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[Cancer Research 62, 6489-6499, November 15, 2002]
© 2002 American Association for Cancer Research

Regular Articles

ß-Catenin Interacts with Low-Molecular-Weight Protein Tyrosine Phosphatase Leading to Cadherin-mediated Cell-Cell Adhesion Increase1

Maria Letizia Taddei, Paola Chiarugi, Paolo Cirri, Francesca Buricchi, Tania Fiaschi, Elisa Giannoni, Doriana Talini, Giacomo Cozzi, Lucia Formigli, Giovanni Raugei and Giampietro Ramponi2

Dipartimento di Scienze Biochimiche [M. L. T., P. Ch., P. Ci., F. B., T. F., E. G., D. T., G. C., G. Rau., G. Ram.], and Dipartimento di Anatomia, Istologia e Medicina Legale [L. F.], Università degli Studi di Firenze, 50134 Florence, Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ß-catenin plays a dual role as a major constituent of cadherin-based adherens junctions and also as a transcriptional coactivator. In normal ephitelial cells, at adherens junction level, ß-catenin links cadherins to the actin cytoskeleton. The structure of adherens junctions is dynamically regulated by tyrosine phosphorylation. In particular, cell-cell adhesion can be negatively regulated through the tyrosine phosphorylation of ß-catenin. Furthermore, the loss of ß-catenin-cadherin association has been correlated with the transition from a benign tumor to an invasive, metastatic cancer. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitous PTP implicated in the regulation of mitosis and cytoskeleton rearrangement. Here we demonstrate that the amount of free cytoplasmic ß-catenin is decreased in NIH3T3, which overexpresses active LMW-PTP, and this results in a stronger association between cadherin complexes and the actin-based cytoskeleton with respect to control cells. Confocal microscopy analysis shows that ß-catenin colocalizes with LMW-PTP at the plasmamembrane. Furthermore, we provide evidence that ß-catenin is able to associate with LMW-PTP both in vitro and in vivo. Moreover, overexpression of active LMW-PTP strongly potentiates cadherin-mediated cell-cell adhesion, whereas a dominant-negative form of LMW-PTP induces the opposite phenotype, both in NIH3T3 and in MCF-7 carcinoma cells. On the basis of these results, we propose that the stability of cell-cell contacts at the adherens junction level is positively influenced by LMW-PTP expression, mainly because of the ß-catenin and LMW-PTP interaction at the plasmamembrane level with consequent dephosphorylation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadherins are transmembrane glycoproteins implicated in cell-cell adhesion. The extracellular domains of cadherins from adjacent cells are engaged in homophilic calcium dependent interactions, whereas the cytoplasmic domains are connected to several proteins, generically termed catenins (1) . ß and {gamma} catenins bind directly to cadherins in a mutually exclusive fashion and form a complex with a catenin, which in turn is connected to the actin network either directly or indirectly (2 , 3) . Another catenin, p120Cas, has also been shown to complex directly with cadherins (4) . Association of the cadherin-catenin complex with the cytoskeleton is essential to establishing tight cell-cell interaction, through formation of the adherens junctions, specialized structures important for the establishment of cell adhesion and polarization. These structures are highly dynamic because they undergo rapid disassembly and reassembly during several processes, such as embryonic development and wound healing (5) , and destabilization of these junctions contributes to tumor invasion and metastasis (6) . The integrity of adherens junctions appears dynamically regulated by tyrosine phosphorylation (7 , 8) . Previous data obtained by Batt and Roberts (9) showed that the level of tyrosine phosphorylation is modified for a large number of proteins in response to cell density. Increased tyrosine phosphorylation of ß-catenin, {gamma} catenin, and p120Cas has been correlated with the decrease of cell-cell adhesion, which occurs on malignant transformation (10, 11, 12, 13, 14) or stimulation with mitogenic growth factors (15, 16, 17, 18, 19) .

Tyrosine phosphorylation of ß-catenin leads to the disruption of the contact with E-cadherin, leading to an increase in the free, uncomplexed cytoplasmic pool of the protein (20) . Recently, the role of tyrosine phosphorylation of ß-catenin has been further investigated; phosphorylation on tyrosine 654 of ß-catenin decreases its binding to E-cadherin and stimulates the association of ß-catenin to the TATA binding protein (TBP; Refs. 21 and 22 ).

Furthermore, a recent study demonstrates that ß-catenin is highly tyrosine phosphorylated in melanoma cells. On the other hand, treatment of cells with geldamycin, a drug that stimulates tyrosine dephosphorylation of ß-catenin, results in an increased ß-catenin/E-cadherin association and a decreased cell mobility (23) .

Moreover, the loss of ß-catenin-E-cadherin association correlates with tumor progression and metastasis. For this reason, an imbalance in the tyrosine phosphorylation level of ß-catenin can be associated with carcinoma formation and tumor invasiveness (24) .

On the other hand, ectopic expression of phosphotyrosine phosphatases strengthens cell-cell adhesion (20) . Furthermore, because increased levels of PTPs3 have been observed in confluent cell cultures, PTPs have been proposed as acting as mediators of cell contact-dependent growth inhibition (25 , 26 , 27) .

Suzuki et al. (28) demonstrated that sodium orthovanadate, a phosphatase inhibitor, induces the reentry into the cell cycle of contact-inhibited human umbilical vascular endothelial cells (HUVECs). Moreover, Pani et al. (29) showed that the growth arrest caused by cell confluence is accompanied by a decrease in the steady-state levels of intracellular ROS. This decrease is associated with an increase in tyrosine phosphatase activity, because tyrosine phosphatases are sensitive to redox regulation. Indeed, PTPs are easily inactivated by oxidation of a critical cysteine residue located in the catalytic site.

Many receptor-like and cytosolic PTPs have been implicated in the regulation of cadherin/catenins complexes: RPTP{kappa} associates with both ß and {gamma} catenin (30) , and RPTP{lambda} forms complexes with ß-catenin (31 , 32) , and RPTPµ binds and dephosphorylates p120 catenin (33) . Furthermore, there are several lines of evidence of the physical association between the nonreceptor PTP PTP1B and N-cadherin (34) , SHP-2 and VE-cadherin (35) , and of the binding and dephosphorylating activity of SHP-1 on p120 catenin (36) . In particular, recent data suggest ß-catenin as a possible substrate of phosphotyrosine phosphatases such as PTP-LAR (20) , PTP{kappa} (30) , and PTP1B (34) .

LMW-PTP is a widely expressed enzyme involved either in the modulation of mitogenic signals triggered by several growth factors such as platelet-derived growth factor and insulin, and in cytoskeleton rearrangement (37, 38, 39) . Recently, it has been demonstrated that LMW-PTP catalytic activity undergoes a double regulation: in fact, it has been demonstrated by our group that LMW-PTP is regulated by tyrosine phosphorylation and by a redox mechanism (40 , 41) .

Previous results have also demonstrated that the LMW-PTP level increases in confluent cells with respect to growing cells (42) , which suggests a possible role of the enzyme in contact-growth inhibition and in the stabilization of cell-cell junctions as cells reach confluence. In this report, we provide evidence that the regulation of cell-cell contacts in confluent cells that is attributable to tyrosine phosphorylation of ß-catenin is influenced by LMW-PTP; the interaction of ß-catenin with LMW-PTP leads to a strong connection between cadherin and the actin-based cytoskeleton. The interaction between ß-catenin and LMW-PTP could play a central role in the maintenance of the architecture of solid tissue and may be important to the opposing of tumor progression and invasiveness.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Unless specified, all of the reagents were obtained from Sigma. NIH3T3 mouse fibroblasts, A431 human epithelial cells, and MCF-7 breast adenocarcinoma cells were purchased from ATCC. Unless specified, all of the antibodies were from Santa Cruz Biotechnology; Fluor 488 goat antirabbit IgG was from Molecular Probes and goat antimouse IgG Texas Red conjugate was from Calbiochem; 5'-F-IAA, DCF-DA, and affinity-purified rabbit antibodies to fluorescein were obtained from Molecular Probes; G418 was from Calbiochem; BCA protein assay reagent was from Pierce; protein A-Sepharose and Enhanced Chemi-Luminescence kit were from Amersham Pharmacia; nitrocellulose was from Sartorius.

Cell Culture and Transfections.
NIH3T3 and A431 cells were routinely cultured in DMEM supplemented with 10% FCS, in 5% CO2 humidified atmosphere. MCF-7 cells were routinely cultured in Ham’s F-12 supplemented with 10% FCS, in 5% CO2 humidified atmosphere. Ten µg of pRcCMV-wtLMW-PTP or pRcCMV-dnLMW-PTP were transfected in NIH3T3 cells using the calcium phosphate method. Stable transfected clonal cell lines were isolated by selection with G418 (400 µg/ml). Mock-transfected cell lines were obtained by transfecting 2 µg of pRcCMVneo alone. The clonal cell lines were screened for expression of the transfected genes by Western blot using polyclonal anti-LMW-PTP rabbit antibodies, which do not cross-react with murine endogenous LMW-PTP. Expression levels of wtLMW-PTP- and dnLMW-PTP-overexpressing clones selected for the experiments are comparable and estimated to be 10 times the level of the endogenous protein.

For transient transfections, MCF-7 cells were plated in 60-mm tissue culture dishes and grown to 80% confluence. Transfections were performed with 5 µg of pRcCMV-wtLMW-PTP, pRcCMV-dnLMW-PTP, or pRcCMVneo using DOTAP Liposomal Transfection Reagent (Roche), according to the manufacturer’s protocol. Twenty-four h after transfection, the medium was replaced with fresh medium, and cells were used for additional experiments. Transfection efficiency was checked using pEGFP-N1 vector (Clontech) as control.

Immunoprecipitation and Western Blot Analysis.
Cells were grown to confluence in DMEM supplemented with 10% FCS. Cells were then extracted in 10-cm culture dishes with 1 ml of cytoskeleton extraction buffer [300 mM sucrose, 10 mM PIPES (pH 6.8), 50 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Triton X-100, 1.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 mg/ml DNase, and 0.1 mg/ml RNase] as described previously (43) . This extraction buffer allows separation of the cytoskeleton-associated (insoluble) pool of catenins from the cytoplasmic (soluble) pool of these proteins. Cells were removed from the dishes with a rubber scraper and centrifuged at 13,000 rpm for 15 min. The soluble fraction was removed from the insoluble pellet. For total-cell extract immunoprecipitation, cultures were extracted with 1 ml of lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 5 mM EDTA, 10% glycerol, 1% (v/v) Triton X-100, 1 mM orthovanadate, 1.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin] for 20 min and harvested as above. After a 15-min centrifugation at 13,000 rpm, the insoluble pellet was removed, and the soluble extract was processed for immunoprecipitation. The soluble fraction was immunoprecipitated for 4 h at 4°C with 2 µg of the specific antibodies. Immunocomplexes were collected on protein A-Sepharose, separated by SDS/PAGE, and transferred onto nitrocellulose. Immunoblots were incubated in 3% BSA, 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 Chemi-Luminescence kit.

Immunofluorescence.
NIH3T3 and A431 cells were grown on coverslips in DMEM supplemented with 10% FCS. Cells were washed with PBS and fixed in 3% paraformaldehyde for 20 min at 4°C. Fixed cells were permeabilized with three washes with TBST [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, and 0.1% Triton X-100] and then were blocked with 5.5% horse serum in TBST for 1 h at room temperature. Cells were incubated overnight at 4°C with anti-LMW-PTP antibodies, or other primary antibodies, diluted 1:100 in TBS [50 mM Tris/HCl (pH 7.4), 150 mM NaCl]. Cells were then washed once with TBST and once with TBST plus 0.1% BSA and incubated for 1 h at room temperature with fluorescein or rhodamine-conjugated secondary antibodies in TBST with 3% BSA. Negative controls were performed by incubating cells with the blocking solution without primary antibodies but with secondary antibodies alone. After extensive washes in TBST, cells were mounted with glycerol plastine and observed under a laser scanning confocal microscope (Bio-Rad MRC 1024 Es; Hercules, CA). A series of optical sections (512 x 512 pixels) were taken through the depth of the cells with a thickness of 1 µm at intervals of 0.2 µm. Twenty optical sections were examined and then projected as a single composite image by superimposition (Confocal Assistant version 4.02).

Trypsin Digestion.
Confluent monolayers of cells were incubated in PBS alone or in PBS containing 0.005% trypsin in the presence of 2 mM Ca2+ or in the presence of 2 mM EGTA for 20 min at 37°C. Untreated or trypsin-treated cells were washed with PBS, dissolved directly in SDS-PAGE sample buffer, and analyzed by immunoblotting with anti-N-cadherin or anti-P-cadherin antibodies.

Aggregation Assay.
Cells were grown to confluence in 35-mm tissue culture dishes. Cell-cell aggregation assay was performed as described previously (43) . Cultures were trypsinized (0.04% trypsin in PBS containing 1 mM Ca2+) to prepare single-cell suspensions and were resuspended in complete medium. Two-ml samples containing 2 x 106 single cells were incubated at 37°C with constant shaking at 90 rpm for a total of 40 min. Every 10 min, 200-µl aliquots were withdrawn and the number of single cells was counted using a hemocytometer. The rate of aggregation was calculated as the percentage of decrease in the number of single cells using the formula [(No - N1)/No] x 100, where No is the number of single cells at time 0 and N1 is the number of single cells detected in the cultures at different times of incubation.

Cell-Cell Adhesion Assay.
Labeled probe cells were prepared by incubating confluent cells in complete medium supplemented with 5 µl/ml [2-3H]glycerol (16.5 Ci/mmol) overnight at 37°C under an atmosphere of 5% CO2. Target monolayer cultures were prepared by plating 5 x 104 NIH3T3 cells/well in a 96-multiwell tissue culture plate. To measure cell-cell adhesion, probe cells were trypsinized with 0.04% trypsin in PBS containing 1 mM Ca2+, resuspended in complete medium (3 x 104 cells in 200 µl/well), and incubated with target cells for 15 min at 37°C in an atmosphere of 5% CO2. Unbound cells were removed by aspiration, and the wells were washed with PBS. Cells were removed by solubilization in 0.2 N NaOH, and radioactivity was determined by liquid scintillation counting.

Antibody Binding.
Adhesion of cells to anti-N- or anti-P- or anti-E-cadherin antibodies immobilized on tissue culture dishes was performed as described previously (44) . Briefly, anti-N- or anti-P-cadherin antibodies (5 µg/well) were incubated overnight at 4°C in 96-well plates. Wells were then incubated with 1% BSA in PBS for 1 h at 37°C, followed by two washes with PBS. Confluent cells were trypsinized and 3 x 104 cells/well were incubated with anti-N- or anti-P- or anti-E-cadherin antibodies immobilized on multiwell plates for 15 min at 37°C under 5% CO2. Pretreatment of cells was performed with 100 µg/ml anti-N- or anti-P-cadherin or anti-E-cadherin antibodies incubated for 30 min at 37°C with target cells before the adhesion assay.

Adhesion of cells to anti-N- or anti-P- or anti-E-cadherin antibodies 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 once with PBS and solubilized with 0.1 M sodium citrate (pH 4.2) at 200 µl/well. The absorbance at 595 nm was evaluated using a microplate reader.

In Vitro Binding Assay.
GST-wtLMW-PTP and GST-dnLMW-PTP plasmids were constructed by cloning wtLMW-PTP and dnLMW-PTP cDNA in the BamHI/EcoRI sites of pGEX-KT vector (Pharmacia). GST-wtLMW-PTP and GST-dnLMW-PTP fusion proteins were expressed in Escherichia coli and were purified as described previously (45) . As previously described (30) , equal amounts of cell lysates from confluent NIH3T3 cells were incubated overnight at 4°C with 5 µg of GST-wtLMW-PTP or GST-dnLMW-PTP or with 8 µg of GST alone, immobilized on glutathione-Sepharose, in gentle agitation. Complexes were washed three times with lysis buffer. Bound proteins were separated by SDS-PAGE for Western blotting analysis.

In Vivo 5'-F-IAA Labeling.
Confluent and exponentially growing wtLMW-PTP NIH3T3 cells cultured in complete medium were lysed in RIPA buffer [50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% (v/v) Triton X-100, 2 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin], and 5'-F-IAA was added from freshly prepared stock to a final concentration of 5 µM. As demonstrated previously (41) , the active site cysteines of LMW-PTP are specifically targeted by the sulfidryl-modifying reagent iodoacetic acid. The lysates were maintained for 10 min at 37°C for the labeling step and then were treated for immunoprecipitation with anti-LMW-PTP antibodies. Redox state was assayed by antifluorescein immunoblot.

Measurement of Intracellular ROS.
Sparse and dense cultures of wtLMW-PTP NIH3T3 cells were seeded in 10-cm plates and cultured in complete medium. Medium was then replaced with DMEM without phenol red and after 1 h cells, were incubated with 5 µg/ml DCF-DA, an oxidant-sensitive fluorescent dye. Cells were then detached from the substrate by trypsinization and analyzed immediately by flow cytometry using a Becton Dickinson FACScan flow cytometer equipped with an Argon laser lamp (FL-1; emission, 480 nm; band pass filter, 530 nm) and the Lysis II Software.

LMW-PTP Assay.
The PTP activity was measured as reported previously (40) . Briefly, sparse and dense wtLMW-PTP NIH3T3 cultures were lysed in RIPA buffer and LMW-PTP was immunoprecipitated from lysates. Immunoprecipitates were then resuspended in 100 µl of 0.1 M sodium acetate (pH 5.5)-10 mM EDTA. PTP activity assay was performed using 100 µl of immunoprecipitates mixed with 100 µl of 10 mM p-nitrophenylphosphate at 37°C for 1 h. The production of p-nitrophenol was measured colorimetrically at 410 nm. The results were normalized on the basis of LMW-PTP content evaluated by Western blot analysis and then quantitated by densitometric analysis. This analysis was performed with the Bio-Rad Gel Doc 2000 Chemi Doc, using Quantity One quantitation Software.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Level of Cytoplasmic ß-Catenin Is Influenced by LMW-PTP Expression.
The presence of phosphorylated tyrosine residues on ß-catenin is correlated with the loss of cadherin-mediated cell adhesion and ß-catenin migration to the cytoplasm (20 , 46) . Furthermore, recent results demonstrate that tyrosine phosphorylation of ß-catenin correlates with carcinoma formation and tumor invasiveness (23 , 24) . Several PTPs such as PTPµ, PTP{kappa}, PTP1B, and LAR-PTP cause the dephosphorylation of members of the catenin and cadherin family (33 , 34 , 47 , 48) with consequent increase in cohesiveness. LMW-PTP is a PTP implicated in cytoskeleton remodeling and especially in cell-extracellular matrix adhesion (39) . To examine whether the level of phosphorylated ß-catenin could be influenced by LMW-PTP expression, we evaluated the amount of ß-catenin and its phosphorylation level in the cytosolic fraction of NIH3T3 cells that overexpressed the active form of LMW-PTP (wtLMW-PTP NIH3T3) and its mutated dominant-negative form (dnLMW-PTP NIH3T3). The results reveal a decrease of free cytoplasmic ß-catenin in cells overexpressing wtLMW-PTP, whereas the overexpression of dnLMW-PTP causes the opposite effect (Fig. 1A)Citation . The amount of cytosolic ß-catenin is known to be the pool that is tyrosine phosphorylated in the cell, different from the cadherin- associated one. For this reason, we analyzed the phosphotyrosine level of ß-catenin in our cell lines also (Fig. 1B)Citation . It is likely that the release of ß-catenin from the cadherin-associated fraction is influenced by LMW-PTP.



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  		Fig. 1. Level of cytoplasmic ß-catenin. wtLMW-PTP, dnLMW-PTP, and mock-transfected cells were grown to confluence and then lysed with cytoskeleton extraction buffer. One mg of total proteins was immunoprecipitated with anti-ß-catenin antibodies (cytoplasmic fraction of ß-catenin). The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-ß-catenin antibodies (A). The blot was then reprobed with anti-phosphotyrosine (PY20) antibodies (B). Results represent the average of four experiments. kDa, Mr in thousands.

 
Because phosphorylation of ß-catenin and its consequent release in the cytoplasm is correlated with a loss of cadherin function (because of the role played by ß-catenin to link cadherins to the actin cytoskeleton network) and with tumor invasion and metastasis, we analyzed the association between N-cadherin and actin in wtLMW-PTP, dnLMW-PTP, and mock-transfected fibroblasts. Fig. 2ACitation shows that the amount of actin coimmunoprecipitating with N-cadherin is reduced in dnLMW-PTP-expressing cells with respect to mock-transfected cells, whereas wtLMW-PTP expression causes the opposite effect. All together, these results demonstrate that the association of ß-catenin with cadherins is influenced by LMW-PTP, which contributes to the stability of adherens junctions through a more stable link between cadherins and the actin cytoskeleton.



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  		Fig. 2. Association between N-cadherin and actin in wtLMW-PTP, dnLMW-PTP, and mock-transfected NIH3T3 cells. Confluent wtLMW-PTP, dnLMW-PTP, and mock-transfected NIH3T3 cells were lysed with lysis buffer. One mg of total proteins from Triton X-100 soluble pool was immunoprecipitated with anti-N-cadherin antibodies. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-actin antibodies (A) or anti-N-cadherin antibodies (B). The results are representative of four experiments. kDa, Mr in thousands.

 
As the next step, we analyzed, using a laser confocal microscope, the cellular localization of ß-catenin in dnLMW-PTP, wtLMW-PTP, and mock-transfected NIH3T3 cells. This experiment was carried out to verify whether the subcellular distribution of ß-catenin could be influenced by the overexpression of LMW-PTP. Overexpression of dnLMW-PTP causes the cytoplasmic pool of ß-catenin to increase as indicated by the red fluorescence scattered throughout the cytoplasm (Fig. 3C)Citation . By contrast, wtLMW-PTP-overexpressing cells show a well-organized membrane-bound ß-catenin, as revealed by the immunostaining that is uniformly distributed along the plasmamembrane (Fig. 3B)Citation . Control sample probed with secondary antibodies alone confirms the specificity of the signal (Fig. 3D)Citation . Thus, expression of dnLMW-PTP induces the accumulation of ß-catenin in the cytoplasm. This event, most likely, causes the cadherin-catenin complexes to dissociate.



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  		Fig. 3. ß-catenin level in wtLMW-PTP, dnLMW-PTP, and mock-transfected NIH3T3 cells. Confluent monolayers of mock-transfected (A), wtLMW-PTP (B), and dnLMW-PTP NIH3T3 cells (C) were grown on glass coverslips, fixed, and labeled with antibodies against ß-catenin. Red fluorescence, ß-catenin. D, negative control without primary antibodies. Bar, 20 µm.

 
Colocalization of ß-Catenin with LMW-PTP.
Because the amount of cytoplasmic ß-catenin is influenced by LMW-PTP, we analyzed the distribution of these two proteins with a laser confocal microscope in A431 cells. This human epithelioma cell line is particularly suitable for studying the ß-catenin and LMW-PTP localization because of the great ability to form well-defined adherens junctions at sites of cell-cell contacts. As shown in Fig. 4Citation , LMW-PTP is localized all around plasmamembrane and, thus, also along cell-cell contacts of adjacent cells, as demonstrated using anti-LMW-PTP antibodies (Fig. 4Citation , green fluorescence). Because adherens junctions are the predominant localization of the cadherin-catenin complexes, we performed, on the same sample, an immunofluorescence staining using anti-ß-catenin antibodies (Fig. 4Citation , red fluorescence). The superimposition that arises from the red ß-catenin and the green LMW-PTP labels shows the degree of colocalization; it is possible to find colocalization of the two proteins at cell-cell adhesion contacts as demonstrated by the yellow fluorescence. As positive control, we analyzed the colocalization of ß-catenin with its known partner E-cadherin (Fig. 4, D–F)Citation . These data indicate that ß-catenin and LMW-PTP colocalize at plasmamembrane level, thus suggesting a possible involvement of LMW-PTP in regulating ß-catenin action at adherens junctions.



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  		Fig. 4. Colocalization of ß-catenin and LMW-PTP. Confluent monolayers of A431 cells were grown on glass coverslips, fixed, and then processed for immunofluorescence. Cells were labeled with anti-ß-catenin antibodies (red fluorescence, A) and simultaneously with anti-LMW-PTP antibodies (green fluorescence, B). C, the colocalization of LMW-PTP and ß-catenin (yellow fluorescence). D–F, positive controls of the colocalization of ß-catenin and E-cadherin. G and H, negative controls without primary antibodies.

 
ß-Catenin Associates with LMW-PTP.
The colocalization of ß-catenin with LMW-PTP, evidenced by the confocal microscopy analysis, led us to investigate whether these two proteins could physically interact within the cell. First, we tested, by a GST-pull down assay, the possible association between ß-catenin and LMW-PTP. ß-catenin was found to bind to GST-dnLMW-PTP and GST-wtLMW-PTP protein, whereas no association was observed with GST alone (Fig. 5A)Citation . The amount of association is comparable when GST-wtLMW-PTP and GST-dnLMW-PTP are considered, which suggests that the interaction is independent of the catalytic activity of the enzyme. When cells reach confluence, adherens junctions are strengthened and stabilized. For this reason, we investigated whether ß-catenin and LMW-PTP associate also in intact cells during exponential growth and contact inhibition. To this purpose, lysates from confluent and growing NIH3T3 were immunoprecipitated with anti-LMW-PTP antibodies. Western blot analysis using anti-ß-catenin antibodies revealed the presence of the protein in the anti-LMW-PTP immunoprecipitates (Fig. 5B)Citation . The level of LMW-PTP association with ß-catenin was cell-density dependent because it increased in lysates from dense cells with respect to lysates from sparse cells. These data are in agreement with the increasing LMW-PTP expression level that was previously observed during cell confluence (42) and that was confirmed by the Western blot in Fig. 5BCitation . On the contrary, the amount of total ß-catenin did not change during cell confluence. Taken together, these results suggest that LMW-PTP and ß-catenin physically interact in the cell and that this interaction increases during cell confluence.



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  		Fig. 5. Association of LMW-PTP with ß-catenin. A, lysates from confluent NIH3T3 cells were incubated overnight at 4°C in gentle agitation with 5 µg of GST-wtLMW-PTP or GST-dnLMW-PTP or with 8 µg of GST alone, immobilized on glutathione Sepharose. After three washes with lysis buffer, complexes were separated by SDS-PAGE and a Western blot with anti-ß-catenin antibodies was performed. B, NIH3T3 fibroblasts were plated onto 10-cm tissue culture dishes at 2.5 x 105 and 2 x 106 cells/dishes in complete medium. Forty-eight h after plating, cells were lysed with lysis buffer. Equal amounts of total proteins were immunoprecipitated using anti-LMW-PTP antibodies. Immunocomplexes were separated by SDS-PAGE, and a Western blot with anti-ß-catenin antibodies was then performed. The blot was then reprobed with anti-LMW-PTP antibodies. For normalization, 25 µg of total proteins of each sample were separated by SDS-PAGE and a Western blot with anti-ß-catenin antibodies was performed. The results are representative of five experiments. kDa, Mr in thousands.

 
LMW-PTP Causes an Increase in Cadherin-mediated Cell-Cell Adhesion.
In normal epithelial cells, ß-catenin associates with E-cadherin, and tight cell-cell contacts are maintained. On the other hand, a loss of E-cadherin expression or an increased level of ß-catenin phosphorylation leads to a decrease in cell-cell adhesion (10 , 12 , 49) . This loss of ß-catenin-E-cadherin association correlates with cancer progression and may contribute to a more metastatic phenotype (24) . For this reason, the effect of LMW-PTP overexpression on the formation of cell-cell contacts has been further investigated.

Clones expressing equal amounts of dn- or wtLMW-PTP and comparable levels of N- and P-cadherins, which are the most representative cadherins in NIH3T3 fibroblasts (50) , were selected to be studied for their adhesive properties. Fig. 6ACitation shows the expression level of LMW-PTP in dnLMW-PTP, wtLMW-PTP, and mock-transfected cells and the N- and P-cadherins level in the same clones. To assess whether our clones have conserved, after transfection, the specific properties of these molecules with no alterations of their surface expression, we examined their sensitivity to trypsin digestion in the presence or absence of calcium. Indeed, one of the main characteristics of cadherins is their sensitivity to trypsin in the absence of calcium and the resistance to trypsin degradation in the presence of calcium. The results clearly indicate that calcium is able to protect, almost completely, cadherins from trypsin digestion in all of the cell lines. Accordingly, in the absence of calcium the Mr 135,000 band, corresponding to N-cadherin, and the Mr 120,000 band, corresponding to P-cadherin, are no longer apparent (Fig. 6B)Citation . We then used the selected clones in an aggregation assay as described in "Materials and Methods." Fig. 6CCitation shows that overexpression of wtLMW-PTP leads to a decrease in the number of nonaggregated cells within 40 min, in comparison with mock-transfected cells, whereas overexpression of dnLMW-PTP induces the opposite phenotype. Photographs, taken during the aggregation assay, show that overexpression of wtLMW-PTP leads to the formation of large aggregates, whereas only small aggregates are formed in dnLMW-PTP-overexpressing cells (Fig. 6D)Citation . To clarify this phenomenon, [2-3H]glycerol-radiolabelled NIH3T3 cells, expressing either wild-type or dnLMW-PTP and mock-transfected cells as a control, were assayed for their ability to adhere to a confluent monolayer of NIH3T3 cells. The presence of a confluent cell monolayer eliminates the adhesion that is attributable to extracellular matrix and that is not dependent on cell-cell adhesion. Overexpression of wtLMW-PTP causes an increase in cell-cell adhesion with respect to mock-transfected cells, whereas dnLMW-PTP overexpression induces the opposite effect (Fig. 6E)Citation . To demonstrate whether the observed adhesion is specifically dependent on N- and P-cadherins localized on fibroblasts plasmamembrane, we performed an adhesion assay using antibodies directed against the extracellular portion of the cadherin transmembrane receptor. In agreement with our previous experiments, Fig. 6, F and GCitation , shows that wtLMW-PTP overexpression causes cell-cell interactions to be strongly increased with respect to mock-transfected cells, whereas dnLMW-PTP overexpression causes the opposite effect. Furthermore, the results indicate that this interaction is dependent on both surface N- and surface P-cadherins because it is completely abolished if cells are saturated with anti-N- and anti-P-cadherin antibodies before the aggregation assay is carried out.




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  		Fig. 6. Cell-cell adhesion properties of wt- and dnLMW-PTP-transfected NIH3T3 cells. A, LMW-PTP level and N- and P-cadherins level in wtLMW-PTP, dnLMW-PTP, and mock-transfected NIH3T3 cells. Total proteins (30 µg) from confluent wtLMW-PTP, dnLMW-PTP. and mock-transfected NIH3T3 cells, lysed in lysis buffer, were assayed by Western blot with anti-LMW-PTP or anti-N-cadherin or anti-P-cadherin antibodies. kDa, Mr in thousands. B, trypsin digestion of dnLMW-PTP, wtLMW-PTP, and mock-transfected NIH3T3 cells. Monolayers of wtLMW-PTP, dnLMW-PTP, and mock-transfected NIH3T3 cells were incubated without (Lanes 1–3) or with 0.005% trypsin for 20 min at 37°C in the presence of 2 mM Ca2+ (Lanes 4–6) or 2 mM EGTA (Lanes 7–9). Digestion products were dissolved in sample buffer and analyzed by Western blot with anti-N-cadherin or P-cadherin antibodies. kDa, Mr in thousands. C, single cells from wtLMW-PTP, dnLMW-PTP, and mock-transfected NIH3T3 cells were allowed to aggregate under constant shaking as described in "Material and Methods." After 5, 10, 20, and 40 min, cells were withdrawn, and the number of single cells was counted. The rate of aggregation was calculated by the percentage of decrease in the number of single cells using the formula [(No - N1)/No] x 100. Each value is the average of four independent experiments. *, significantly different from the control (P < 0.05, Student’s t test). D, phase-contrast micrographs of cell aggregates of wtLMW-PTP- and dnLMW-PTP-NIH3T3-overexpressing cells. Cell aggregates are shown after 40 min of incubation at 37°C under gentle shaking. Bar, 20 µm. E, 3 x 104 wtLMW-PTP, dnLMW-PTP, and mock-transfected cells/well, labeled with [2-3H]glycerol, were incubated in complete medium for 15 min at 37°C with monolayers of confluent NIH3T3 cells. After a wash with PBS, cells were removed by solubilization in NaOH, and radioactivity was evaluated by liquid scintillation counting. The values are normalized on the basis of the radioactivity incorporated by each clone. Results represent the average of three independent experiments. *, significantly different from the control (P < 0.05, Student’s t test). F and G, 3 x 104 wtLMW-PTP, dnLMW-PTP, and mock-transfected NIH3T3 cells/well were incubated in complete medium for 15 min at 37°C with 5 µg/well anti-N-cadherin antibodies (F) or P-cadherin antibodies (G) immobilized on tissue culture 96-well plates. Before the adhesion to anti-N- or P-cadherin antibodies coated-wells, cells were incubated with or without 100 µg/ml anti-N- or -P-cadherin antibodies for 30 min at 37°C. The number of cells adhering to anti-N- or -P-cadherin antibodies was evaluated with crystal violet staining. Results represent the average of three measurements. *, significantly different from the control (P < 0.05, Student’s t test). H, 3 x 104 MCF-7 cells transiently transfected with wtLMW-PTP, dnLMW-PTP, and mock cells were incubated for 30 min at 37°C with 5 µg/well anti-E-cadherin antibodies immobilized on tissue-culture 96-well plates. Before the adhesion to anti-E-cadherin antibody-coated wells, cells were incubated with or without 100 µg/ml anti-E-cadherin antibodies for 30 min at 37°C. The number of cells adhering to anti-E-cadherin antibodies was evaluated with crystal violet staining. Results represent the average of four measurements. *, significantly different from the control (P < 0.05, Student’s t test).

 
We next investigated the cell adhesion properties of MCF-7 cells transfected with wtLMW-PTP and dnLMW-PTP to observe whether LMW-PTP could also affect the adhesive capabilities of neoplastic cells. MCF-7 breast adenocarcinoma cells, which possess well-organized E-cadherin/ß catenin-complexes (51) , were transiently transfected with the active and dominant-negative form of LMW-PTP, and an adhesion assay with anti-E-cadherin antibodies was performed 24 h after transfection. By means of pEGFP-N1 transfection, we evaluated the transfection efficiency at ~50%, and an equal expression level of wtLMW-PTP and dnLMW-PTP was checked by Western blot analysis (data not shown). As shown in Fig. 6HCitation , wtLMW-PTP overexpression caused cell-cell interactions to be strongly increased with respect to mock-transfected cells, whereas dnLMW-PTP overexpression caused the opposite effect. These effects were dependent on E-cadherin because they were completely abolished if cells were saturated with anti-E-cadherin antibodies before the aggregation assay was carried out. Results indicate that LMW-PTP is able to interfere with the adhesive properties of this neoplastic cell line also, which demonstrates that the effect of LMW-PTP is not restricted to a single cell line but is a widespread phenomenon.

Taken together, these data suggest that LMW-PTP can potentiate the intracellular adhesion causing a strong association between cadherin and ß-catenin, not only in normal cells, but also in transformed cells, which, thus, suggests a possible inhibitory role of LMW-PTP in tumor progression

Redox Regulation During Cell Confluence.
Recently, the mitogenic properties of oxidants have been emphasized, and a possible involvement of ROS in proliferative disorders such as cancer and atherosclerosis has been postulated. It is well known that decreased ROS production during cell confluence is correlated to a decrease in the levels of protein tyrosine phosphorylation associated with an increased PTP activity (29 , 52) . Recently, Chiarugi et al. (41) demonstrated that the LMW-PTP activity is regulated by exogenous and endogenous production of H2O2 after platelet-derived growth factor treatment. To assess whether, during cell confluence, the increase in the expression level of LMW-PTP was not the only functional regulator of the enzyme, we investigated whether the density-dependent decrease in ROS production could lead to LMW-PTP enzymatic activation through the reduction of the SH groups in the catalytic site of the phosphatase. The redox regulation of LMW-PTP in confluent and growing NIH3T3 cells was investigated by 5'-F-IAA labeling. The results show that LMW-PTP is much more oxidized in growing cells with respect to the ones undergoing contact inhibition (Fig. 7A)Citation . Moreover, cytofluorimetric analysis indicates a decrease in ROS levels when cells are grown to confluence with respect to exponentially growing ones (Fig. 7B)Citation . These results suggest that the decrease of intracellular ROS observed in dense cultures could be responsible for the increase of the reduced form of LMW-PTP.



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  		Fig. 7. LMW-PTP level and redox state in sparse and dense NIH3T3 fibroblasts. A, LMW-PTP was immunoprecipitated from sparse and dense cells as reported in Fig. 1ACitation . Immunocomplexes were treated with 5'-F-IAA as indicated in "Material and Methods." An antifluorescein immunoblot was then performed. Normalization has been obtained by reprobing the blot with anti-LMW-PTP antibodies. Band intensities of both antifluorescein and anti-LMW-PTP immunoblots were quantitated. The histogram in B represents the ratio between these two values, taking as 100% the value of the reduced form of LMW-PTP in dense cultures. B, intracellular ROS in sparse and dense cultures: 1 x 105 and 1 x 106 wtLMW-PTP NIH3T3 cells were seeded onto 35-mm cell culture dishes and cultured in complete medium. After 48 h, cells were treated with DCF-DA, and a cytofluorimetric analysis was performed. C, LMW-PTP specific activity: 2.5 x 105 and 2 x 106 wtLMW-PTP NIH3T3 cells were seeded onto 10-cm dishes in complete medium. After 48 h, LMW-PTP was immunoprecipitated and a PTP activity assay was performed using PNPP. Values were normalized on the basis of the LMW-PTP level in sparse and dense cultures evaluated by Western blot analysis. The results are representative of three experiments. *, significantly different from the value for sparse cells (P < 0.05, Student’s t test).

 
We next analyzed the PTP activity of LMW-PTP in sparse and dense cultures using p-nitrophenylphosphate (PNPP) as a substrate. Results demonstrate a 2-fold increase in LMW-PTP specific activity in dense cells compared with sparse cultures (Fig. 7C)Citation . These results clearly show that, when cells reach confluence, in addition to an up-regulation of LMW-PTP expression, the LMW-PTP specific activity also increases, which suggests a possible positive role for LMW-PTP in controlling the growth arrest and cell-cell contacts induced by cell confluence.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tyrosine phosphorylation of ß-catenin has been proposed as a mechanism for regulating the stability of the adherens junctions. Indeed, increased phosphorylation of ß-catenin has been associated with a decrease in adhesive junctional strength, because of the dissociation of cadherin-cytoskeleton interaction (53) . It has also been shown that increased tyrosine phosphorylation of ß-catenin results in its dissociation from the cadherin complex and in increased amount of ß-catenin free cytoplasmic pool (20 , 46) . Indeed, although ß-catenin tyrosine phosphorylation is correlated with a loss of cadherin function, the connection between ß-catenin and cadherin is not always lost (12 , 14 , 19 , 49 , 54 , 55) . Furthermore, the loss of ß-catenin/E-cadherin association correlates directly with tumor invasion and metastasis, and deregulation of ß-catenin tyrosine phosphorylation has been altered in melanoma cells expressing proteasome-resistant ß-catenin (23 , 24) . The phosphorylation level of ß-catenin and other members of the cadherin-associated complex, such as p120ctn or plakoglobin, is attributable to the equilibrium between the action of tyrosine kinases and phosphotyrosine phosphatases. Exposure to a number of growth factors results in direct (through receptor tyrosine kinases) or indirect (e.g., through Src tyrosine kinases) tyrosine phosphorylation of both catenin and p120 family members. Furthermore, transfection of a v-src oncogene (10 , 49) causes tyrosine phosphorylation of ß-catenin and loss of adhesive abilities. On the other hand, interaction with PTPs, such as PTPµ, PTP{kappa}, PTP1B, and LAR-PTP results in the dephosphorylation of ß catenin and p120 family members, with a subsequent increase in cell cohesiveness (20 , 30 , 33 , 34) . In accordance with these data, the inhibition of tyrosine phosphorylation has been shown to promote adherens junction formation in src-expressing cells (56) .

Our results are along the lines of this general view. We provide evidence that cell-cell contacts at the adherens junction level and ß-catenin/cadherin association are influenced by LMW-PTP expression.

Indeed, the amount of free cytoplasmic ß-catenin is reduced in LMW-PTP-overexpressing cells compared with cells expressing dnLMW-PTP (Fig. 1)Citation . In agreement, we observed an increased level of association between cadherins and the actin network by wtLMW-PTP overexpression (Fig. 2)Citation . Roura et al. (21) demonstrated that tyrosine phosphorylation of ß-catenin on residue 654 decreases the association between ß-catenin and E-cadherin. Furthermore, it is well known that tyrosine phosphorylation of ß-catenin causes a loss of cadherin function, because cadherins dissociate from the actin cytoskeleton. We suggest that LMW-PTP acts on cadherin-associated ß-catenin at the adherens junction level, thus determining the retention of ß-catenin at the membranes and causing a stronger association between cadherin and ß-catenin. The confocal microscope confirms the molecular data because wtLMW-PTP- overexpressing NIH3T3 cells possess well-organized cadherin/catenin complexes in which ß-catenin is prevalently associated in proximity to cell-cell contacts. On the contrary, dnLMW-PTP-overexpressing cells show less structured complexes, and ß-catenin appears to be dissociated from the plasmamembrane and more dislocated in the cytosol (Fig. 3)Citation . Confocal microscopy experiments provided further evidence of the possible interaction between ß-catenin and LMW-PTP, showing the colocalization of ß-catenin and LMW-PTP at the plasmamembrane (Fig. 4)Citation . Furthermore, we have shown, both in vitro and in vivo, the association between ß-catenin and LMW-PTP (Fig. 5)Citation . In the cell, the association of ß-catenin with LMW-PTP is dependent on cell density, because it is greatly increased when cells reach confluence. This event is attributable to the fact that the expression of LMW-PTP increases with cell density as shown in Fig. 5BCitation . This result supports the idea that LMW-PTP plays a crucial role in maintaining cadherin/catenin complexes in a functional state when cells reach high density and need to stabilize cell-cell contacts. Furthermore, the expression levels of many other PTPs, such as PTPµ, PTP{kappa}, LAR PTP, and DEP-1, are up-regulated during cell confluence (30 , 57, 58, 59) , in agreement with the increase in tyrosine phosphatase activity observed in confluent cells and associated with the stabilization of cell-cell contacts. In this view, PTPs would antagonize kinases in regulating adherens junctions; in fact, tyrosine kinases phosphorylate ß-catenin, dissociating cadherins from the actin cytoskeleton and, thus, leading to a decrease in intercellular contacts (10 , 12 , 16 , 49) . For this reason, it has been suggested that a correlation exists between tyrosine phosphorylation, the disruption of contacts between cadherins and cytoskeleton, and the increase of free ß-catenin.

In addition, we demonstrated that when cells are grown to high density, the specific activity of LMW-PTP is increased, because of redox regulation (Fig. 7)Citation . Recently, growing evidence has indicated ROS as mitogens for mammalian cells, and ROS have been shown to operate on the stimulation with several growth factors and cytokines. For this reason, in a manner analogous to reversible protein phosphorylation, the reversible oxidation of several proteins involved in signal transduction may be important in the regulation of cellular response (60) . Among these, PTPs are a potential target for both exogenous and endogenous ROS. In fact, tyrosine phosphatases are sensitive to redox regulation and are easily inactivated by the oxidation of a critical cysteine residue located in the catalytic site (61) . Indeed, PTP1B is rapidly and transiently inactivated by ROS after epidermal growth factor (EGF) receptor stimulation (62) . Previous data obtained by Chiarugi et al. (41) showed that LMW-PTP is oxidized and, hence, inactivated, by endogenous and exogenous H2O2. In the cellular model used in this work, ROS intracellular level decreases with cell density, similarly to data previously reported by Pani et al. (29) . This leads to an increase in the reduced/active form of LMW-PTP and to an enhancement of its specific activity in dense cells. Recently, Fiaschi et al. (63) observed that, during both myogenesis and neurogenesis, LMW-PTP levels and activity are increased. These data indicate that processes that require growth arrest and exit from the cell cycle, such as cell differentiation and cell contact inhibition, are influenced by LMW-PTP.

Furthermore, we investigated in detail whether cell-cell contact regulation could be influenced by LMW-PTP. We show that cellular aggregation (Fig. 6, C–D)Citation and cell-cell adhesion (Fig. 6, E–H)Citation are positively regulated by overexpression of LMW-PTP. Recently, Stein et al. (64) demonstrated that LMW-PTP is recruited to ephrin receptor complexes at cell-cell contact sites. These data suggest that the adhesiveness of cells is positively influenced by LMW-PTP, probably because of the modification of the tyrosine phosphorylation levels of molecules present at the adherens junctions. Indeed, the level of the free cytoplasmic pool of ß-catenin is decreased in LMW-PTP-expressing cells, an event that correlates with an increased adhesive junctional strength and an increased association between cadherin and actin-based cytoskeleton. The increased dissociation between cadherins and ß-catenin observed in several cancer cell lines (24) leads us to think that LMW-PTP possibly plays an inhibitory role in events such as malignant transformation and metastasis. In addition, we showed that LMW-PTP has a positive effect on cell-cell adhesion formation in the neoplastic cell line MCF-7, which, thus, supports the general idea that LMW-PTP could protect from tumor progression. In fact, the integrity of the cadherin-catenin complex is crucial because down-regulation of any of its components results in the loss of the tumor-suppressive action of adherens junctions (6) . On the basis of these results, we can conclude that cell-cell adhesion is strengthened by LMW-PTP through a general reorganization of the association between cadherins and cytoskeleton. This is probably caused by the control of ß-catenin tyrosine phosphorylation at the adherens junction level. We think that the dephosphorylation of ß-catenin induced by LMW-PTP causes ß-catenin to remain associated with cadherins and to maintain a strong connection between actin and cadherin, thus strengthening the intercellular association.

These results are particularly relevant suggesting an involvement of LMW-PTP in processes that require the remodeling of cell-cell contacts such as tumor progression and metastasis.


   ACKNOWLEDGMENTS
 
We thank Daniele Nosi for technical assistance. We are grateful to Drs. Guido Camici, Fabrizio Chiti, and Fabio Marra for critically reading the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Consiglio Nazionale delle Ricerche (C. N. R.; Grant 97.03810.CT14, target project on Biotechnology, Strategic project "Controlli post-trascrizionali dell’espressione genica," and MURST-C. N. R. Biotechnology program L.95/95), by the Italian Association for Cancer Research (AIRC), and in part by the Ministero della Università e Ricerca Scientifica e Tecnologica (MURST-PRIN 2000), and by Cassa di Risparmio di Firenze. Back

2 To whom requests for reprints should be addressed, at Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, viale Morgagni 50, 50134 Firenze, Italy. Phone: 39-055-413765; Fax: 39-055-4222725; E-mail: ramponi{at}scibio.unifi.it Back

3 The abbreviations used are: PTP, protein tyrosine phosphatase; DCF-DA, dichlorofluorescein-diacetate; LMW, low molecular weight; dnLMW-PTP, dominant-negative LMW-PTP; 5'-F-IAA, iodoacetamide-fluorescein; GST, glutathione S-transferase; PNPP, nitrophenylphosphate; ROS, reactive oxygen species; wtLMW-PTP, wild-type LMW-PTP; RIPA, radioimmunoprecipitation assay; RPTP, receptor protein tyrosine phosphatase. Back

Received 3/11/02. Accepted 9/20/02.


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 DISCUSSION
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