Regulation of Fibroblast Motility by the Protein Tyrosine Phosphatase PTP-PEST*

Andrew J. GartonDagger and Nicholas K. Tonks§

From Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208

    ABSTRACT
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Abstract
Introduction
References

The protein tyrosine phosphatase PTP-PEST is a cytosolic enzyme that displays a remarkable degree of selectivity for tyrosine-phosphorylated p130Cas as a substrate, both in vitro and in intact cells. We have investigated the physiological role of PTP-PEST using Rat1 fibroblast-derived stable cell lines that we have engineered to overexpress PTP-PEST. These cell lines exhibit normal levels of tyrosine phosphorylation of the majority of proteins but have significantly lower levels of tyrosine phosphorylation of p130Cas than control cells. Initial cellular events occurring following integrin-mediated attachment to fibronectin (cell attachment and spreading) are essentially unchanged in cells overexpressing PTP-PEST; similarly, the extent and time course of mitogen-activated protein kinase activation in response to integrin engagement is unchanged. In contrast, the reduced phosphorylation state of p130Cas is associated with a considerably reduced rate of cell migration and a failure of cells overexpressing PTP-PEST to accomplish the normally observed redistribution of p130Cas to the leading edge of migrating cells. Furthermore, cells overexpressing PTP-PEST demonstrate significantly reduced levels of association of p130Cas with the Crk adaptor protein. Our results suggest that one physiological role of PTP-PEST is to dephosphorylate p130Cas, thereby controlling tyrosine phosphorylation-dependent signaling events downstream of p130Cas and regulating cell migration.

    INTRODUCTION
Top
Abstract
Introduction
References

PTP-PEST is a ubiquitously expressed mammalian, cytosolic protein tyrosine phosphatase (PTP)1 that was cloned in our laboratory (1) and by others (2-4). Several signaling proteins have been shown to be capable of binding to PTP-PEST, including Shc (5, 6), paxillin (7), Grb2 (8), and Csk (9), but the physiological significance of these interactions and their relevance to the function of PTP-PEST is unclear. In order to investigate the physiological substrate specificity of members of the PTP family, we developed a novel method involving expression of substrate-trapping mutant forms of PTPs (10, 11). These mutants retain a high affinity for substrates but are catalytically impaired. Therefore, they form complexes with appropriate target substrates that are amenable to isolation and characterization. We have used this method to identify substrates of PTP-PEST and thus gain insight into its physiological function. We have shown that the adaptor protein p130Cas is a specific substrate for PTP-PEST in vitro and in intact cells (11). Similar results have also been obtained in studies performed on fibroblasts derived from PTP-PEST-/- mice (12). This high degree of selectivity of PTP-PEST for p130Cas is derived from two distinct high affinity interactions. The catalytic domain of PTP-PEST itself displays intrinsic specificity for tyrosine-phosphorylated p130Cas (11), but this specificity is greatly enhanced by an interaction between the SH3 domain of p130Cas and a proline-rich sequence surrounding Pro-337 in the phosphatase (13). These data strongly suggest that a major role of PTP-PEST is to regulate the level of tyrosine phosphorylation of p130Cas within the cell.

p130Cas was initially identified as a major tyrosine-phosphorylated protein in cells transformed by the oncogenes v-crk (14, 15) and v-src (16, 17). In addition, p130Cas is rapidly phosphorylated following mitogenic stimulation by a wide variety of agonists (18-22), during B-cell activation via antigen receptor ligation (23, 24), and following cell attachment to fibronectin (25-29). Although these observations implicate tyrosine phosphorylation of p130Cas in the control of a variety of cellular processes, the precise role of p130Cas remains largely obscure. The structure of p130Cas suggests that it is involved in the formation of multiprotein complexes via several types of protein-protein interaction. Thus, p130Cas contains an SH3 domain, which has been shown to interact with proline-rich sequences in several proteins (13, 25, 30-32), a proline-rich segment, representing a potential SH3 domain binding sequence, a central domain with multiple copies of the motif YXXP which, when tyrosine-phosphorylated, represent potential binding sites for a variety of SH2 domains (33), and a C-terminal segment containing high affinity binding sites for the SH2 and SH3 domains of Src (34).

In order to investigate further the cellular function of PTP-PEST, we have generated Rat1 fibroblast lines that overexpress the wild-type phosphatase protein. These cells display a specific defect in p130Cas phosphorylation following cell attachment to fibronectin and exhibit a considerably reduced rate of migration. The data suggest that regulation of the tyrosine phosphorylation status of p130Cas by PTP-PEST is a critical control element of cell motility.

    EXPERIMENTAL PROCEDURES

Generation of Cell Lines-- cDNA encoding full-length PTP-PEST was subcloned into the retroviral transfer vector pWZL(hygro), from which gene expression is driven by the viral long terminal repeat promoter, and the hygromycin resistance gene is expressed from the same transcript via an internal ribosomal entry site. PTP-PEST-encoding retrovirus (as well as a control retrovirus containing empty pWZL(hygro) vector) was then generated by calcium phosphate-mediated transfection of the recombinant vector into the Bosc23 replication-incompetent ecotropic virus-packaging cell line. Specifically, 20 µg of vector DNA was added to a 50% confluent 10-cm diameter tissue culture plate of Bosc23 cells growing at 37 °C in 10 ml of Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum; 10 ml of fresh medium was then added 16 h after transfection. Virus-containing culture supernatants were harvested after a further 32 h of incubation, passed through a 0.45-µm syringe-loaded filter (Millipore), then added to 30% confluent cultures of Rat1 fibroblasts (5 ml of virus supernatant per 10-cm diameter plate of cells) in the presence of 4 µg/ml Polybrene (Sigma). An identical second round of infection was performed using fresh virus supernatant obtained by replenishing the medium of the transfected Bosc23 cells and incubation for a further 5 h at 37 °C. Fresh medium (DMEM with 5% fetal bovine serum) was added to the cells 16 h after the second round of infection, and the cells were grown for a further 24 h. Drug selection was initiated by splitting the infected population of cells into growth medium containing 100 µg/ml hygromycin B (Life Technologies, Inc.); the medium was changed every 2 days, and drug-resistant colonies were picked 10 days after the initial addition of hygromycin. All cell lines were routinely grown in DMEM containing 5% fetal bovine serum and 100 µg/ml hygromycin B. Expression of PTP-PEST was found to be stable over at least 40 passages.

Antibodies and Reagents-- Rabbit polyclonal (CSH8) and mouse monoclonal (AG10) antibodies to human PTP-PEST have been described previously (13). Antibodies to p130Cas (rabbit polyclonal B+F, mouse monoclonal 8G4 (35)) were provided by Dr. Amy Bouton (University of Virginia). Mouse monoclonal anti-phosphotyrosine antibody G104 was generated as described previously (11). Antibodies to paxillin (mouse monoclonal, clone 349), p130Cas (mouse monoclonal, clone 21), Crk (mouse monoclonal, clone 22), and Shc (rabbit polyclonal) were from Transduction Laboratories; antibody to p125FAK (rabbit polyclonal sc-558) was from Santa Cruz Biotechnology; mouse monoclonal antibody to vinculin (VIN-11-5) was from Sigma, polyclonal rabbit antibody specific for activated ERK1 and ERK2 MAP kinases was from Promega. Human plasma fibronectin was from Life Technologies, Inc., and polylysine and soybean trypsin inhibitor were from Sigma.

Cell Adhesion to Fibronectin-- Cells were grown to approx 80% confluence and then serum-starved for 16 h in DMEM with 0.5% fetal bovine serum in the absence of hygromycin. Removal of hygromycin from the medium was necessary to maintain cell viability during serum starvation and had no effect on the level of expression of PTP-PEST. The cells were removed from the plates by the addition of 0.05% trypsin, 0.53 mM EDTA and were subsequently suspended in serum-free DMEM containing 125 µg/ml soybean trypsin inhibitor (5 ml of medium per 10-cm diameter tissue culture dish). The cells were recovered by centrifugation, washed three times with serum-free DMEM, suspended in this medium at a concentration of 106 cells/ml, placed into tissue culture dishes on a slowly rotating platform (the dishes were pre-coated with 1% bovine serum albumin to prevent cell attachment at this point), and allowed to recover for 45 min at 37 °C. The resultant cell suspension was added to fibronectin-coated tissue culture dishes (5 × 106 cells per 10-cm diameter dish) or fibronectin-coated glass coverslips. Coating with fibronectin was achieved by incubation for 16 h at 4 °C in PBS containing 10 µg/ml fibronectin; excess matrix protein was then removed by rinsing with PBS. The coated surfaces were blocked with 1% bovine serum albumin in PBS for 1 h at 37 °C and washed twice in DMEM at 37 °C prior to addition of cells.

Cell Lysis, Immunoprecipitation, and Immunoblotting-- Cells were lysed for 30 min at 4 °C in 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 10 mM NaF, and insoluble material was removed by centrifugation at 4 °C for 10 min at 14,000 × g. Immunoprecipitation was performed on a rocking platform for 2 h at 4 °C using antibodies that were pre-coupled to 10 µl of protein A-Sepharose beads (Amersham Pharmacia Biotech). The beads were collected by a brief centrifugation (10 s at 5000 × g), washed three times with 1 ml of 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml of aprotinin, 10% glycerol, 0.5% Triton X-100, resuspended in SDS-PAGE sample buffer, and heated at 95 °C for 5 min. Immunoprecipitated proteins were then analyzed by SDS-PAGE and visualized by immunoblotting using horseradish peroxidase-coupled secondary antibodies and enhanced chemiluminescence reagents from Amersham Pharmacia Biotech.

Indirect Immunofluorescence-- Cells attached to glass coverslips were rinsed in PBS and fixed at room temperature for 15 min in 2% paraformaldehyde in PBS. The cells were then washed twice in PBS and solubilized on ice for 5 min in 0.2% Triton X-100, 3% goat serum in PBS. After washing three times at room temperature with PBS containing 3% goat serum, primary antibody solutions were added (in PBS containing 3% goat serum), and the samples were incubated for 1 h at room temperature. Excess antibody was removed by washing a further four times as above. Appropriate secondary antibodies conjugated to either fluorescein isothiocyanate or Texas Red (obtained from Cappel) were then added, and the samples were incubated for a further 30 min at room temperature. Finally, the samples were rinsed four times with PBS containing 3% goat serum and once with PBS, and the stained coverslips were mounted onto microscope slides using ProLong antifade reagent from Molecular Probes Inc. Samples were analyzed using a Zeiss Axiophot epifluorescence microscope; images were collected digitally using a CCD camera and image processing software from Oncor Image.

Cell Migration Assays-- Cell migration was assessed using a monolayer wound healing assay. Cells were first grown to confluence in plastic tissue culture dishes, and a wound was made in the cell monolayer using a sterile 1-ml pipette tip. Cell movement into the cleared area was then assessed by visual inspection of the cells during growth at 37 °C in DMEM containing 5% fetal bovine serum and 100 µg/ml hygromycin. Cells growing on coverslips were also subjected to wounding and allowed to recover for 16 h at 37 °C, at which time they were fixed and analyzed by indirect immunofluorescence as described above.

PTP-PEST Expression in v-Crk-transformed Cells-- 3Y1 rat fibroblast cells that stably express the v-Crk oncoprotein were grown in DMEM containing 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Human PTP-PEST cDNA was introduced into 3Y1v-Crk cells on glass coverslips by calcium phosphate-mediated transfection. The vector (PMT2) expresses PTP-PEST under the control of the adenovirus major late promoter. Transfected cells were allowed to grow for 2 days before fixation and immunofluorescence analysis.

    RESULTS

Fibroblasts Overexpressing PTP-PEST Exhibit a Specific Reduction in Phosphotyrosine Content of p130Cas-- We have previously demonstrated that PTP-PEST displays selectivity for tyrosine-phosphorylated p130Cas as a substrate both in vitro and in intact cells (11, 13), suggesting that a major function of PTP-PEST in vivo is to regulate the tyrosine phosphorylation state, and thus the function, of p130Cas. In order to identify the cellular processes that are controlled through regulation of the phosphorylation status of p130Cas by PTP-PEST, we generated Rat1 fibroblast lines that stably express human PTP-PEST protein and compared their properties with control cell lines into which an empty vector construct was introduced. Immunoblot analysis of cell lysates derived from these clones, using a monoclonal antibody (AG10) that recognizes a segment within PTP-PEST which is identical in the rat and human PTP-PEST proteins (residues 216-305), demonstrated that the level of PTP-PEST expression was increased by 5-10-fold in the PTP-PEST-overexpressing clones, compared with the endogenous levels in the vector control clones (Fig. 1, lower panels). Immunoblot analysis of these cell lysates with an anti-phosphotyrosine antibody indicated that the phosphorylation state of the majority of tyrosine-phosphorylated proteins present in Rat1 fibroblasts under normal growth conditions was not significantly affected by this level of overexpression of PTP-PEST (Fig. 1, upper panels). However, the phosphotyrosine content of a protein of Mr approx  130,000 was significantly reduced in lysates of PTP-PEST overexpressing cells. There was some variability in the Tyr(P) content of protein(s) of Mr approx  65,000-70,000. Thus, although two of the vector control clones (65 and 73) exhibited slightly higher Tyr(P) content at 65,000-70,000 when compared with the PTP-PEST-overexpressing cell lines, this difference was not observed in control clone 67 (Fig. 1, upper panels). In addition, this difference was not observed consistently in different lysates prepared from the same cell lines. The identity of this 65,000-70,000 protein is not known, but it appears not to be paxillin since direct analysis of the Tyr(P) content of paxillin demonstrated that its tyrosine phosphorylation was not altered following overexpression of PTP-PEST (Fig. 2).


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Fig. 1.   Effect of PTP-PEST overexpression on phosphotyrosine levels in Rat1 fibroblast cells. Aliquots (30 µg of protein) of lysates prepared from Rat1 fibroblast cells stably expressing PTP-PEST (right panels) or empty vector (left panels) were analyzed by SDS-PAGE and immunoblotting with antibodies to phosphotyrosine (monoclonal antibody G104, upper panels) or PTP-PEST (monoclonal antibody AG10, lower panels). Three clonal cell lines of each type were analyzed, as indicated by the numbers above the upper panels. The position of the arrow indicates the approximate molecular weight of tyrosine-phosphorylated p130Cas.


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Fig. 2.   Specific reduction in phosphotyrosine content of p130Cas in cells overexpressing PTP-PEST. Proteins were immunoprecipitated from aliquots (0.3 mg of protein) of lysates prepared from Rat1 fibroblast cells stably expressing PTP-PEST (right panels) or empty vector (left panels). The antibodies used for immunoprecipitation were as follows: rabbit polyclonal serum B+F (p130Cas), affinity purified rabbit polyclonal sc-558 (FAK), and mouse monoclonal antibody 349 (paxillin). In each case, the immunoprecipitated (IP) protein was divided into two equal aliquots, which were analyzed by SDS-PAGE and immunoblotting with either anti-phosphotyrosine antibody G104 (pY) or with the antibodies used for immunoprecipitation of the protein, as indicated.

Since PTP-PEST recognizes p130Cas as a substrate with a high degree of selectivity (11, 13), it appeared likely that the protein of Mr 130,000 in which we observed Tyr(P) content was reduced in PTP-PEST-overexpressing cells was p130Cas. This was confirmed by direct assessment of the Tyr(P) content of p130Cas following immunoprecipitation (Fig. 2). In contrast, neither the focal adhesion kinase p125FAK nor paxillin, which have been reported to associate in stable complexes with PTP-PEST (7), exhibited detectable alteration in Tyr(P) content (Fig. 2). Similarly, no differences were observed in the levels of phosphorylation of Shc (which has also been reported to bind to PTP-PEST under certain conditions (5, 6)) or of Src (data not shown). Similar observations were made in five different PTP-PEST-overexpressing cell lines. These results reinforce the previously described high degree of specificity of PTP-PEST for p130Cas and demonstrate, for the first time, that this specificity for p130Cas is maintained at physiological levels of tyrosine phosphorylation within the cell.

Analysis of Fibronectin-induced Signaling Events in PTP-PEST-expressing Cells-- Integrin-mediated cell adhesion to the extracellular matrix protein fibronectin rapidly induces a high level of tyrosine phosphorylation of p130Cas (25-28, 36, 37) suggesting a role for p130Cas in integrin signaling. Therefore, we compared various fibronectin-induced signaling processes in control and PTP-PEST overexpressing Rat1 fibroblasts. We observed that during attachment and initial spreading on fibronectin-coated plates PTP-PEST-overexpressing lines adhere to fibronectin at similar rates (cells attached within 10 min of replating) and displayed similar initial rates of spreading on fibronectin when compared with control cells (data not shown). In addition, the morphology of the spread cells was similar in both cell types, and immunofluorescence analysis demonstrated that there was no significant effect of PTP-PEST expression on the rapid accumulation of phosphotyrosine and of focal adhesion proteins, such as paxillin, into focal adhesion structures following cell attachment to fibronectin (Fig. 3). Under these conditions, very little accumulation of p130Cas in focal adhesion structures was observed. The majority of the p130Cas protein was localized to the cytoplasm, particularly in the perinuclear region, and there was no discernible difference between PTP-PEST-overexpressing and control cells (data not shown).


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Fig. 3.   Fibronectin-induced formation of focal adhesion complexes in cells overexpressing PTP-PEST. Cells overexpressing PTP-PEST (clone 50, lower panels) or empty vector (clone 73, upper panels) were serum-starved and attached to fibronectin-coated glass coverslips as described under "Experimental Procedures." Following incubation at 37 °C for 1 h the cells were fixed and stained with the appropriate antibodies for immunofluorescence analysis. The primary antibodies used were as follows: affinity purified rabbit polyclonal CSH8 (PTP-PEST), mouse monoclonal G104 (phosphotyrosine, pY), mouse monoclonal 349 (paxillin).

Anti-Tyr(P) immunoblots of lysates prepared from control and PTP-PEST-overexpressing fibroblasts during their attachment to fibronectin revealed patterns of Tyr(P)-containing proteins distinct from that observed in lysates of randomly growing cells (compare Fig. 4 upper panel with Fig. 1). This is because the fibronectin replating experiment was performed using serum-starved cells in the absence of soluble growth factors; these conditions result in a reduction in the level of tyrosine phosphorylation of many proteins within these cells in comparison to that observed in samples derived from cells grown in the presence of serum. Detachment of serum-starved fibroblasts and incubation in suspension resulted in extensive dephosphorylation of the majority of tyrosine-phosphorylated proteins present in the initial attached population of cells, regardless of the expression level of PTP-PEST within the cells. Replating these cells onto fibronectin-coated dishes rapidly (within 20 min) induced the tyrosine phosphorylation of a similar array of proteins in both cell types, with the exception of a prominent tyrosine-phosphorylated protein of Mr approx  130,000 which was significantly reduced in intensity in lysates of cells overexpressing PTP-PEST (Fig. 4, upper panels). We identified this 130-kDa protein as p130Cas by immunoprecipitation with a p130Cas-specific antibody, followed by anti-Tyr(P) immunoblotting (Fig. 5). Since the initial response of cells to fibronectin was largely unchanged in cells overexpressing PTP-PEST, these observations suggest that even though p130Cas is one of the major Tyr(P)-containing proteins induced by cell attachment to fibronectin, tyrosine phosphorylation of p130Cas may be dispensable for the majority of initial fibronectin-induced cellular events, including cell attachment and spreading.


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Fig. 4.   Fibronectin-induced protein tyrosine phosphorylation and MAP kinase activation in cells overexpressing PTP-PEST. Cells overexpressing PTP-PEST (clone 50, right panels) or empty vector (clone 73, left panels) were serum-starved (lanes A), suspended (lanes S), seeded onto fibronectin-coated tissue culture plates, and incubated at 37 °C for the indicated times (in min) as described under "Experimental Procedures." Lysates prepared from these cells were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody (pY) G104 (upper panels) or with a polyclonal antibody specific for the phosphorylated, active forms of p44 and p42 MAP kinase (ERK 1 and 2) (lower panels). The position of the arrow (upper panels) indicates the approximate molecular weight of tyrosine-phosphorylated p130Cas.


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Fig. 5.   Effect of PTP-PEST overexpression on fibronectin-stimulated tyrosine phosphorylation of p130Cas. Cells overexpressing PTP-PEST (clone 50) or empty vector (clone 73) were serum-starved, suspended in serum-free medium (lanes S), and then seeded onto fibronectin-coated tissue culture plates and incubated at 37 °C for 60 min (lanes FN). A, proteins were immunoprecipitated from aliquots (0.5 mg of protein) of lysates prepared from these cells, using the same antibodies as in Fig. 2. In each case, the immunoprecipitated protein (IP) was divided into two equal aliquots, which were analyzed by SDS-PAGE and immunoblotting with either anti-phosphotyrosine antibody G104 (pY, left panels) or with the antibodies used for immunoprecipitation of the protein (right panels) as indicated. B, aliquots of the same lysates (0.8 mg of protein) were also analyzed by immunoprecipitation using a monoclonal Crk antibody (clone 22) (upper panels). The immunoprecipitated protein was divided into two equal aliquots, which were analyzed by SDS-PAGE and immunoblotting with antibodies to p130Cas (monoclonal 8G4, left panels) or with the Crk antibody (right panels). Aliquots of the cell lysates were also immunoblotted with the Crk and p130Cas antibodies to ensure that all lysates contained similar concentrations of the two proteins (lower panels).

Since paxillin and p125FAK constitute two of the major Tyr(P)-containing proteins present in fibroblasts following adhesion to fibronectin, the phosphorylation state of these proteins was also analyzed directly by anti-Tyr(P) immunoblotting of the immunoprecipitated proteins (Fig. 5). The extent of fibronectin-stimulated tyrosine phosphorylation of both paxillin and p125FAK was unchanged in cells expressing PTP-PEST compared with the control cells (Fig. 5), further demonstrating the high degree of specificity of PTP-PEST for p130Cas.

Many previous studies have suggested that the adaptor protein Crk is a major downstream effector of tyrosine-phosphorylated p130Cas following fibronectin stimulation. The sequence of p130Cas contains 15 copies of the motif YXXP (38) which, when tyrosine-phosphorylated, corresponds to the consensus site for binding of the Crk SH2 domain (33). Furthermore, cell attachment to fibronectin has been shown to result in the formation of stable complexes between tyrosine-phosphorylated p130Cas and the SH2 domain of Crk (24, 27, 36). Therefore, we investigated the formation of Crk-p130Cas complexes in fibronectin-stimulated control and PTP-PEST overexpressing cells by immunoprecipitation of the Crk protein, followed by immunoblotting the precipitates with a p130Cas antibody. Lysates prepared from fibronectin-stimulated control (vector-expressing) cells contained significantly higher levels of stable complexes between Crk and p130Cas than lysates prepared from PTP-PEST-overexpressing cells; no Crk-p130Cas complexes were detected in lysates prepared from cells held in suspension (Fig. 5B). These results indicate that PTP-PEST is capable of regulating Crk-dependent, fibronectin-induced signaling events downstream of p130Cas, via reducing the level of tyrosine phosphorylation of Crk SH2 domain binding sites on p130Cas.

In addition to the rapid induction of protein tyrosine phosphorylation that occurs in cells during their attachment to fibronectin, ERK1 and ERK2 MAP kinases are concomitantly activated, rapidly and transiently, under these conditions (26, 39-41). The function of activated MAP kinases in integrin-induced cellular processes is currently unclear, as is the mechanism whereby integrin receptor ligation leads to their activation. One potential mechanism is via binding of the adaptor protein Grb2 to Tyr(P)-925 on p125FAK, recruitment of Grb2-associated SOS (a potent guanine nucleotide exchange factor for Ras) to focal adhesion complexes containing tyrosine-phosphorylated p125FAK, and subsequent activation of Ras (39, 42, 43). However, several reports now suggest alternative pathways for activation of ERK1/ERK2 MAP kinases in many cell types (44-46). These include the formation of signaling complexes based on tyrosine-phosphorylated p130Cas, via the binding of Crk or Nck adaptor proteins and recruitment of SOS (36, 37, 47). Therefore, we compared fibronectin-induced activation of MAP kinase in control and PTP-PEST-expressing fibroblasts to investigate the possible involvement of p130Cas tyrosine phosphorylation. Both cell types responded to fibronectin attachment by rapidly (within 10-20 min) and transiently activating ERK1/ERK2 (Fig. 4, lower panels); the level of activation was found to be similar to that observed following serum stimulation (data not shown). The rate and extent of MAP kinase activation were similar in both cell types indicating that, at least in Rat1 fibroblasts, MAP kinase activation in response to fibronectin attachment is largely independent of p130Cas tyrosine phosphorylation. Furthermore, this observation demonstrates that overexpression of PTP-PEST in fibroblasts affects only a limited subset of fibronectin-induced signaling pathways.

Effect of PTP-PEST Expression on Cell Motility-- As outlined above, ectopic expression of PTP-PEST in Rat1 fibroblasts had no apparent effect on the majority of the initial cellular events induced by fibronectin, even though p130Cas exhibited a markedly reduced level of tyrosine phosphorylation. However, many important integrin receptor-mediated cellular processes, including the movement of cells across the extracellular matrix, occur over an extended time course; such processes are also potentially influenced by fibronectin-induced tyrosine phosphorylation events and might therefore be significantly affected by PTP-PEST overexpression. We assessed the motility of the PTP-PEST expressing Rat1 fibroblasts by visual inspection of wound closure following wounding of a cell monolayer. Cells overexpressing PTP-PEST were found to migrate into the wound much less rapidly than control, vector-expressing cells (Fig. 6A). Thus, control cells migrated into the center of the cleared area to close the wound within 24 h, whereas PTP-PEST-overexpressing cells required 3-4 days to migrate a similar distance. This difference in cell motility was observed in all clones examined, i.e. three vector control clones and three PTP-PEST overexpressing clones (Fig. 6B), and in five independent experiments.


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Fig. 6.   Effect of PTP-PEST overexpression on cell motility. Cells overexpressing PTP-PEST (clone 50, right panels) or empty vector (clone 73, left panels) were grown to confluence in tissue culture dishes (A), and an area (approximately 2 mm wide) was cleared of cells by scraping with a sterile pipette tip. The cells were then photographed immediately (upper panels) or following incubation in growth medium for 24 h at 37 °C (lower panels). B, results of cell motility assays were quantitated from the resultant photographs by counting the number of cells present after 24 h within areas corresponding to cell migration distances of 0.2-0.6 mm and 0.6-1.0 mm. Results shown represent the average number of cells per mm2 counted at the indicated distances from the edge of the initial wound; quadruplicate cell counts were performed at each distance per clone, and error bars represent standard deviations. Results are representative of 5 independent experiments. Shaded bars represent vector control clones; striped bars represent PTP-PEST-overexpressing clones. C, wounds were made in cell monolayers growing on glass coverslips, and the cells were incubated in growth medium for 15 h at 37 °C, fixed, and stained for immunofluorescence using antibodies to p130Cas (rabbit polyclonal B+F, upper panels) and vinculin (monoclonal VIN 11-5, lower panels).

The potential involvement of p130Cas in controlling cell motility was investigated further by indirect immunofluorescence analysis of cells during their migration into a wound. Staining of vector control cells with appropriate antibodies revealed that p130Cas, as well as the focal adhesion proteins paxillin, vinculin, and p125FAK, was localized to the leading edge of migrating cells in lamellar structures (Fig. 6C). This subcellular location is clearly consistent with the idea that p130Cas plays a role in controlling cell movement across the extracellular matrix. In contrast, p130Cas was found throughout PTP-PEST expressing cells, with prominent staining in the perinuclear region and very little of the protein discernible at the cell periphery. Furthermore, in these cells vinculin and paxillin were localized in focal adhesion structures, with no significant accumulation at the leading edge of the cell (Fig. 6C). These observations suggest that the ability of PTP-PEST to interfere with cell motility may result from its ability to prevent the redistribution of p130Cas from a cytoplasmic location to a specific cytoskeletal location, namely the leading edge of the cell, in cells that are migrating across the matrix. This inhibition of p130Cas redistribution by PTP-PEST presumably results from the reduced level of tyrosine phosphorylation of p130Cas observed in PTP-PEST-overexpressing cells, since several studies have highlighted the involvement of p130Cas tyrosine phosphorylation in the redistribution of p130Cas from soluble to insoluble fractions of cell lysates (38, 48, 49). Thus, by dephosphorylating p130Cas, PTP-PEST can prevent its association with appropriate Tyr(P)-dependent binding partners, including the Crk adaptor protein (Fig. 5B), and these Tyr(P)-dependent interactions appear to be essential for the efficient recruitment of p130Cas to the leading edge of the migrating cell.

PTP-PEST Affects the Subcellular Location of p130Cas in v-Crk-transformed Fibroblasts-- The ability of PTP-PEST to influence the function of p130Cas by affecting its subcellular location was analyzed further by transient transfection of PTP-PEST into v-Crk-transformed rat fibroblasts. In these transformed cells p130Cas is highly tyrosine-phosphorylated and is predominantly localized to prominent focal adhesion structures (Fig. 7, upper panels). In contrast, in cells overexpressing PTP-PEST, p130Cas displays a diffuse cytoplasmic staining pattern, with little detectable staining of focal adhesions (Fig. 7, lower panels). This effect of PTP-PEST appears to be selective for the p130Cas protein since the location of paxillin within the cell was unaffected by PTP-PEST overexpression. Thus, paxillin was predominantly observed in focal adhesion structures regardless of the level of PTP-PEST expression within the cell (Fig. 7). These results demonstrate that PTP-PEST is capable of overcoming the potent effects of the v-Crk oncoprotein on p130Cas and support the idea that PTP-PEST controls specifically the phosphorylation state, and thereby the subcellular location, of the p130Cas protein.


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Fig. 7.   Effect of PTP-PEST overexpression on the subcellular location of p130Cas in v-Crk-transformed fibroblasts. cDNA encoding PTP-PEST was transfected into 3Y1v-Crk rat fibroblasts growing on glass coverslips, and the cells were allowed to grow for 48 h before fixation followed by immunofluorescence analysis using antibodies to PTP-PEST (affinity purified rabbit polyclonal antibody CSH8), p130Cas (mouse monoclonal, clone 21), and paxillin (mouse monoclonal, clone 349). Cells shown are representative either of transfected cells that overexpress PTP-PEST (lower panels) or of cells from the same coverslip that were not transfected (upper panels).


    DISCUSSION

The results described in this paper suggest that PTP-PEST is a regulator of integrin-mediated movement of fibroblasts across the extracellular matrix via its regulation of the tyrosine phosphorylation state of p130Cas. Although previous reports have suggested a potential involvement of p130Cas in cell motility (50-52), our data are the first to reveal a requirement for tyrosine phosphorylation of p130Cas in this process. The precise role of p130Cas in controlling cell movement is not clear. However, data recently obtained from mouse embryos lacking the p130Cas protein suggest that it is involved in organization of the actin cytoskeleton in fibroblasts and cardiocytes (53).

Integrin-mediated cell movement across the extracellular matrix is a highly dynamic process requiring continual assembly and disassembly of focal contacts, together with extensive remodeling of the actin cytoskeleton (54-57). Many of the proteins found in focal contacts, including p130Cas, are highly tyrosine-phosphorylated; in fact, focal contacts represent one of the major sites of tyrosine phosphorylation within the cell, as assessed by immunofluorescence analysis using anti-Tyr(P) antibodies (58) (see Fig. 3). Furthermore, at least two protein tyrosine kinases, p125FAK and Src, are localized to focal contacts (59, 60), and both kinases have been implicated in the control of cell movement (52, 61-63). These observations have led to the suggestion that tyrosine phosphorylation plays a significant role in controlling the assembly and disassembly of focal adhesion complexes, thereby regulating cell motility. However, the control of cell movement is a reversible, dynamic process suggesting that its regulation by tyrosine phosphorylation must involve both kinases and phosphatases. Our results suggest that the protein tyrosine phosphatase PTP-PEST regulates cell motility by modulating specifically the extent of tyrosine phosphorylation of p130Cas.

The observation that p130Cas is localized at the leading edge of migrating fibroblasts suggests a model for the observed effects of PTP-PEST expression on cell movement. By limiting the accumulation of tyrosine-phosphorylated p130Cas, the ability of p130Cas to interact with appropriate SH2 domain-containing binding partners is impaired in cells overexpressing PTP-PEST. It appears likely that some of these interactions are essential for targeting tyrosine-phosphorylated p130Cas to the correct cytoskeletal location. Thus, it has been shown that the SH2 domain of v-Crk is required for the v-Crk-induced translocation of tyrosine-phosphorylated p130Cas to a detergent-insoluble fraction (48). Also, in p130Cas-overexpressing COS cells, fibronectin-induced relocation of p130Cas to focal adhesion complexes requires both the SH3 domain of p130Cas (which can bind directly to the focal adhesion kinase p125FAK (25, 30, 49)) and a C-terminal segment which contains binding sites for the SH2 and SH3 domains of Src (34, 64). Our observation that cells overexpressing PTP-PEST display a defect in the formation of complexes between p130Cas and Crk following fibronectin stimulation (Fig. 5B) suggests that such complexes are involved in regulating cell movement. Indeed, recent evidence obtained using COS cells transfected with mutant forms of Crk and p130Cas suggested an involvement of p130Cas-Crk complexes in COS cell migration on vitronectin-coated membranes (50).

Recently, p130Cas-/- fibroblasts have been reported to display a defect in their response to an activated, transforming variant of the Src tyrosine kinase; they fail to form colonies in soft agar, a defect that can be rescued by re-expression of p130Cas (53). These results suggest that the involvement of p130Cas in regulating the organization of actin cytoskeleton structures may also influence the response of cells to transforming oncogenes, a suggestion that is supported by several earlier observations implicating a direct regulatory role for p130Cas in cellular transformation (65-67). These data further suggest that PTP-PEST may play a key role in suppressing cellular transformation through its ability to dephosphorylate p130Cas. Indeed, such a role is supported by our observation that overexpression of PTP-PEST dramatically influences the location of p130Cas within v-Crk-transformed cells, suggesting that PTP-PEST potentially influences the response of cells to a variety of transforming oncogenes.

The results in this paper represent the first description of the physiological role of PTP-PEST. The data suggest that PTP-PEST is involved in controlling the rate of cell movement via the dephosphorylation of p130Cas. Our results also provide new insight into the function of p130Cas in integrin-mediated processes. Thus, it appears that tyrosine phosphorylation of p130Cas is largely dispensable for many of the cellular responses to fibronectin, including cell attachment, cell spreading, and MAP kinase activation. However, tyrosine phosphorylation of p130Cas is required for the efficient movement of cells across the extracellular matrix and therefore is likely to represent an important mechanism for regulating the dynamic process of cell migration in vivo.

    ACKNOWLEDGEMENTS

We thank Kim Pennino for assistance in characterizing the G104 monoclonal anti-phosphotyrosine antibody used in this work; Amy Bouton (University of Virginia) for p130Cas antibodies, Rat1 fibroblast and 3Y1v-Crk fibroblast cells; and Scott Lowe (Cold Spring Harbor Laboratory) for providing the pWZL(hygro) vector and Bosc23 cell line.

    FOOTNOTES

* This work was funded by Grant CA 53840 from the National Institutes of Health.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.

Dagger Current address: OSI Pharmaceuticals Inc., 106 Charles Lindbergh Blvd., Uniondale, NY 11553-3649.

§ To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724-2208. Tel.: 516-367-8846; Fax: 516-367-6812; E-mail:tonks{at}cshl.org.

The abbreviations used are: PTP, protein tyrosine phosphatase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein.
    REFERENCES
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Abstract
Introduction
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