Low molecular weight protein-tyrosine phosphatase controls the rate and the strength of NIH-3T3 cells adhesion through its phosphorylation on tyrosine 131 or 132.

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement. Our previous results demonstrated that LMW-PTP is able to bind and dephosphorylate activated PDGF receptor, thus inhibiting cell proliferation. Recently we have shown that LMW-PTP is specifically phosphorylated by c-Src in a cytoskeleton-associated fraction in response to PDGF, and this phosphorylation increases LMW-PTP activity about 20-fold. LMW-PTP strongly influences cell adhesion, spreading, and chemotaxis induced by PDGF stimulation, by regulating the phosphorylation level of p190Rho-GAP, a protein that is able to regulate Rho activity and hence cytoskeleton rearrangement. In the present study we investigate the physiological role of the two LMW-PTP tyrosine phosphorylation sites, using LMW-PTP mutants on tyrosine 131 or 132. We demonstrate that each tyrosine residue is involved in specific LMW-PTP functions. Both of them are phosphorylated during PDGF signaling. Phosphorylation on tyrosine 131 influences mitogenesis, dephosphorylating activated PDGF-R and cytoskeleton rearrangement, acting on p190RhoGAP. Phosphorylation on tyrosine 132 leads to an increase in the strength of cell substrate adhesion, down-regulating matrix metalloproteases expression, through the inhibition of Grb2/MAPK pathway. In conclusion, LMW-PTP tyrosine phosphorylation on both Tyr(131) or Tyr(132) cooperate to determine a faster and stronger adhesion to extracellular matrix, although these two events may diverge in timing and relative amount.

Protein tyrosine phosphorylation plays a key role in the regulation of many cellular processes in eukaryotes such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). Accumulating evidence indicates that the contribution of protein-tyrosine phosphatases (PTPs) 1 to the control of cell phosphorylation state is as relevant as that of phosphotyrosine protein kinases. The PTPs superfamily is composed of over 70 enzymes that, despite very limited sequence similarity, share a common active site motif CX 5 R and an identical catalytic mechanism. On the basis of their function, structure, and sequence, PTPs can be classified in four main families: 1) tyrosine specific phosphatases, 2) VH1-like dual specificity PTPs, 3) the cdc25, and 4) the low molecular weight phosphatase (2).
The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an 18-kDa enzyme that is expressed in many mammalian tissues (3). Our previous studies on the molecular biology of LMW-PTP in NIH-3T3 cells evidenced a well defined role of this enzyme in PDGF-induced mitogenesis. The most relevant phenotypic effect of LMW-PTP overexpression was the strong reduction of cell growth rate in response to PDGF stimulation. We have previously shown that activated PDGF-R is a LMW-PTP substrate (4) and that LMW-PTP is involved in the control of specific pathways triggered by PDGF-R activation. In particular, LMW-PTP is able to modulate both myc expression, interfering with Src pathway, and fos expression through a MAPK-independent pathway mediated by the STAT proteins (5). More recently, we have found that in NIH-3T3 cells LMW-PTP is constitutively localized in both cytoplasmic and cytoskeleton-associated fractions. These two LMW-PTP pools are differentially regulated because only the cytoskeleton-associated LMW-PTP fraction is specifically phosphorylated by c-Src after PDGF stimulation (6). As a consequence of its phosphorylation, LMW-PTP shows an average 20-fold increase in its in vitro catalytic activity (7) instead of the 2-fold activation that was previously reported (8,9).
Furthermore, we have shown that the cytoskeleton-associated LMW-PTP influences cell adhesion, migration, and spreading and that these effects are dependent on LMW-PTP tyrosine phosphorylation (10). We have shown that cytoskeleton-associated LMW-PTP influences the phosphorylation state of p190Rho-GAP, a protein that is able to regulate Rho activity and, hence, cytoskeleton rearrangement in response to PDGF stimulation. In addition, we have demonstrated that, in vivo, LMW-PTP is regulated by c-Src phosphorylation, because phosphorylated LMW-PTP presents an increased activity on a physiologic substrate such as p190Rho-GAP (10). We suggest that LMW-PTP is able to perform multiple roles in PDGF-induced mitogenesis. In fact, cytosolic LMW-PTP binds and dephospho-rylates PDGF-R (4), thus modulating part of its signaling cascade, whereas cytoskeleton-associated LMW-PTP acts on phosphorylated p190Rho-GAP consequently playing a role in PDGF-mediated cytoskeleton rearrangement (10).
We have previously demonstrated that, in vitro, the tyrosine phosphorylation of LMW-PTP by c-Src is directed to both tyrosine 131 and 132, although they appear to have different effects: tyrosine 131 phosphorylation determines a strong increase in LMW-PTP specific activity, whereas phosphorylation of tyrosine 132 leads to in vitro Grb2 binding (7). The relative in vivo contribution of each tyrosine phosphorylation remained to be determined. In this paper we analyze the role of Tyr 131 and Tyr 132 LMW-PTP mutants in cell growth, migration, and adhesion to determine the specific function of the two phosphorylation sites. Our findings suggest that, in vivo, both LMW-PTP tyrosines are efficiently phosphorylated during PDGF signaling, leading to different effects. Phosphorylation on Tyr 131 , leads to the regulation of Rho-mediated cell adhesion rate through the dephosphorylation of p190Rho-GAP. On the other side, the phosphorylation on Tyr 132 leads to an increase in the strength of cell adhesion through the down-regulation of MMPs expression, probably through the inhibition of the Grb2/MAPK pathway.

EXPERIMENTAL PROCEDURES
Materials-Unless specified all reagents were obtained from Sigma. NIH-3T3 cells were purchased from American Type Culture Collection; human recombinant platelet-derived growth factor (PDGF) BB was from Peprotech; the enhanced chemiluminescence kit was from Amersham Pharmacia Biotech; all antibodies were from Santa Cruz, except those against phosphoMAPKs that were from New England Biolabs. BCA protein assay reagent is from Pierce, and PP1 Src family kinase inhibitor was from BIOMOL Research Laboratories, Inc.
Site-specific Mutagenesis and Cloning of LMW-PTP Mutants in Eukaryotic Expression Vector-Oligonucleotide-directed mutagenesis was performed using the Unique Restriction Elimination Site kit of Amersham Pharmacia Biotech. The 26-base-long target mutagenesis primer contains an ACA codon (alanine) substituting the original TAT codon (tyrosine) for each tyrosine. The mutated LMW-PTP coding sequence was completely sequenced by the Sanger method and subcloned in the HindIII and ApaI restriction sites of pRcCMV eukaryotic expression vector, harboring the neomycin resistance gene.
Cell Culture and Transfections-NIH-3T3 cells were routinely cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum (FCS) in 5% CO 2 humidified atmosphere. 10 g of pRcCMV-wtLMW-PTP or pRcCMV-Y131A-LMW-PTP or pRcCMV-Y132A-LMW-PTP or pRcCMV-Y131A/Y132A-LMW-PTP were transfected in NIH-3T3 cells using the calcium phosphate method. Stable transfected clonal cell lines were isolated by selection with G418 (400 g/ml). Control cell lines were obtained by transfecting 2 g of pRcCMVneo alone. The clonal lines were screened for expression of the transfected genes by 1) Northern blot analysis and 2) enzyme-linked immunosorbent assay using polyclonal anti-LMW-PTP rabbit antibodies, which do not cross-react with murine endogenous LMW-PTP.
Cell Growth Assay-2 ϫ 10 4 NIH-3T3 cells were seeded in 24-multiwell plates and serum starved for 24 h before receiving 30 ng/ml PDGF-BB for 24 and 48 h. Fresh PDGF was added daily. Cellular growth was stopped by removing the medium and by the addition of a 0.5% crystal violet solution in 20% methanol. After 5 min of staining the fixed cells were washed with PBS and solubilized with 200 l/well of 0.1 M sodium citrate, pH 4.2. The absorbance at 595 nm was evaluated using a microplate reader.
Immunoprecipitation and Western Blot Analysis-1 ϫ 10 6 cells were seeded in 10-cm plates in Dulbecco's modified Eagle's medium supplemented with 10% FCS. Cells were serum starved for 24 h before receiving 30 ng/ml PDGF-BB. Cells were then lysed for 20 min on ice in 500 l of RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethanesulphonylfluoride, 10 g/ml aprotinin, 10 g/ml leupeptin). Lysates were clarified by centrifugation and immunoprecipitated for 4 h at 4°C with 0.1 g of the specific antibodies. Immune complexes were collected on protein A-Sepharose (Amersham Pharmacia Biotech), separated by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose (Sartorius). Immunoblots were incubated in 3% bovine serum albumin, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, and 0.1% Tween-20 for 1 h at room temperature, probed first with specific antibodies and then with secondary antibodies conjugated with horseradish peroxidase, washed, and developed with the enhanced chemiluminescence kit.
Cell Lysate Fractionations-Cell lysate fractions were obtained as already described (6). Briefly, PDGF-stimulated NIH-3T3 were lysed in RIPA buffer, and the lysates were clarified by centrifugation for 30 min at 20000 ϫ g. Pellets were washed twice with 1 ml RIPA and then resuspended in complete RIPA buffer, which is RIPA buffer plus 0.5% sodium deoxycholate and 0.1% sodium dodecilsulphate, by shaking for 1 h at room temperature and newly clarified by centrifugation at 20,000 ϫ g for 30 min. RIPA or complete RIPA buffer fractions were then used for immunoprecipitation analysis.
Cell Adhesion Assay-Cell adhesion was assessed as described elsewhere (11). Briefly, 3 ϫ 10 4 cells are seeded for the indicated time in a 96-well dish precoated for 2 h with 10 g/ml of human fibronectin and washed twice with PBS. Cell adhesion was stopped by removing the medium and by the addition of a 0.5% crystal violet solution in 20% methanol. After 5 min of staining the fixed cells were washed with PBS and solubilized with 200 l/well of 0.1 M sodium citrate, pH 4.2. The absorbance at 595 nm was evaluated using a microplate reader. The adhesion assay was performed in complete medium. All cell adhesion assays were performed in triplicate.
Cell Motility Assay-Cell migration was assessed as described elsewhere with minor modifications (5). Migration of NIH-3T3 cells was assayed with the Transwell system of Costar, equipped with 8-m pore polyvinylpirrolidone-free polycarbonate filters (diameter, 13 mm) precoated with human type I collagen (20 g/ml) and placed between the chemoattractant (lower chamber) and the upper chamber. The lower chamber was filled with medium supplemented with 10 ng/ml of PDGF-BB. Serum-free Dulbecco's modified Eagle's medium cultured cells were suspended by trypsinization, and 1.5 ϫ 10 5 cells in 200 l were added to the top wells and incubated at 37°C in 5% CO 2 for 6 h. After incubation, the cells attached to the upper side but not migrated through the filter were mechanically removed using cotton swabs. The filters were fixed in 96% methanol and stained with Diff Quick staining solutions. Chemotaxis was evaluated by counting the cells that had migrated to the lower surface of the polycarbonate filters. For each filter the number of cells in six randomly chosen fields was determined, and the counts were averaged (mean Ϯ S.D.).
Cell Detachment Assay-The strength of cell adhesion was measured as reported elsewhere (12). Briefly, confluent monolayers of cells treated or not with 0.5 M of PP1 Src family kinase inhibitor were washed twice with PBS and then incubated for 10 min at 37°C in calcium and magnesium free PBS containing 0.05% trypsin. 0.5 mg/ml soybean trypsin inhibitor was added, and the cells were incubated at 37°C for 5 min. The dishes were washed twice with PBS, and the attached cells were evaluated by crystal violet staining. After 5 min of staining the fixed cells were washed with PBS and solubilized with 200 l/well of 0.1 M sodium citrate, pH 4.2. The absorbance at 595 nm was evaluated using a microplate reader. The value in ordinate (see Fig. 5) is the ratio between the cells attached after and before the treatment.
Zymography-Metalloprotease zymography was assessed as described elsewhere with minor modifications (13). Briefly, culture medium from confluent cell monolayers treated or not with 0.5 M of PP1 Src family kinase inhibitor was collected, and 20 l were added to sample buffer (SDS 0.4%, 2% glycerol, 10 mM Tris-HCl, pH 6.8, 0.001% bromphenol blue). The sample were run on a 8% SDS gel containing 0.1% gelatin. After electrophoresis the gel was washed twice with 2.5% Triton X-100 and once with reaction buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl 2 . The gel was incubated overnight at 37°C with freshly added reaction buffer and stained with Laemli Comassie blue solution.
MAPK Activation-3 ϫ 10 4 cells were plated on a 6-well dish in complete medium. After 48 h the cells were washed twice in PBS, lysed in RIPA buffer, and centrifuged to remove the insoluble debris. The total protein content was evaluated by the BCA protein assay and 20 g of lysates were resolved on a 12% SDS-polyacrylamide gel electrophoresis. The resolved protein were transferred to nitrocellulose and probed overnight with anti-phospho-p44/42 MAPK monoclonal antibodies.
PTP Activity Assay-The PTP activity was measured as previously reported (7). Briefly, 1,5 ϫ 10 6 cells were collected in 300 l of 0.1 M sodium acetate, pH 5.5, 10 mM EDTA, 1 mM ␤-mercaptoethanol and sonicated for 10 s. The lysates were clarified by centrifugation, and 50 l were used in the PTP activity assay with 50 l of 10 mM p-nitrophenylphosphate at 37°C for 30 min. The production of p-nitrophenol was measured colorimetrically at 410 nm. The results were normalized on the basis of total protein content.

Expression of Y131A and Y132A LMW-PTP Mutant in NIH-3T3 Cells-To study the role of LMW-PTP phosphorylation on
Tyr 131 and Tyr 132 , we generated by site specific mutagenesis two LMW-PTP tyrosine to alanine single mutants (Y131A and Y132A). We overexpressed these LMW-PTP mutants in NIH-3T3 cells, selecting two different clones for each mutant for further experiments (Y131A, clones P and M; Y132A, clones X and XII). Because we have previously demonstrated that LMW-PTP exists in cytoplasmic and cytoskeleton associated distinct pools, we analyzed the subcellular distribution and the tyrosine phosphorylation of the mutant forms of LMW-PTP. As far as the double mutant Y131A/Y132A-LMW-PTP is concerned, we have already reported that it is targeted to cytoskeleton but not tyrosine phosphorylated upon PDGF treatment (10). The results show that both mutants are efficiently targeted to cytoskeleton (Fig. 1A) and differently phosphorylated on tyrosine (Fig. 1B). This finding indicates that, in vivo, both tyrosine 131 and 132 are efficiently phosphorylated in NIH-3T3 cells after PDGF stimulation. As we reported previously, the activity on p-nitrophenylphosphate of Y131A and Y132A LMW-PTP mutants was 40 and 70%, respectively, with respect to wtLMW-PTP. The LMW-PTP, which is a C12A mutant, is totally inactive (7). Because the tyrosine phosphorylation level of cytoskeleton-associated LMW-PTP seems to be dependent on LMW-PTP catalytic activity, we suggest that LMW-PTP could undergo autodephosphorylation in vivo.
Furthermore, to exclude the possibility that the LMW-PTP mutants are inactive in vivo, we quantitated their catalytic activity in NIH-3T3 cells. The results are very similar to those obtained in vitro; taken as 100% the catalytic activity of the wtLMW-PTP, we obtained about 40 and 90% for Y131A-LMW-PTP and Y132A-LMW-PTP, respectively. These findings confirm that in vivo both LMW-PTP single tyrosine mutants are functional phosphatases, at least on synthetic substrates such as p-nitrophenylphosphate.
Effect of Single Tyrosine Mutants of LMW-PTP in PDGFinduced Mitogenesis-We analyzed the serum-induced cell growth rate of Y131A-LMW-PTP and Y132A-LMW-PTP transfected cells in comparison with wtLMW-PTP and mock transfected cells (Fig. 2A). The results reveal that the Y132A-LMW-PTP, which maintains the possibility of phosphorylation on tyrosine 131, induces a decrease in cell growth rate, similar to the wtLMW-PTP, with respect to mock transfected cells. On the contrary, the Y131A-LMW-PTP, which maintains only the tyrosine in position 132, determines a slight increase in the cell growth rate with respect to mock transfected cells. Phosphorylation on Tyr 131 but not on Tyr 132 gives rise to a behavior similar to the wtLMW-PTP, suggesting that only the phosphorylation of LMW-PTP in position 131 mediates the inhibitory effect of this enzyme on serum-induced mitosis. We analyzed the tyrosine phosphorylation level of PDGF-R in Y131A-NIH-3T3 and Y132A-NIH-3T3 cells in comparison with wtLMW-PTP and mock transfected cells. The result, shown in Fig. 2B, demonstrates that although the Y132A mutant behaves as wtLMW-PTP, dephosphorylating the PDGF-R even more efficiently than the wild type enzyme, the Y131A mutant appears to be inefficient in this action. To determine whether the mutation of tyrosine 132 impairs the PDGF-R binding of LMW-PTP or only its ability to dephosphorylate the receptor, we coimmunoprecipitated LMW-PTP and the PDGF-R in all cell lines. We were not able to detect any interaction between the activated PDGF-R and Y131A-LMW-PTP (data not shown). Taken together, these findings suggest that both tyrosine 131 and 132 are phosphorylated in vivo upon PDGF administration, but these events lead to different LMW-PTP effects on mitosis regulation and PDGF receptor activation.
Role of LMW-PTP Tyr(P) 131 in the Regulation of Rho-dependent Cytoskeleton Rearrangement-wtLMW-PTP overexpression induces a marked increase in integrin-mediated cell adhesion rate and PDGF-induced migration (10). To analyze the role of tyrosine phosphorylation on Tyr 131 or Tyr 132 in this phenomenon, we evaluated cell adhesion on fibronectin of cells expressing wtLMW-PTP or the two single tyrosine mutants in comparison with mock transfected cells. Fig. 3A shows that Y131A mutant behaves similarly to mock transfected cells. On the contrary, the Y132A mutant leads to a marked potentiation of fibronectin-mediated cell adhesion rate. This effect is even stronger than in wtLMW-PTP. In addition we evaluated the chemotaxis induced by PDGF-BB in all cell lines. Fig. 3B shows that again the phosphorylation in Tyr 131 mainly mediates the increase of cell chemotaxis toward PDGF induced by LMW-PTP overexpression.
LMW-PTP is involved in Rho-mediated cytoskeleton rearrangement such as chemotaxis and cell adhesion, as it specifically dephosphorylates the GTPase activating protein of Rho, called p190Rho-GAP, thus leading to an up-regulation of Rho activity (14,15). To analyze the activity of Y131A and Y132A LMW-PTP mutants on p190Rho-GAP, we tested the tyrosine phosphorylation level of p190Rho-GAP in all cell lines upon PDGF treatment. Fig. 4 shows that the Y132A-LMW-PTP mutant is able to dephosphorylate p190Rho-GAP even better than wtLMW-PTP, but the Y131A-LMW-PTP mutant is almost ineffective on this substrate. These findings indicate that only the phosphorylation of tyrosine 131 is essential for the function of LMW-PTP in Rho-mediated cytoskeleton rearrangement leading to cell migration toward PDGF-BB and adhesion to extracellular matrix, through the regulation of p190Rho-GAP tyrosine phosphorylation.
Role of LMW-PTP Tyr(P) 132 in the Regulation of Cell Detachment-The transfection of Y131A-LMW-PTP mutant leads to the expression of a protein that is efficiently targeted to the cytoskeleton architecture (Fig. 1A) and phosphorylated upon PDGF treatment (Fig. 1B). Nevertheless, in Y131A-NIH-3T3 cells we failed to observe any variations in cell growth rate, PDGF-induced migration, and cell adhesion, with respect to mock transfected cells. We routinely observed, in all clones expressing Y131A-LMW-PTP mutant a resistance to trypsin detachment during cell culturing. Hence, we analyzed the strength of cell substratum adhesion by a detachment assay (12) in exponentially growing cells treated or not with the PP1 Src family kinase inhibitor (16) (Fig. 5). The overexpression of wtLMW-PTP leads to a slight decrease of the strength of cell substratum adhesion in 10% FCS growing cells (Fig. 5), as indicated by the decrease in the resistance to detachment reported in the ordinate of the plot. Although the Y132A-NIH-3T3 cells showed a behavior similar to wtLMW-PTP-NIH-3T3 cells, the Y131A-NIH-3T3 cells exhibited a dramatic increase in the resistance to detachment (Fig. 5). The NIH-3T3 cells overexpressing the LMW-PTP Y131A/Y132A double mutant, that is not phosphorylated upon PDGF treatment (10), show a phenotype similar to mock-transfected cells. Hence, this in-crease in the resistance to cell detachment is likely due to the phosphorylation of LMW-PTP on tyrosine 132. In fact, the increased adhesion strength of Y131A-NIH-3T3 is abolished in serum-starved cells (data not shown), where the LMW-PTP is not phosphorylated by c-Src (6), and in PP1 treated cells (Fig.  5), suggesting a role for LMW-PTP phosphorylation on tyrosine 132 in the modulation of of cell adhesion strength.
As reported in literature, the adhesion of cells to the extracellular matrix is controlled by an equilibrium between integrin receptors interaction with ECM components and the activity of membrane protease acting on them. Matrix metalloprotease expression increases when remodelling of ECM is required (17, 18) and is primarily regulated at a transcriptional level (19,20). Recently it has been demonstrated that there is a modulation of the expression of MMPs in re- The RIPA lysates were immunoprecipitated with anti-PDGF-r antibodies, and an anti-phosphotyrosine immunoblot was performed. The same blot was stripped and reprobed with anti-PDGF-R antibodies to check for equal loading (data not shown). The result is a representative Western blot; band intensities from three independent experiments were quantitated with laser scanning densitometer, and the mean Ϯ S.D. is shown in C. The results are normalized to the phosphorylation level of mock transfected cells taken as a unit. Ip, immunoprecipitation; WB, Western blot; dn, dominant negative; wt, wild type. sponse to many growth factors such as PDGF or epidermal growth factor (21,22). To assess whether the increased resistance to cell detachment of Y131A-NIH-3T3 cells could be due to variations in MMPs production in response to growth factors, we analyzed by gelatin zymography the MMPs activity of Y131A, Y132A, and Y131A/Y132A-NIH-3T3 cells in comparison with mock transfected and wtLMW-PTP-expressing cells. We observed in growing cells (Fig. 6A) a general down-regulation of MMPs activity in Y131A-NIH-3T3 cells with respect to mock transfected cells, whereas both Y132A-NIH-3T3 cells and wtLMW-PTP-expressing cells showed an increased MMPs production. In the same condition MMPs activity of Y131A/Y132A-NIH-3T3 cells is similar to mock transfected cells (Fig. 6A). In both serum-starved cells (data not shown) and PP1-treated cells, where LMW-PTP is not phosphorylated (6, 10), all cell lines show a similar MMPs activity (Fig. 6B). These findings correlate with the cell detachment phenotypes observed in all cell lines. Hence, the data obtained by cell detachment and MMPs activity experiment indicate a role for LMW-PTP phosphorylation on tyrosine 132 in regulating cell adhesion.
Role of LMW-PTP Tyr(P) 132 in Grb2/APK Pathway Regulation-We have previously reported that, in vitro, the phosphorylation of Tyr 132 (but not Tyr 131 ) creates a docking site for Grb2 binding (7). To assess whether Grb2 adapter protein really binds LMW-PTP in vivo, we analyzed by coimmunoprecipitation the interaction between Grb2 and the phosphatase, in wtLMW-PTP, Y131A and Y132A mutant-expressing cells. The result of the anti-LMW-PTP immunoblot of Grb2 immunoprecipitates is shown in Fig. 7; we detect the interaction between LMW-PTP and Grb2 mainly in PDGF-treated Y131A-NIH-3T3 cells, although there is a detectable association also in wtLMW-PTP-NIH-3T3 cells. In any case the interaction between Grb2 and LMW-PTP is dependent on the activation of PDGF signaling, in agreement with the dependence of LMW-PTP tyrosine phosphorylation by c-Src upon PDGF treatment (6). This result indicates that the phosphorylation of tyrosine 132 really represents in vivo a Grb2 binding site, as we hypothesized on the basis of structural data and in vitro binding assay (7).
It has been recently reported that many MMPs are under transcriptional control by growth factors, cytokines, and other environmental factors such as contact with ECM (23)(24)(25). The Grb2/Ras/MAPK pathway is implicated in the transduction of the signal starting from tyrosine kinase receptors (26 -29), leading to the induction of membrane proteases. LMW-PTP could be involved in the regulation of this pathway through the recruitment of the Grb2 adapter protein by its phosphotyrosine 132 docking site. We analyze the p44/p42 MAPK activation in NIH-3T3 cells expressing wtLMW-PTP or the tyrosine mutants Y131A and Y132A, during exponential cell growth. Fig. 8 shows that p44/p42 MAPK activation is down-regulated in the Y131A-LMW-PTP mutant-expressing cells in comparison with wtLMW-PTP-expressing cells. The data reported in Fig. 8 suggest that the phosphorylation of tyrosine 132 of LMW-PTP leads to a MAPK down-regulation, and this is probably due to its binding to the SH2 domain of Grb2. In fact, the Grb2/Sos complex is able to activate the Ras/MAPK pathway when the SH2 domain of Grb2 binds the phosphorylated tyrosines of the activated tyrosine kinase receptors. The Grb2/LMW-PTP association through phosphotyrosine 132 could prevent the Grb2/ Sos complex binding to the receptor, thus determining a downregulation of the MAPK pathway. The p44/p42 MAPK activation levels (Fig. 8) in the analyzed cell clones are in agreement with the MMPs activities shown in Fig. 7. Taken together these data suggest that the phosphorylation of Tyr 132 leads to a regulation of the strength of the cell substratum adhesion through the control of MMPs expression via a Grb2/ MAPK regulated pathway.

FIG. 3. Effect of wtLMW-PTP and single tyrosine mutants on integrin mediated cell adhesion and PDGF-induced migration.
A, 3 ϫ 10 4 cells of each indicated type were seeded in a 96-well plate precoated with fibronectin, serum starved for 24 h, and then treated with 30 ng/ml of PDGF-BB. The cells were allowed to attach for the indicated times, and the adhesion was evaluated with crystal violet staining. B, 1.5 ϫ 10 5 cells of the indicated type were seeded into a 2.5-cm Transwell. 10 ng/ml of PDGF-BB was added to the lower chamber, and the cell migration was evaluated after Diff-Quick staining and reported in the histogram as a percentage of the control unstimulated cells. S.D. is indicated. wt, wild type.

DISCUSSION
Many cellular processes such as cell migration, adhesion, and proliferation require the collaborative interaction between growth factors and ECM stimuli (17,30). In addition cell adhesion to ECM is a balance between focal adhesion formation and proteolysis of extracellular matrix. The addition of a growth factor that induces cell migration has a double role in promoting cell substratum adhesion through the regulation of Rho-mediated cytoskeleton rearrangement and the induction of MMPs expression, which promotes the degradation of base-ment membranes and stromal extracellular matrix. These concurrent events allow cells to adopt an adhesive state permissive to migration (17).
Our previous results on LMW-PTP function in PDGF-induced mitogenesis indicate that the tyrosine phosphorylation of LMW-PTP is essential in Rho-mediated cytoskeleton rearrangement (10). Herein, we have investigated the specific role of the single LMW-PTP tyrosine phosphorylation sites in this phenomenon. In this report we show that the LMW-PTP tyrosines in positions 131 and 132 are independently phosphorylated in response to PDGF. These effects lead to differential cell behavior. It is possible that in the wtLMW-PTP, where both 131 and 132 sites are accessible and thus phosphorylable by the c-Src tyrosine kinase, the amount of Tyr(P) 131 is counterbalanced by the phosphorylation of Tyr 132 . On the basis of structural data (7), it is likely that only one tyrosine is phosphorylated on a LMW-PTP molecule by c-Src, because of the steric block of the phosphorylated residue. In this light, it is possible that the LMW-PTP mutant, which does not contain the tyrosine 132 (which is mutated to alanine), lacking any competition between the two phosphorylation sites, is fully phosphorylated in the remaining tyrosine 131. The reverse condition leads to the full phosphorylation of tyrosine in position 132 in the Y131A-LMW-PTP mutant. This situation give us a tool to study the differential role of the two tyrosines in LMW-PTP regulation of PDGF signaling, because both LMW-PTP mutants give rise to an enhancement of the native phosphorylation in each tyrosine of the phosphatase.
Firstly, the phosphorylation on tyrosine 131 seems to mainly mediate the inhibition of wtLMW-PTP in growth factor-induced mitosis (Fig. 1). In fact the Y132A-LMW-PTP-expressing cells show a decreased cell growth rate with respect to wtLMW-PTP cells. The effect of the tyrosine phosphorylation in position 131 on PDGF-induced mitosis is in agreement with the activation of PDGF receptor (Fig. 2). In fact, the tyrosine phosphorylation level of PDGF receptor is down-regulated in Y132A-LMW-PTP-expressing cells with respect to wtLMW-PTP transfected cells. We observed that the Y132A-LMW-PTP mutant overexpressing cells show an increased fibronectin mediated cell adhesion (Fig. 3A) and PDGF-induced cell migration (Fig. 3B), with respect to wtLMW-PTP-expressing cells. These phenotypic effects are in agreement with the increased activity of Y132A-LMW-PTP mutant on phosphorylated p190Rho-GAP with respect to wtLMW-PTP (Fig. 4). These data indicate that the role of LMW-PTP exerted in the regulation of PDGF-induced mitogenesis and Rho-dependent cytoskeletal rearrangement, such as chemotaxis toward growth factors and cell adhesion rate on ECM, is mainly mediated by the phosphorylation of LMW-PTP on tyrosine 131.
On the contrary, Y131A-LMW-PTP mutant fails to dephosphorylate both PDGF receptor and p190Rho-GAP upon PDGF stimulation (Figs. 2B and 4). It is very likely that the phosphorylation of LMW-PTP on Tyr 132 is not involved in neither PDGF-induced cell growth nor in the phenotypic effects of Rho-mediated cytoskeleton rearrangement ( Figs. 2A and 3). Nevertheless, the Y131A-LMW-PTP mutant is indeed a functional protein that is correctly targeted to the cytoskeleton structure (Fig. 1A) and tyrosine phosphorylated (Fig. 1B) in response to PDGF. In addition, the in vivo catalytic activity of the Y131A-LMW-PTP mutant on p-nitrophenylphosphate is almost 40% with respect to the wild type enzyme. Furthermore, our data indicate an intriguing inhibitory role of the phosphorylation in position 132 on LMW-PTP function in PDGF-induced mitogenesis, chemotaxis, and cell adhesion. It should be noted that in every analyzed phenomenon the wtLMW-PTP shows a softened behavior with respect to Y132A-LMW-PTP-expressing cells. In fact, the wtLMW-PTP, which can be phosphorylated in both 131 and 132 sites, is less active on activated PDGF-R and phosphorylated p190RhoGAP with respect to Y132A-LMW-PTP, which can be phosphorylated only in position 131. As a consequence, wt-LMW-PTP is a less effective enzyme than Y132A-LMW-PTP in PDGF signaling, suggesting that the phosphorylation of Tyr 132 in LMW-PTP could represent a negative regulation of enzyme activity during PDGF signaling. The behavior of the Y131A-LMW-PTP mutant overexpressing cells has suggested the involvement of the phosphorylation on Tyr 132 in the matrix remodelling process. We analyzed the resistance to cell detachment from the substratum as an indication of the strength of cell adhesion. The phosphorylation of LMW-PTP on Tyr 132 only leads to a strong decrease of the production of matrix membrane metalloproteases, as indicated by a zymography analysis (Fig. 7). Studies on the regulation of the MMPs and serine-protease urokinase (17,18) expression have revealed that the MMPs and urokinase genes are regulated at a transcriptional level (19,20). The promoter regions of the genes for these proteases show conserved motifs for AP1 and NF-B transcription factors (31, 32), which confer regulability by growth factors and cytokines (33,34). The signal transduction pathway that leads to protease transcription control, involves the Grb2/ Sos1/Ras/Raf/MEK1/MAPK route (27,28). In this light, we investigated the p44/p42 MAPK activation level in exponentially growing NIH-3T3 cells expressing wt-LMW-PTP and in cells expressing the single tyrosine mutants (Fig. 8). The phosphorylation levels of p44 and p42 correlate with the activity of metalloproteases because the Y131A-LMW-PTP mutant overexpressing cells showed a decrease in MAPK stimulation together with a decrease in secreted MMPs level.
We have already reported that the sequence C-terminal to Tyr 132 conforms to the motifs specifically recognized by the Grb2 SH2 domain (YXN) (7) and that in vitro only the phosphorylation in Tyr 132 leads to Grb2 adapter binding by GST-LMW-PTP. Grb2 is a phosphotyrosine-binding protein that transduces the signal starting from activated tyrosine kinase receptors to the Sos/Ras/Raf/MAPK pathway. It has been reported that other PTPs, such as SHP1, SHP2, and RPTP␣, possess the Grb2 consensus binding site (35,36). We demonstrated herein that the phosphorylation in Tyr 132 mediates in vivo the binding between LMW-PTP and Grb2 after PDGF stimulation (Fig. 6). In fact, the Y131A-LMW-PTP mutant is able to bind Grb2 even more efficiently than the wild type phosphatase, whereas the Y132A-LMW-PTP mutant lack any detectable association, suggesting a specificity for Tyr 132 and not for Tyr 131 in the interaction between phospho-LMW-PTP and Grb2.
On the basis of structural data and computer modelling (7), we suggest that the interaction of LMW-PTP with Grb2 is most likely at the level of its SH2 domain, which is engaged with LMW-PTP Tyr(P) 132 and hence no more available to be recruited by phospho-PDGF receptor. The formation of the complex between LMW-PTP and Grb2 could lead to the subtraction of Grb2/Sos to the PDGF-R signal transduction system and may lead to MAPK pathway down-regulation. The final effect is a down-regulation of the growth factor-induced expression of MMPs and a decrease in the strength of the adhesion of cell to the ECM. We underline that the association between Grb2 and LMW-PTP is not restricted to the Y131A-LMW-PTP-expressing cells (a mutant in which the phosphorylation of Tyr 132 should be enhanced) but is present also in wtLMW-PTP transfected cells. These data suggest that the phosphorylation of Tyr 132 is not an artifact of the mutant that lacks Tyr 131 but is a naturally occurring phenomenon.
The relative phosphorylation amount of each LMW-PTP tyrosine is indicated by the behavior of wtLMW-PTP transfected cells. We suggest that in wtLMW-PTP, Tyr 131 is the main phosphorylation site in PDGF-induced mitogenesis, chemotaxis, cell adhesion, and, marginally, cell detachment, because the wtLMW-PTP behaves like Y132A-LMW-PTP mutant in any of the analyzed phenomena. This is in agreement with our previous in vitro results (7), which report that the c-Src tyro-sine kinases mainly phosphorylate LMW-PTP in position 131. Herein, we demonstrate that at least for the key event of Grb2 binding the wtLMW-PTP behaves as Y131A mutant, suggesting that in vivo the tyrosine in position 132 is really phosphorylated in the wtLMW-PTP, although with a lower extent with respect to the Y131A mutant, which enhances this effect. We demonstrate that the behavior of Y131A-LMW-PTP mutant on cell detachment and MMPs activity is correlated with the phosphorylation of the position 132, as indicated by the behavior of the Y131A/Y132A double mutant and by the inhibition of the LMW-PTP phosphorylation by Src inhibition. We suggest that in some physiological conditions Tyr 132 could become the main phosphorylation site of LMW-PTP. In this occurrence the effect of LMW-PTP on cellular behavior could lead to a decreased cell detachment and MMPs expression, thus increasing the strength of cell adhesion, as in Y131A-LMW-PTP-expressing cells.
The addition of a growth factor that induces cell migration has a double role in promoting cell substratum adhesion through the regulation of Rho-mediated cytoskeleton rearrangement and the induction of MMPs expression, which promote the degradation of basement membranes and stromal extracellular matrix. These concomitant events allows cells to assume an adhesive condition permissive to migration (17). We propose that LMW-PTP is a bifunctional phosphatase that regulates both the strength and the rate of formation of cell adhesions through the alternative phosphorylation of LMW-PTP in tyrosine 131 or 132. These events are potentially diverging in terms of timing and relative amount, but they concurrently regulate cell adhesion and migration.