were attenuated in
cells expressing the Y1009F mutant PDGFR-
, indicating that SHP-2
could be critical for regulation of FAK kinase activity. Moreover,
cells treated with the phosphotyrosine phosphatase inhibitors
phenylarsine oxide and orthovanadate migrated only very inefficiently
toward PDGF-BB and failed to respond to PDGF-BB stimulation with
induction of FAK activity. p125 FAK was originally identified as an
abundantly tyrosyl-phosphorylated protein in v-Src-transformed cells
(30). FAK associates with integrins in focal contact sites and
clustering of integrins via binding to their extracellular matrix
ligands leads to activation of FAK. Targeted knock-out of the
fak gene in mice generates mutant embryos with a general
defect in mesoderm development, and cells from the embryos show reduced
motility in vitro (29). The number of focal contacts is
increased in the mutant embryos, indicating that FAK activity is
required for turnover of focal contacts. How FAK activity regulates the
turnover of focal contacts remains to be shown. We suggest that regular cycles of activation and inactivation of FAK kinase activity could be a
mechanism for the turnover of focal contacts. SHP-2 has been shown to
regulate p125 FAK activity in insulin-stimulated cells (31). Moreover,
a direct correlation between the level of FAK tyrosine phosphorylation
and assembly of focal adhesion in focal contacts has been reported
(32). In suspended cells, complex formation between SHP-2 and FAK has
been identified (33). The complex formation appears to diminish in
conjunction with attachment, and we have not been able to identify
PDGF-induced SHP-2·FAK complex formation by immunoblotting in our
cell model. In this work, we have measured the level of FAK activity by
in vitro kinase activity of immunoprecipitated FAK in the
different conditions. It was striking that the pattern of deregulated
FAK activity was similar between cells expressing the mutant Y1009F
PDGFR-
and cells expressing the wild type PDGFR-
treated with
phosphatase inhibitors. Under these conditions, we failed to detect
changes in the net amount of phosphotyrosine in FAK, as measured by
immunoblotting using anti-phosphotyrosine antibodies (data not shown),
which implies that the level of phosphotyrosine in FAK may not reflect
FAK kinase activity, as suggested previously (34).
Other PDGFR-
-coupled signal transduction molecules have been shown
to be critical for membrane edge ruffling and chemotaxis of cells
toward PDGF. Thus, PI 3-kinase has been implicated in regulation of
actin and in migration induced by different growth factors, most likely
via regulation of the monomeric GTP-binding protein Rac (35), which is
linked to rearrangement of the actin cytoskeleton (36). Moreover, PI
3-kinase has been shown to regulate FAK activity in PDGF-stimulated
cells (37), which would be of consequence for cellular migration. By
treating cells with low doses of PI 3-kinase inhibitor wortmannin or
LY294002 in combination with the phosphotyrosine phosphatases PAO and
orthovanadate, we could show that migration was regulated
synergistically by PI 3-kinase and SHP-2, indicating that these
signaling molecules regulate independent pathways. It is likely that
these pathways converge at some point, e.g. by affecting FAK
function, but our data indicate that they originate independently and
that SHP-2 does not regulate PI 3-kinase activity.
Furthermore, PLC-
has been shown to have a regulatory role in
PDGF-stimulated migration (8, 9). Using a thick filter assay, where the
migration distance is measured, we have previously examined the effect
of a double mutation at Tyr1009 and Tyr1021,
replacing the tyrosine residues with phenylalanine residues, thereby
creating the mutant receptor Y1009F/Y1021F. PAE cells expressing
Y1009F/Y1021F migrate as efficiently as the wild type PDGFR-
cells
(7), which implies that PLC-
has a regulatory role that balances the
loss of SHP-2. The complexity of this regulation is indicated by the
data reported by Hansen et al. (9), who described a mutant
PDGFR-
Y943F, which allows increased tyrosine phosphorylation of
PLC-
and mediates cellular migration with increased efficiency. How
PLC-
exerts its modulatory role in chemotaxis is not clear.
Different second messengers downstream of PLC-
, such as
Ca2+, or members of the protein kinase C family could be
involved in mediation of this effect. Treatment of the Y934F mutant
receptor cells with the protein kinase C inhibitor bisindolylmaleimide attenuated chemotaxis toward PDGF; in contrast, wild type PDGFR-
cells were able to migrate in the presence of the protein kinase C
inhibitor (9). In accordance, our data show that loss of PLC-
binding and inositol phosphate formation does not affect PDGF-induced
migration (cf. Fig. 4A). Thus, the role of
PLC-
in growth factor-induced chemotaxis appears to be dependent on the balance of negative and positive signal transduction pathways affecting cellular migration.
In a recent study by Yu et al. (33), fibroblasts isolated
from mouse embryos with targeted inactivation of the shp-2
gene were shown to display an increased number of focal adhesions, deregulated tyrosine phosphorylation of FAK, and decreased cellular motility on fibronectin. Our data agree with and extend the study by Yu
et al., by showing that SHP-2 has a critical role, possibly by regulating FAK kinase activity, in growth factor-stimulated actin
reorganization and chemotaxis.