From the Department of Biochemistry and Molecular
Biology, ¶ Medical and Molecular Genetics, § Walther
Oncology Center, Indiana University School of Medicine and Walther
Cancer Institute, Indianapolis, Indiana 46202-5254
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ABSTRACT |
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Shp-2, a widely expressed cytoplasmic tyrosine phosphatase with two SH2 domains, is believed to participate in signal relay downstream of growth factor receptors. We show here that this phosphatase also plays an important role in the control of cell spreading, migration, and cytoskeletal architecture. Fibroblast cells lacking a functional Shp-2 were impaired in their ability to spread and migrate on fibronectin compared with wild-type cells. Furthermore, Shp-2 mutant cells displayed an increased number of focal adhesions and condensed F-actin aggregation at the cell periphery, properties reminiscent of focal adhesion kinase (FAK)-deficient cells. This is consistent with our previous observations in vivo that mice homozygous for the Shp-2 mutation died at midgestation with similar phenotype to FAK and fibronectin-deficient embryos, having severe defects in mesodermal patterning, particularly the truncation of posterior structures. Biochemical analysis demonstrated that FAK dephosphorylation was significantly reduced in Shp-2 mutant cells in suspension. Furthermore, regulated association of Src SH2 domain with FAK and paxillin during cell attachment and detachment on fibronectin was disrupted in Shp-2 mutant cells. This report defines a unique role of the Shp-2 tyrosine phosphatase in cell motility, which might guide the design of a new strategy for pharmaceutical interference of tumor metastasis.
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INTRODUCTION |
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Cell adhesion to extracellular matrix
(ECM)1 is crucial for
multiple biological functions, which include cell growth,
differentiation, migration, tumor metastasis, and embryonic development
(1-3). Integrins are a major family of transmembrane proteins that
mediate cellular association with ECM. The engagement of cell surface integrins with ligands leads to recruitment of a number of
intracellular proteins to specialized sites of the cytoplasmic face in
focal adhesions (4). Although the molecular mechanism of
integrin-mediated signal transduction is not well defined, tyrosine
phosphorylation of several cytoplasmic proteins, including focal
adhesion kinase (FAK), paxillin, tensin, and Cas, is a critical
biochemical aspect in this process (5-10). FAK, a nonreceptor
protein-tyrosine kinase (PTK), has received most attention in recent
years and a close relative of FAK has also been identified that was
variously named as proline-rich tyrosine kinase 2 (PYK2), cellular
adhesion kinase (CAK
), and related adhesion focal tyrosine
kinase (RAFTK) (11-13).
It was previously thought that FAK might have a primary role in the formation of focal adhesion, based on the cellular localization of activated FAK at focal adhesion sites (14). However, recent studies suggest that FAK is more likely to modulate the turnover of focal adhesion and to regulate cell migration. FAK-deficient cells exhibited an elevated number of focal adhesions accompanied by a decreased rate of cell migration (15). Overexpression of FAK augmented cell migration and activation of mitogen-activated protein kinase, whereas inactivation of FAK suppressed cell migration and proliferation (16-18). Increased FAK activity has been correlated with the invasiveness of tumors (19). Integrins, whereas acting as a bridge between ECM and cytoskeleton, might also transduce biochemical signals into cells. PTKs, such as Src and FAK, seem to be important players in integrin-initiated signaling (14, 20). This model would predict the requirement for protein-tyrosine phosphatases (PTPs) in the regulation of signals downstream of integrins. However, a role of a specific cytoplasmic PTP has not been documented in this process.
Shp-2 is a widely expressed cytoplasmic PTP that contains two tandem
SH2 domains at the NH2 terminus (21, 22). Several lines of
evidence indicate that Shp-2 is the mammalian homologue of the gene
product of Drosophila corkscrew (Csw), which also encodes a SH2-containing PTP. Csw participates in signaling downstream of the Torso and Sevenless receptor PTKs, as revealed by genetic analyses in Drosophila (23, 24). Biochemical studies in
mammalian cells suggested that Shp-2 might participate in transmission
of signals from growth factor receptors (25-27). The PTP physically interacts via its SH2 domains with a number of ligand-activated receptor PTKs as well as cytoplasmic signaling proteins and presumably functions to promote mitogenic signals (28-30). To define the
biological function of mammalian Shp-2, a targeted mutation was
introduced into the murine Shp-2 locus in embryonic stem
(ES) cells that results in a deletion of exon 3, encoding for amino
acid residues 46-110 in the NH2-terminal SH2 (SH2-N)
domain of Shp-2 (31). Homozygous Shp-2 mutant mice die around day
8.5-10.5 of gestation, with multiple defects in mesodermal patterning.
Notably, the abnormalities of mesodermal patterning in Shp-2/
animals are similar to the phenotype of FAK- and fibronectin
(FN)-deficient embryos (32, 33), suggesting that Shp-2 and FAK were in
a common signaling pathway controlling cell motility. In this report,
we present evidence that Shp-2 plays a critical role in the regulation
of cell spreading, migration, and cytoskeletal organization.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Antibodies--
Wild-type (Shp-2+/+),
heterozygous and homozygous Shp-2 mutant (Shp-2+/ and Shp-2
/
)
embryonic fibroblast cell lines were described in detail previously
(34). Monoclonal antibody (mAb) against FAK was kindly provided by Dr.
J. T. Parsons (University of Virginia) and also purchased from
Santa Cruz Biotechnology, Inc. Polyclonal antibodies against FAK and
Shp-2, mAbs against phosphotyrosine (PY20), and fluorescein
isothiocyanate-labeled secondary anti-mouse IgG were purchased from
Santa Cruz Biotechnology, Inc. Tetramethylrhodamine B
isothiocyanate-labeled secondary anti-mouse IgG and mAb against
vinculin were obtained from Sigma. mAb against paxillin was purchased
from Transduction Laboratories, and mAbs to
5 and
1 integrins were from PharMingen, Inc.
Anti-phosphotyrosine mAb (4G10) was obtained from Upstate Biotechnology
Inc.
Cell Spreading on FN-- Fibroblast cells were collected by trypsinization and washed twice with Dulbecco's modified Eagle's medium (DMEM) containing 0.2% soybean trypsin inhibitor (Sigma). Cell spreading was assessed as described previously (35). Briefly, cells (5 × 105) were resuspended in DMEM and added to 30-mm tissue culture dishes that were precoated with FN (10 µg/ml) overnight at 4 °C. Cells were allowed to spread for the indicated times at 37 °C, chilled on ice for 10 min, and then photographed. Spread cells were defined as cells with extended processes, lacking a rounded morphology and not phase-bright, whereas nonspread cells were rounded and phase-bright under microscope.
Cell Adhesion-- Fibroblast cells collected upon trypsinization were washed in serum-free DMEM containing 0.2% trypsin inhibitor. Cells were resuspended at 106 cells/ml in DMEM, and 100 µl was added to each well of 96-well plates that had been coated overnight at 4 °C with 10 µg/ml FN and blocked with bovine serum albumin (1 µg/ml). To allow cell attachment, these plates were incubated for certain period of times at 37 °C in CO2 incubator. Nonadherent cells were removed by washing with PBS, and attached cells were then stained with 0.5% crystal violet in 20% methanol. After washing with PBS, crystal violet staining was eluted with 0.1 M sodium citrate (pH 4.2), and the optical absorbency was measured at 595 nm using a microplate reader (36).
Cell Motility Assay-- Cell migration was determined using modified chambers containing polycarbonate membranes (tissue culture-treated, 6.5-mm diameter, 8-µm pores, transwell; Costar, Cambridge, MA) (16). Both sides of the membrane were coated with FN (10 µg/ml) for 1 h at 37 °C. Trypsinized cells were first washed once with DMEM containing 0.2% soybean trypsin inhibitor and then washed twice with DMEM. Cells were added to the upper chamber at 6 × 104 cells/well, and the lower chamber was filled with DMEM containing 4 µg/ml of FN. After incubation at 37 °C for the indicated times, the membrane was fixed in methanol, and cells on the upper surface were mechanically removed. Migrated cells on the lower side of membranes were stained with Giemsa stain and enumerated under a microscope at × 200 magnification. Two random microscopic fields were counted per well, and all experiments were performed in duplicate.
Detection of Actin Organization and Immunofluorescent Staining-- Cells were plated in six-well plates on FN-coated glass coverslips (Becton Dickinson Labware), washed twice with PBS, and fixed in 3.7% paraformaldehyde for 20 min. Fixed cells were washed twice with PBS, permeabilized by treatment with 0.2% Triton X-100 in PBS for 5 min, and then blocked in PBS containing 0.2% gelatin and 0.02% NaN3. For actin organization, cells were stained with rhodamine-conjugated phalloidin (5 µg/ml in PBS, Sigma). For immunofluorescence staining, cells were incubated with 1/50 diluted antibodies against vinculin and paxillin or 1/100 diluted anti-phosphotyrosine antibodies for 40 min at 20 °C in a humidified chamber. Coverslips were then treated with secondary antibodies at the recommended dilution and washed with PBS. For examination by fluorescent microscopy, slides were covered with 4',6'-diamidine-2'-phenylindole dihydrochloride antifade, which stained the nuclei blue. Microscopic analysis of fluorescent in situ hybridization images was done using an Aristoplan fluorescence microscope (Leitz, Rockleigh, NJ) equipped with appropriate filters and using 63× and 100× oil-immersion objectives. Image capturing was performed with a cooled charged-coupled device camera (Photometrics, Tucson, AR) and a software package developed by Vysis, Inc. (Downers Grove, IL). Separate gray images of the three colors (red, green, and blue) were taken using individual fluorescein isothiocyanate, Texas Red, and 4',6'-diamidine-2'-phenylindole dihydrochloride filters, transferred to a Macintosh computer, then pseudocolored and superimposed using a commercial software package by Vysis, Inc. to yield the final image.
Immunoprecipitation and Immunoblotting-- Cells were washed with ice-cold PBS and lysed in modified radioimmunoprecipitation assay buffer (37). Lysates were centrifugated at 15,000 × g for 10 min, and supernatants were subjected to immunoprecipitation with specific antibodies at 4 °C for 2-4 h. Resulting immune complexes were incubated with protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) at 4 °C for 1-2 h. Beads were washed four times in 1 ml of ice-cold immunoprecipitation buffer (150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium orthovanadate, 0.5% Triton X-100, 0.5% Nonidet P-40) and subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred to nitrocellulose membranes, and membranes were blocked in TBST (10 mM Tris at pH 8.0, 150 mM NaCl, 0.2% Tween 20) plus 5% bovine serum albumin (Sigma) or 5% non-fat milk (Bio-Rad). Blots were incubated first with primary antibodies for 1.5 h, washed three times in TBST buffer, and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Proteins were detected with the enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech).
In Vitro Protein Binding Assay-- Glutathione S-transferase (GST) fusion protein containing the c-Src SH2 domain was purified as described previously (38). GST-SrcSH2 fusion protein (5 µg) immobilized on glutathione-Sepharose beads was mixed with cell lysates (1 mg of total protein) at 4 °C for 2 h and washed three times with the immunoprecipitation buffer. Protein complexes were separated by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting as described above.
Expression of Wild-type Shp-2 in Shp-2/
Cells--
A
construct was engineered by inserting a 2.2-kilobase pair Shp-2
cDNA fragment into pcDNA3.1/hygro(+) vector (Invitrogen) at a
BamHI site, for expression of a full-length wild-type Shp-2 protein. Subconfluent Shp-2
/
cells were exposed for 5 h to
PerFectTMLipids (Invitrogen) containing 10 µg of the
recombinant DNA construct or the vector in a 60-mm cell culture dish.
Cells were first cultured in DMEM plus 10% fetal calf serum for
24 h and then selected with hygromycin B (Sigma) at the dosage of
200 µg/ml. Hygromycin-resistant clones were isolated and screened for
expression of wild-type Shp-2 protein by immunoblot analysis using
anti-Shp-2 antibody.
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RESULTS |
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Delayed Spreading of Shp-2 Mutant Fibroblast Cells on FN--
The
establishment and growth properties of wild-type (Shp-2+/+),
heterozygous (Shp-2+/), and homozygous (Shp-2
/
) mutant fibroblast
cell lines were described previously (34). As demonstrated in our
previous experiments (31, 34, 39), Shp-2+/
cells and animals have the
wild-type phenotype, suggesting that the mutant Shp-2 protein without
the intact SH2-N domain does not function in a dominant negative manner
but rather is a loss-of-function molecule.
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Reduced Cell Migration--
Using the modified chamber for cell
mobility assay, we compared the migration of wild-type and Shp-2/
cells. Fibroblasts were seeded in the upper chamber and allowed to
migrate into the lower chamber through the small pores of a membrane (8 µm). Migrated cells were enumerated after 3, 6, and 9 h.
Compared with wild-type cells, the ability of Shp-2
/
cells to
migrate on FN was significantly reduced (Fig.
2A). The experiments have been
repeated with several other wild-type and mutant fibroblast cell lines,
and consistent results have been obtained (data not shown).
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Similar Cell Adhesion to FN between Wild-type and Shp-2 Mutant
Fibroblasts--
The data shown above indicate that Shp-2/
cells
have reduced capacity to spread and migrate on FN, which could result
from a defect in cell adhesion on FN. To test this hypothesis, we
examined wild-type and mutant cells in their adhesion to FN. Cells were seeded in 96-well plates previously coated with FN and incubated for
10, 20, 30, and 60 min. After removal of nonadherent cells by washing
with PBS, attached cells were stained with crystal violet and
A595 was measured. The results are shown in Fig.
3. No significant differences between
Shp-2+/+, -+/
, and -
/
cells were observed in their adhesion to
FN. This result argues that reduced spreading and migration of
Shp-2
/
cells was not due to an inability of the cells to adhere to
FN.
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F-actin Rearrangement and Enhanced Focal Adhesion--
Cells
plated on FN were stained with rhodamine-conjugated phalloidin for
examination of filamentous actin (F-actin). In comparison with
wild-type cells (Fig. 5, a and
b), Shp-2/
cells (Fig. 5, c and d)
displayed an increased density of F-actin staining and numerous
microspikes at the cell periphery, hallmarks of the early stage of
nonpolar cell spreading. To characterize the formation of focal
adhesions, cells plated on FN were stained with antibodies against
vinculin or paxillin. There was a much higher number of focal adhesion
contacts in Shp-2
/
cells than in wild-type cells as revealed by
vinculin staining (Fig. 5, f and i). Furthermore, vinculin-positive patches were scattered across the ventral surface in
Shp-2
/
cells, in contrast to the typical focal adhesion patches at
the cell periphery in wild-type cells. The distribution of paxillin was
similar to the arrangement of F-actin fibers, which was condensed
around cell perimeters in mutant cells as compared with wild-type cells
(Fig. 5, e and h). As revealed by
anti-phosphotyrosine antibody staining (Fig. 5, g and
j), more abundant tyrosine-phosphorylated proteins
distributed in numerous podosome-like focal adhesion sites were
detected in Shp-2
/
cells than in wild-type cells, correlating well
with the observed enhanced formation of focal adhesion in mutant
cells.
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Alteration in the Association of Src SH2 Domain with FAK, Paxillin,
and Shp-2--
Previous experiments suggested that Src is physically
associated with and phosphorylates several proteins, including FAK and paxillin, in focal adhesions (41-44). The Src SH2 domain is
responsible for mediating the protein-protein interaction. To
understand the biochemical basis for the alteration of focal adhesion
in Shp-2 mutant cells, we examined the physical association of FAK and paxillin with Src SH2 domain in vitro. Purified GST-SrcSH2
fusion protein was incubated with cell lysates, and the bound proteins were subjected to immunoblot analysis using specific antibodies. As
shown in Fig. 6, the immobilized
GST-SrcSH2 fusion protein precipitated FAK, paxillin, and Shp-2.
Interestingly, FAK and paxillin were associated with SrcSH2 in
wild-type and Shp-2+/ cells only when they were attached to ECM.
These proteins were not precipitable by SrcSH2 in cells kept in
suspension for 2 h, but reassociation was detected after replating
cells on FN for 20 and 40 min. These results suggest that the physical
interaction between these proteins is regulated during cell attachment
and detachment. However, this regulation was apparently disrupted in
Shp-2
/
cells; FAK and paxillin were precipitated by the SrcSH2 fusion protein in mutant cells even in suspension. Interestingly, both
the wild-type and mutant Shp-2 proteins were detected in the SrcSH2
precipitates either in suspension or attachment. Thus, it is evident
that a functional Shp-2 with intact SH2 domains might be required for
its physiological activity in cells.
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Rescue of the Mutant Phenotype by Expression of Wild-type
Shp-2--
The results described above point to a putative function of
Shp-2 in the control of cell mobility by working in concert with FAK. A
targeted mutation in the Shp-2 locus severely
decreased cell migration in homozygous mutant cells, a
phenotype similar to FAK/
cells. Because a truncated Shp-2 protein
with a deletion of 65 amino acids in the SH2-N domain was expressed,
one might argue that the abnormal phenotype could be due to an aberrant activity of the mutant molecule. To rule out that possibility, we
transfected the wild-type Shp-2 cDNA into Shp-2
/
fibroblast cells. Several clones expressing different levels of wild-type Shp-2
were isolated, as demonstrated by immunoblot analysis using anti-Shp-2
antibody. Cell migration over FN was then assayed in the same way as in
Fig. 2. Upon reintroduction of wild-type Shp-2 protein, an enhanced
cell motility on FN was observed, and more importantly, the increased
rate in cell migration was proportional to the expression levels of
wild-type Shp-2 in mutant cells (Fig. 8).
These results establish an important role of Shp-2 in the stimulation
of cell motility.
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DISCUSSION |
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In the present study, we have shown that Shp-2, an ubiquitously expressed cytoplasmic tyrosine phosphatase with two SH2 domains at the NH2 terminus, participates in integrin-initiated signaling events. Fibroblast cells in which a functional Shp-2 molecule is absent displayed significantly reduced ability to spread over FN. Impaired cell migration on FN was observed in Shp-2 mutant fibroblasts as well as totipotent ES cells. The defect in cell mobility was rescued by re-introduction of wild-type Shp-2 protein into Shp-2-deficient fibroblast cells. These results indicate that Shp-2 tyrosine phosphatase functions to promote cell migration.
It is interesting to note that the expression of integrins
5 and
1 on cell surface was not changed,
and cell adhesion to FN was not altered by the Shp-2 mutation.
Therefore, the defect in cell spreading and migration might be due to
an alteration in cytoskeletal organization in Shp-2 mutant cells.
Indeed, we observed that Shp-2
/
cells displayed an increased number
of focal adhesion and condensed F-actin staining at the cell periphery. This phenotype is quite similar to FAK-deficient cells, and it would
suggest that Shp-2 might work in concert with FAK in the control of
dynamics of focal adhesions. Support to this hypothesis came from our
observations described in Figs. 6 and 7. FAK underwent a regulated
association with Src SH2 domain during cell attachment and detachment,
which was apparently correlated with its tyrosine-phosphorylation status. FAK was highly phosphorylated on tyrosine when cells attached to ECM and became rapidly dephosphorylated upon detachment. In Shp-2
mutant cells, both FAK dephosphorylation and its dynamic interaction
with Src SH2 domain were significantly reduced. Therefore, Shp-2 might
be involved in the deactivation of FAK, which is required for the
regeneration of active FAK in the turnover of focal adhesion during
cell migration. A typical cell movement across a two-dimensional substrate could be divided into three concerted steps, membrane protrusion, cell traction, deadhesion and tail retraction. Adhesion at
the leading edge and deadhesion at the rear portion of cells are
required for protrusion and tail retraction, respectively (45). The
dynamic turnover of focal adhesions is likely to play a critical role
in cell spreading and migration over ECM. Condensed distribution of
focal adhesion sites at the edge of FAK
/
and Shp-2 mutant
fibroblasts might reflect a defect in their turnover, which leads to a
reduction in cell motility. These results would allow us to raise an
intriguing proposal regarding the dynamic interplay between a PTK and a
PTP in the control of cell mobility and focal adhesion. Shp-2 might be
involved in the turnover of focal adhesions by mediating the
dephosphorylation of several proteins, including FAK and paxillin,
which influences cell spreading, migration, and cytoskeletal
architecture. Although the biochemical mechanism for the specific
function of Shp-2 in focal adhesion is to be defined, our results
indicate that an intact SH2-N domain is required for Shp-2 activity.
Notably, Kaplan et al. (35) reported that the function of
c-Src in promoting cell spreading requires its SH2 and SH3 domain but
not the kinase catalytic activity. Experiments are in progress to
determine whether Shp-2 can affect cell motility by a
phosphatase-independent mechanism.
We have reported previously that homozygous Shp-2 mutant mice died at
midgestation with multiple developmental defects in mesodermal
structures (31). The development of axial structures, node, notochord,
and the anterior-posterior (A-P) axis, was severely perturbed in
homozygous mutant embryos. Shp-2 mutant embryos exhibited variable
degrees of posterior truncations apparently associated with the
abnormal development of the node/tail bud and the notochord (31). These
results suggest that Shp-2 might be involved in mediating the proper
organization and migration of mesodermal cells during gastrulation. The
abnormal phenotype of Shp-2 mutant mice is consistent with a dominant
negative study in Xenopus embryos (47). Injection of a
catalytically inactive mutant Shp-2 molecule caused defects in
mesodermal induction, particularly the truncation of terminal
structures. Shp-2 mutant embryos share several abnormal features in the
axial mesodermal tissues with FAK/
and FN-deficient embryos, which
died at the same stage of gastrulation (32). In addition, targeted
mutations in the FGF-R1, csk, and
GAP/Nf1 (double mutant) loci also gave rise to a similar
phenotype in mesodermal patterning in homozygous mutant animals (46,
48-50). These results would suggest an integration of signals in the
control of cell growth, movement, and differentiation during mammalian development.
The past 5 years have witnessed a rapid progress in our understanding of SH2-containing PTPs in cell regulation. Genetic and biochemical evidence indicates that Shp-1 and Shp-2 are important players in cell proliferation and differentiation, in a negative or a positive manner. We and others have shown that Shp-2 apparently plays a positive role in mediating mitogenic stimulation of extracellular-signal regulated kinases (29, 30, 47), whereas acting as a negative effector in c-Jun NH2-terminal kinase activation under stress (34). This report establishes that Shp-2 operates in signal relay downstream of integrins in guiding cell migration. Therefore, Shp-2 appears to participate in transmission and/or integration of signals emanating from receptors for cytokines and ECM.
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ACKNOWLEDGEMENTS |
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We thank Drs. Mark Kaplan, Ron Wek, and Peter Roach for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NIHR29GM53660 and Council for Tobacco Research Grant 4345R1 (to G. S. F.) and by a grant from the Indiana University Cancer Center (to G. S. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a career development award from the American
Diabetes Association. To whom correspondence should be addressed: Walther Oncology Center, Indiana University School of Medicine, 1044 W. Walnut St., Rm. 302, Indianapolis, IN 46202-5254. Tel.: 317-274-7515;
Fax: 317-274-7592; E-mail: gfeng{at}iupui.edu.
The abbreviations used are: ECM, extracellular matrix; FAK, focal adhesion kinase; FN, fibronectin; PTP, protein-tyrosine phosphatase; PTK, protein-tyrosine kinase; DMEM, Dulbecco's modified Eagle's medium; ES cell, embryonic stem cell; GST, glutathione S-transferase.
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REFERENCES |
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