Protein-tyrosine Phosphatase Shp-2 Regulates Cell Spreading, Migration, and Focal Adhesion*

De-Hua YuDagger , Cheng-Kui QuDagger §, Octavian Henegariu, Xiaolan LuDagger , and Gen-Sheng FengDagger §parallel

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta  (CAKbeta ), 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.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
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References

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 alpha 5 and beta 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

It was first noticed in our routine cell passages that Shp-2-/- cells spread at a slower rate than Shp-2+/+ and Shp-2+/- cells after trypsinization and that the same phenotype was observed in at least five independently isolated Shp-2-/- cell lines. To carefully compare the spreading efficiency, we collected wild-type and mutant fibroblasts in serum-free DMEM and plated cells on FN-coated cell culture dishes. As depicted in Fig. 1A, a striking difference in the appearance of cell spreading on FN was observed between wild-type and Shp-2-/- cells (Fig. 1). Wild-type cells initiated spreading within 10 min after plating, with approximately 50% of them attaining a flat morphology in 30 min, and at 60 min, nearly 90% of wild-type cells appeared well extended and had achieved a flattened morphology. In contrast, Shp-2-/- cells spread at a much reduced rate with only about 60% of the cells spreading well by 60 min. Similar results were observed when cells were cultured in DMEM containing 10% fetal calf serum and plated either on FN-coated or uncoated tissue culture dishes (data not shown). Consistent with the defective cell spreading of Shp-2-/- fibroblasts is an alteration in cell morphology (Fig. 1). Wild-type cells appear more extended and elongated, whereas mutant cells do not spread or elongate extensively and look smaller. However, flow cytometry analysis indicated that there was no difference of cell sizes between wild-type and Shp-2 mutant cellls (data not shown), suggesting that the Shp-2 mutation affects cell spreading but not cell size.


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Fig. 1.   Reduced spreading of Shp-2 mutant fibroblast cells on FN. A, wild-type and mutant (Shp-2-/-) fibroblasts were plated on FN-coated cell culture dishes, incubated at 37 °C, and then photographed at 10, 20, 30, and 60 min. B, quantitative comparison of cell spreading efficiency was obtained by calculating the percentage of spread cells of wild-type and Shp-2-/- origins at each time point.

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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Fig. 2.   Shp-2 mutant cells exhibit deficient migration. A, to measure cell motility, fibroblasts were added to the upper chamber, incubated at 37 °C for indicated times, and allowed to migrate through an 8-µm porous membrane into the lower chamber. Migrated cells on the lower side of the membrane were enumerated. The data represent the mean with S.E. of six samples from three independent experiments. B, one wild-type and two Shp-2-/- ES cell lines were evaluated for their migration capacity over FN for 6 h as described for fibroblasts as above.

To further explore the effect of the Shp-2 mutation on cell motility, we also compared wild-type and Shp-2-/- embryonic stem (ES) cells for their migration capacity. These Shp-2-/- ES cell lines were independently isolated by selection of Shp-2+/- ES cells in a high dosage of G418 as described previously (39). One wild-type and two Shp-2-/- ES cell lines were assessed for migration on FN. As shown in Fig. 2B, mutant ES cells exhibited significantly slower migration rate than wild-type ES cells. Therefore, reduced cell motility was observed in both totipotent stem cells and fibroblasts of Shp-2-/- origin. We have shown previously in the in vitro ES cell differentiation assay that Shp-2 mutant ES cells exhibited an impaired capacity to develop into embryoid bodies that contain various tissue-specific cells, including hematopoietic and cardiac muscle cells (39, 40). Decreased cell motility may account, at least in part, for differentiation problems of Shp-2-/- ES cells in vitro and embryos in vivo.

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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Fig. 3.   Cell adhesion to FN. Wild-type, Shp-2+/-, and Shp-2-/- cells were seeded in 96-well plates precoated with FN and incubated for 10, 20, 30, and 60 min at 37 °C. The efficiency of cell adhesion was assessed as described in the text, and the data represent the means with S.D. from three independent experiments.

Consistent with this observation, similar levels of integrin alpha 5 and beta 1 expression were detected on Shp-2+/+, Shp-2+/-, and Shp-2-/- fibroblast cells by immunoblot analysis (Fig. 4). Integrins alpha 5 and beta 1 are the major isoforms of integrins expressed on the surface of fibroblasts that are responsible for cell adhesion on FN. Therefore, the Shp-2 mutation did not cause any changes in the expression of integrins and cell adhesion behavior. It seems more likely that Shp-2 modulates integrin-mediated intracellular signaling pathways for cytoskeletal organization and control of cell spreading and migration.


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Fig. 4.   The expression of integrins on wild-type and mutant cells. Equal amounts of wild-type, Shp-2+/- and Shp-2-/- cell lysates were subjected to immunoprecipitation and immunoblot analysis using specific anti-integrin alpha 5 and beta 1 antibodies, respectively (PharMingen Inc.).

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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Fig. 5.   Effect of Shp-2 mutation on focal adhesion and cytoskeletal architecture. a, b, e, f, and g, wild-type cells; c, d, h, i, and j, Shp-2-/- cells. In a, b, c, and d, cells were seeded on FN-coated coverslips for 1 h (a, c) and 16 h (b, d) and stained with rhodamine-conjugated phalloidin (Sigma). For examination of focal adhesion, cells were stained with anti-paxillin (e, h) and anti-vinculin antibodies (f, i), respectively. In g and j, the distribution of tyrosine-phosphorylated proteins was examined with anti-phosphotyrosine antibody (4G10, Upstate Biotechnology Inc). Fluorescein isothiocyanate-labeled secondary anti-mouse IgG were from Santa Cruz and tetramethylrhodamine B isothiocyanate-labeled secondary anti-mouse IgG and mAb against vinculin were from Sigma. mAb against paxillin was from Transduction Laboratories.

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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Fig. 6.   Association of FAK, paxillin, and Shp-2 with c-Src via its SH2 domain. Cell lysates were prepared in radioimmunoprecipitation assay buffer (42) from fibroblasts attached to tissue culture plates (Adh.), cells kept in suspension for 2 h (Susp.), and cells that had been allowed to attach to FN for 20 and 40 min. GST-SrcSH2 fusion protein (5 µg) immobilized on glutathione beads was mixed with cell lysates (1 mg of total protein) at 4 °C for 2 h and washed three times. Precipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was first blotted with anti-FAK antibody, striped, and blotted with anti-paxillin antibody. After striping again, it was blotted with anti-Shp-2 antibody.

To determine whether Shp-2 had a functional role in focal adhesion, we examined tyrosine phosphorylation levels of FAK in wild-type, Shp-2+/-, and Shp-2-/- cells under various conditions. Fibroblast cells were trypsinized and kept in suspension in serum-free DMEM containing 0.1% trypsin inhibitor for 0, 5, 15, and 45 min at 37 °C. Cell suspensions were then left on ice for 10 min, and cell lysates were made. FAK was specifically precipitated with anti-FAK antibody, and the immunoprecipitates were subjected to immunoblot analysis using anti-PY antibody. As shown in Fig. 7, rapid FAK dephosphorylation was observed in wild-type and Shp-2+/- cells when they were kept in suspension. In contrast, decrease in the phosphorylation level of FAK was slowed down in Shp-2-/- cells when kept in suspension. The same filter of FAK immunoprecipitates was striped and reblotted with anti-Shp-2 antibody. Indeed, Shp-2 was coprecipitated with FAK in fibroblast cells either in attachment or in suspension. This represents the first biochemical evidence for the physical involvement of Shp-2 in mediating the regulation of focal adhesion and FAK activity.


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Fig. 7.   Tyrosine phosphorylation status of FAK. FAK was precipitated with anti-FAK antibodies from lysates prepared from attached cells, or detached cells kept in suspension in serum-free medium for 5, 15 and 45 min. Immunoprecipitates were resolved in SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and immunoblotted with anti-phosphotyrosine antibody (4G10). The filter was striped and re-blotted with anti-Shp-2 antibody, and then, after striping, blotted with anti-FAK antibodies.

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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Fig. 8.   Rescue of the mutant phenotype by re-introduction of wild-type Shp-2. Shp-2-/- cells were transfected with an expression construct for wild-type Shp-2 and selected in hygromycin B. Isolated clones were screened for wild-type Shp-2 expression by immunoblot analysis. The upper panel shows the expression levels of wild-type and mutant Shp-2 proteins, as revealed by immunoblot analysis. The lower panel shows the result of cell migration assay. Column 1, wild-type cells; column 2, Shp-2-/- cells; column 3, Shp-2-/- cells transfected with the vector; columns 4 and 5, Shp-2-/- cells expressing different levels of wild-type Shp-2.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 5 and beta 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.

    ACKNOWLEDGEMENTS

We thank Drs. Mark Kaplan, Ron Wek, and Peter Roach for critically reading the manuscript.

    FOOTNOTES

* 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.

parallel 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.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Clark, E. A., and Brugge, J. S. (1995) Science 268, 233-239[Medline] [Order article via Infotrieve]
  2. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
  3. Ruoslahti, E. (1991) J. Clin. Inv. 87, 1-5[Medline] [Order article via Infotrieve]
  4. Gilmore, A. P., and Burridge, K. (1996) Structure 4, 647-651[Medline] [Order article via Infotrieve]
  5. Bockholt, S. M., and Burridge, K. (1993) J. Biol. Chem. 268, 14565-14567[Abstract/Free Full Text]
  6. Burridge, K., Turner, C. E., and Romer, L. H. (1992) J. Cell Biol. 119, 893-903[Abstract]
  7. Turner, C. E., Glenney, J. R., Jr., and Burridge, K. (1990) J. Cell Biol. 111, 1059-1068[Abstract]
  8. Polte, T. R., and Hanks, S. K. (1997) J. Biol. Chem. 272, 5501-5509[Abstract/Free Full Text]
  9. Nojima, Y., Mimura, T., Morino, N., Hamasaki, K., Furuya, H., Sakai, R., Nakamoto, T., Yazaki, Y., and Hirai, H. (1996) Hum. Cell 9, 169-174[Medline] [Order article via Infotrieve]
  10. Guan, J. L., and Shalloway, D. (1992) Nature 358, 690-692[CrossRef][Medline] [Order article via Infotrieve]
  11. Avraham, S., London, R., Fu, Y., Ota, S., Hiregowdara, D., Li, J., Jiang, S., Pasztor, L. M., White, R. A., Groopman, J. E., and Avraham, H. (1995) J. Biol. Chem. 270, 27742-27751[Abstract/Free Full Text]
  12. Sasaki, H., Nagura, K., Ishino, M., Tobioka, H., Kotani, K., and Sasaki, T. (1995) J. Biol. Chem. 270, 21206-21219[Abstract/Free Full Text]
  13. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737-745[CrossRef][Medline] [Order article via Infotrieve]
  14. Ilic, D., Damsky, C. H., and Yamamoto, T. (1997) J. Cell Sci. 110, 401-407[Abstract/Free Full Text]
  15. Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., and Yamamoto, T. (1995) Nature 377, 539-544[CrossRef][Medline] [Order article via Infotrieve]
  16. Cary, L. A., Chang, J. F., and Guan, J. L. (1996) J. Cell Sci. 109, 1787-1794[Abstract/Free Full Text]
  17. Schlaepfer, D. D., and Hunter, T. (1997) J. Biol. Chem. 272, 13189-13195[Abstract/Free Full Text]
  18. Gilmore, A. P., and Romer, L. H. (1996) Mol. Biol. Cell 7, 1209-1224[Abstract]
  19. Owens, L. V., Xu, L., Craven, R. J., Dent, G. A., Weiner, T. M., Kornberg, L., Liu, E. T., and Cance, W. G. (1995) Cancer Res. 55, 2752-2755[Abstract]
  20. Parsons, J. T., and Parsons, S. J. (1997) Curr. Opin. Cell Biol. 9, 187-192[CrossRef][Medline] [Order article via Infotrieve]
  21. Feng, G. S., and Pawson, T. (1994) Trends Genet. 10, 54-58[CrossRef][Medline] [Order article via Infotrieve]
  22. Neel, B. G., and Tonks, N. K. (1997) Curr. Opin. Cell Biol. 9, 193-204[CrossRef][Medline] [Order article via Infotrieve]
  23. Perkins, L. A., Larsen, I., and Perrimon, N. (1992) Cell 70, 225-236[Medline] [Order article via Infotrieve]
  24. Allard, J. D., Chang, H. C., Herbst, R., McNeill, H., and Simon, M. A. (1996) Development (Camb.) 122, 1137-1146[Abstract/Free Full Text]
  25. Feng, G. S., Hui, C. C., and Pawson, T. (1993) Science 259, 1607-1611[Medline] [Order article via Infotrieve]
  26. Lechleider, R. J., Sugimoto, S., Bennett, A. M., Kashishian, A. S., Cooper, J. A., Shoelson, S. E., Walsh, C. T., and Neel, B. G. (1993) J. Biol. Chem. 268, 21478-21481[Abstract/Free Full Text]
  27. Vogel, W., Lammers, R., Huang, J., and Ullrich, A. (1993) Science 259, 1611-1614[Medline] [Order article via Infotrieve]
  28. Xiao, S., Rose, D. W., Sasaoka, T., Maegawa, H., Burke, T., Jr., Roller, P. P., Shoelson, S. E., and Olefsky, J. M. (1994) J. Biol. Chem. 269, 21244-21248[Abstract/Free Full Text]
  29. Noguchi, T., Matozaki, T., Horita, K., Fujioka, Y., and Kasuga, M. (1994) Mol. Cell. Biol. 14, 6674-6682[Abstract]
  30. Milarski, K. L., and Saltiel, A. R. (1994) J. Biol. Chem. 269, 21239-21243[Abstract/Free Full Text]
  31. Saxton, T. M., Henkemeyer, M., Gasca, S., Shen, R., Shalaby, F., Feng, G. S., and Pawson, T. (1997) EMBO J. 16, 2352-2364[Abstract/Free Full Text]
  32. Furuta, Y., Ilic, D., Kanazawa, S., Takeda, N., Yamamoto, T., and Aizawa, S. (1995) Oncogene 11, 1989-1995[Medline] [Order article via Infotrieve]
  33. George, E. L., Georges-Labouesse, E. N., Patel-King, R. S., Rayburn, H., and Hynes, R. O. (1993) Development (Camb.) 119, 1079-1091[Abstract/Free Full Text]
  34. Shi, Z. Q., Lu, W., and Feng, G. S. (1998) J. Biol. Chem. 273, 4904-4908[Abstract/Free Full Text]
  35. Kaplan, K. B., Swedlow, J. R., Morgan, D. O., and Varmus, H. E. (1995) Genes Dev. 9, 1505-1517[Abstract]
  36. Zhang, Z., Morla, A. O., Vuori, K., Bauer, J. S., Juliano, R. L., and Ruoslahti, E. (1993) J. Cell Biol. 122, 235-242[Abstract]
  37. Schlaepfer, D. D., Broome, M. A., and Hunter, T. (1997) Mol. Cell. Biol. 17, 1702-1713[Abstract]
  38. Feng, G. S., Ouyang, Y. B., Hu, D. P., Shi, Z. Q., Gentz, R., and Ni, J. (1996) J. Biol. Chem. 271, 12129-12132[Abstract/Free Full Text]
  39. Qu, C. K., Shi, Z. Q., Shen, R., Tsai, F. Y., Orkin, S. H., and Feng, G. S. (1997) Mol. Cell. Biol. 17, 5499-5507[Abstract]
  40. Qu, C. K., and Feng, G. S. (1998) Oncogene 15, in press
  41. Calalb, M. B., Polte, T. R., and Hanks, S. K. (1995) Mol. Cell. Biol. 15, 954-963[Abstract]
  42. Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. (1994) Nature 372, 786-791[Medline] [Order article via Infotrieve]
  43. Vuori, K., Hirai, H., Aizawa, S., and Ruoslahti, E. (1996) Mol. Cell. Biol. 16, 2606-2613[Abstract]
  44. Xing, Z., Chen, H. C., Nowlen, J. K., Taylor, S. J., Shalloway, D., and Guan, J. L. (1994) Mol. Biol. Cell 5, 413-421[Abstract]
  45. Mitchison, T. J., and Cramer, L. P. (1996) Cell 84, 371-379[Medline] [Order article via Infotrieve]
  46. Yamaguchi, T. P., Harpal, K., Henkemeyer, M., and Rossant, J. (1994) Genes Dev. 8, 3032-3044[Abstract]
  47. Tang, T. L., Freeman, R., Jr., O'Reilly, A. M., Neel, B. G., and Sokol, S. Y. (1995) Cell 80, 473-483[Medline] [Order article via Infotrieve]
  48. Henkemeyer, M., Rossi, D. J., Holmyard, D. P., Puri, M. C., Mbamalu, G., Harpal, K., Shih, T. S., Jacks, T., and Pawson, T. (1995) Nature 377, 695-701[CrossRef][Medline] [Order article via Infotrieve]
  49. Deng, C. X., Wynshaw-Boris, A., Shen, M. M., Daugherty, C., Ornitz, D. M., and Leder, P. (1994) Genes Dev. 8, 3045-3057[Abstract]
  50. Imamoto, A., and Soriano, P. (1993) Cell 73, 1117-1124[Medline] [Order article via Infotrieve]


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