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• W2037291219 abstract "The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPα, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-α. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPα expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPα-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPα in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPα, and there was evidence of association between PTPα and Src kinase(s). PTPα expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125FAK. In addition, paxillin, a Src and/or pp125FAK substrate, displayed increased levels of tyrosine phosphorylation in PTPα-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPα-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPα may have a role in regulating cell-substratum adhesion. The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPα, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-α. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPα expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPα-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPα in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPα, and there was evidence of association between PTPα and Src kinase(s). PTPα expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125FAK. In addition, paxillin, a Src and/or pp125FAK substrate, displayed increased levels of tyrosine phosphorylation in PTPα-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPα-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPα may have a role in regulating cell-substratum adhesion. extracellular matrix protein-tyrosine phosphatase epidermal growth factor focal adhesion kinase insulin receptor monoclonal antibody piperazine-N, N′-bis 2-ethanesulfonic acid glutathione S-transferase malachite green microtiter plate 2-(N-morpholino)ethanesulfonic acid control polyacrylamide gel electrophoresis phosphate-buffered saline. Reversible protein phosphorylation is a widely employed mechanism for regulating enzyme activity, assembly, and localization of protein complexes and gene transcription within eukaryotic cells (1Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2211) Google Scholar, 2Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (922) Google Scholar). Protein phosphorylation also controls cytoskeleton organization during cell adhesion to extracellular matrix (ECM)1 or to other cells during processes such as morphogenesis, cell migration, differentiation, and metastases (3Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6355) Google Scholar). Indeed, similar to growth factor receptor-mediated signal transduction, engagement of cell adhesion molecules is followed by the rapid activation of specific protein tyrosine kinases and the ensuing assembly of multimeric protein complexes at sites of cell adhesion (4Schwartz M.T. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar). Moreover, many of the signal transduction molecules activated or phosphorylated in response to ligand binding to growth factor receptors are also regulated by cell adhesion (5Clark E.A. Brugge J.S. Science. 1995; 268: 233-238Crossref PubMed Scopus (2802) Google Scholar, 6Miyamoto S. Teramoto H. Coso O.A. Gutland J.S. Burbelo P.D. Akiyama S.K. Yamada K.M. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1100) Google Scholar). Due to their ability to regulate protein phosphotyrosine levels, PTPs are undoubtedly essential to such processes as proliferation, differentiation, and cell adhesion (7Charbonneau H. Tonks N.K. Annu. Rev. Cell Biol. 1992; 8: 463-493Crossref PubMed Scopus (296) Google Scholar, 8Mauro L.J. Dixon J.E. Trends Biochem. Sci. 1994; 19: 151-155Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 9Fashena S.J. Zinn K. Curr. Biol. 1995; 5: 1367-1369Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). However, the roles of specific PTPs in the regulation of growth factor receptor initiated signal transduction events and cell adhesion remain poorly understood. In this study, we have evaluated the ability of the receptor-like phosphatase PTPα to regulate EGF-receptor-dependent cell signaling processes in the human epidermoid carcinoma cell line A431. Structurally, the widely expressed PTPα is composed of a heavilyN- and O-glycosylated 123- or 132-residue alternatively spliced extracellular domain, a transmembrane region, and two tandem cytoplasmic phosphatase domains, as is characteristic of the majority of receptor-like PTPs (10Jirik F.R. Janzen N.M. Melhado I.G. Harder K.W. FEBS Lett. 1990; 273: 239-242Crossref PubMed Scopus (28) Google Scholar, 11Daum G. Regenass S. Sap J. Schlessinger J. Fischer E.H. J. Biol. Chem. 1994; 269: 10524-10528Abstract Full Text PDF PubMed Google Scholar, 12Sap J. D’Eustachio P. Givol D. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6112-6116Crossref PubMed Scopus (142) Google Scholar, 13Matthews R.J. Cahir E.D. Thomas M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4444-4448Crossref PubMed Scopus (125) Google Scholar). A431 cells have been well characterized with respect to EGF-dependent signal transduction. These cells express high levels of the EGF receptor on their surface (0.5–3.0 × 106/cell) (14Gill G.N. Lazar C.S. Nature. 1981; 293: 305-307Crossref PubMed Scopus (321) Google Scholar), and proliferate in response to autocrine production of transforming growth factor α (15Van de Vijver M. Kumar R. Mendelsohn J. J. Biol. Chem. 1991; 266: 7503-7508Abstract Full Text PDF PubMed Google Scholar). Although EGF enhances the growth of A431 cell-derived tumors in nude mice, when grown as a monolayer in culture, these cells, similar to squamous cell carcinoma cell lines overexpressing EGF receptors, are inhibited by high concentrations of EGF (14Gill G.N. Lazar C.S. Nature. 1981; 293: 305-307Crossref PubMed Scopus (321) Google Scholar, 16Barnes D.W. J. Cell Biol. 1982; 93: 1-4Crossref PubMed Scopus (233) Google Scholar, 17Ginsburg E. Vonderhaar B.K. Cancer Lett. 1985; 28: 143-150Crossref PubMed Scopus (29) Google Scholar, 18MacLeod C.L. Luk A. Castagnola J. Cronin M. Mendelson J. J. Cell. Physiol. 1986; 127: 175-182Crossref PubMed Scopus (51) Google Scholar, 19Santon J.B. Cronin M.T. MacLeod C.L. Mendelson J. Masui H. Gill G.N. Cancer Res. 1986; 46: 4701-4705PubMed Google Scholar, 20King I.C. Sartorelli A.C. Biochem. Biophys. Res. Commun. 1986; 140: 837-843Crossref PubMed Scopus (32) Google Scholar, 21Kamata N. Chida K. Rikimaru K. Horikoshi M. Enomoto S. Kuroki T. Cancer Res. 1986; 46: 1648-1653PubMed Google Scholar). In addition, EGF stimulation of A431 cells causes dramatic change in cell morphology, including extensive membrane ruffling, filipodia extension, and changes in cytoskeletal organization and cell adhesion. These processes culminate in the rounding-up and retraction of these cells from the substratum (22Chinkers M. McKanna J.A. Cohen S. J. Cell Biol. 1979; 83: 260-265Crossref PubMed Scopus (197) Google Scholar, 23Schlessinger J. Geiger B. Exp. Cell Res. 1981; 134: 273-279Crossref PubMed Scopus (101) Google Scholar, 24Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar). Thus, A431 cells have proven invaluable to studies of cell growth, differentiation, and cell adhesion as regulated by receptor tyrosine kinase activity. We found that PTPα expression in A431 cells led to a PTPα D1-dependent increase in cell-substratum adhesion and inhibited EGF-induced cell rounding and lift-off. This PTPα-dependent phenotype was not restricted to EGF stimulated A431 cells, as PTPα expression in BHK-IR cells also led to resistance of these cells to insulin-induced cell rounding and detachment from the substratum. These results suggest that PTPα might be capable of regulating the activities of molecules involved in cell-substratum adhesion. In keeping with this hypothesis, we found that in A431 cells PTPα could be co-immunoprecipitated with Src kinase(s). PTPα expression was also associated with the dephosphorylation and/or activation of specific Src kinases. Moreover, Src kinases immunoprecipitated from PTPα-expressing A431 cells were associated with elevated levels of focal adhesion kinase (FAK), a molecule activated by stimuli such as integrin-dependent cell adhesion to the substratum, v-src transformation, and growth factor or neuropeptide stimulation (reviewed in Ref. 25Zachary I. Rozengurt E. Cell. 1992; 71: 891-894Abstract Full Text PDF PubMed Scopus (384) Google Scholar). PTPα expression was also associated with an increase in the tyrosine phosphorylation of paxillin, a protein localized to focal adhesions and a putative substrate of FAK and/or Src kinases (26Glenney J.R. Zokas L. J. Cell Biol. 1989; 108: 2401-2408Crossref PubMed Scopus (354) Google Scholar, 27Turner C.E. Glenney J.R. Burridge K. J. Cell Biol. 1990; 111: 1059-1068Crossref PubMed Scopus (522) Google Scholar, 28Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (498) Google Scholar). Paxillin obtained from PTPα-expressing cells was complexed with increased levels of Csk, suggesting a possible feedback loop in the regulation of Src activity in these cells and supporting previous observations linking the activation of Src kinases with changes in the intracellular localization of Csk (29Sabe H. Okada M. Nakagawa H. Hanafusa H. Mol. Cell. Biol. 1992; 12: 4706-4713Crossref PubMed Scopus (84) Google Scholar, 30Howell B.W. Cooper J.A. Mol. Cell. Biol. 1994; 14: 5402-5411Crossref PubMed Scopus (122) Google Scholar, 31Sabe H. Hata A. Okada M. Nakagawa H. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3984-3988Crossref PubMed Scopus (212) Google Scholar, 32Thomas S.M. Soriano P. Imamoto A. Nature. 1995; 376: 267-271Crossref PubMed Scopus (303) Google Scholar, 33Sabe H. Shoelson S.E. Hanafusa H. J. Biol. Chem. 1995; 270: 31219-31224Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The dephosphorylation of Src kinases, together with the observed PTPα D1-dependent changes in cell adhesion and the tyrosine phosphorylation and association of FAK and paxillin with Src and Csk kinases, support the hypothesis that PTPα may be involved in the regulation of cell-substratum adhesion. A431 cells were obtained from the American Type Culture Collection and were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, antibiotics, and 50 μm β-mercaptoethanol. The PTPα cDNA was obtained from a human HepG2 cell line cDNA library (Stratagene) as described previously (10Jirik F.R. Janzen N.M. Melhado I.G. Harder K.W. FEBS Lett. 1990; 273: 239-242Crossref PubMed Scopus (28) Google Scholar). The wild type and catalytically inactive forms of PTPα (containing a cysteine-to-alanine mutation at residue 433 within the first catalytic domain, D1 C433A) were subcloned into the eukaryotic expression vector pBCMGNeo. This vector contains the cytomegalovirus immediate-early gene promoter and 79% of the bovine papilloma virus genome, allowing episomal replication of transfected plasmids (34Karasuyama H. Melchers F. Eur. J. Immunol. 1988; 18: 97-101Crossref PubMed Scopus (1076) Google Scholar). Plasmids were introduced into A431 cells by electroporation, and G418 resistant clones were selected. The BHK cell line overexpressing the human insulin receptor (BHK-IR) (35Anderson A.S. Kjeldsen T. Wiberg F.C. Vissing H. Schaffer L. Rasmussen J.S. De Meyts P. Moller N.P.H. J. Biol. Chem. 1992; 267: 13681-13686Abstract Full Text PDF PubMed Google Scholar) was maintained at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose, 10% fetal calf serum, 2 mm l-glutamine, 1 μm methotrexate, and penicillin/streptomycin. This cell line was used to establish stable BHK-IR cell lines overexpressing PTPα in a functionally dependent way as described previously (36Moller N.P.H. Moller K.B. Lammers R. Kharitonenkov A. Hoppe E. Wiberg F.C. Sures I. Ullrich A. J. Biol. Chem. 1995; 270: 23126-23131Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). BHK-IR/PTPα cells were maintained in complete medium in the presence of 100 nm insulin. PTPα-specific antibodies were produced by immunization of New Zealand White rabbits with recombinant PTPα cytoplasmic domain containing residues 167–793 (PTPα-2) or with N-terminal cysteine-linked keyhole limpet hemocyanin conjugated synthetic peptides (37Ziltener H.J. Clark-Lewis I. Hood L.E. Kent S.B.H. Schrader J.W. J. Immunol. 1987; 138: 1099-1104PubMed Google Scholar), corresponding to amino acids 20–60 within the extracellular domain of PTPα (PTPα-ext), or residues 512–558, corresponding to the region separating the two catalytic domains (PTPα-1). Antibodies were affinity purified on thiol-Sepharose peptide or CNBr-Sepharose recombinant PTPα specific affinity columns. The anti-Src mAb 327 (provided by J. Brugge, ARIAD Pharmaceuticals), anti-Fyn mAb (provided by R. Perlmutter, University of Washington, Seattle, WA), anti-Yes antiserum (from J. Bolen, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ), and anti-Csk antiserum (from J. Cooper and B. W. Howell, Fred Hutchinson Cancer Research Center) were used to immunoprecipitate each kinase. The antibody SRC2 (Santa Cruz Biotechnology), specific for the C-terminal peptide sequence 509–533 of Src and the conserved sequences of Fyn and Yes, was also used to immunoprecipitate and immunoblot Src, Fyn, and Yes. Antibodies against phosphotyrosine (4G10), paxillin, FAK, and Src (GD11) were obtained from Upstate Biotechnology Inc. and Transduction Laboratories. Anti-Lyn kinase antibodies were obtained from Santa Cruz Biotechnology Inc. A431 cells were lysed on ice for 30 min in buffer containing 1% Nonidet P-40, 10% glycerol, 50 mmNaCl, 50 mm Tris, pH 7.5, 2 mm EDTA, 1 mm sodium orthovanadate, 10 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 1 mmsoybean trypsin inhibitor, and 100 μm leupeptin. Insoluble cellular debris was removed by ultracentrifugation at 30,000 × g for 30 min. Protein concentrations were estimated with the bicinchoninic acid assay (Pierce). Src kinase activity from transfected A431 clones was assessed by autokinase and enolase assays. Src kinase was immunoprecipitated from 500 μg of cell lysate with 1 μg of mAb 327. Immune complexes were collected with 75 μl of a 30% slurry of rabbit anti-mouse IgG preabsorbed protein A-Sepharose. Beads were washed in cell lysis buffer, radioimmune precipitation buffer (lysis buffer containing 1% Nonidet P-40, 0.5% deoxycholic acid, and 0.1% SDS), and then kinase buffer (100 mm PIPES, pH 7.0, 5 mm MnCl2, and 10 μm vanadate) before resuspension in kinase buffer containing 25 μm ATP and 10 μCi [γ-32P]ATP (3000 Ci/mmole) with or without 10 μg of acid-denatured enolase. Kinase assays were performed at 25 °C for 15 min before termination by addition of 2× Laemmli buffer. 50% of each immunoprecipitate was immunoblotted with anti-Src antiserum (GD11) to ensure that equal quantities of Src were used in each assay. Regions corresponding to the entire cytoplasmic domain of PTPα (PTPα-D1+D2, amino acids 167–793), the first catalytic domain (PTPα-D1, amino acids 167–555), and the C-terminal phosphatase domain (PTPα-D2, amino acids 510–793), were polymerase chain reaction-amplified with Vent DNA polymerase (New England Biolabs). The cDNA sequence of each PTPα fragment was confirmed by DNA sequencing before subcloning into pGex 2T-tag, a modified pGex 2T plasmid, containing an expanded polylinker and sequence encoding the 10-residue hemagglutinin epitope tag derived from influenza virus (38Harder K.W. Owen P. Wong L.K.H. Aebersold R. Clark-Lewis I. Jirik F.R. Biochem. J. 1994; 298: 395-401Crossref PubMed Scopus (182) Google Scholar). Thrombin cleavage of glutathioneS-transferase (GST) fusion proteins generates proteins containing the hemagglutinin epitope at the N terminus. Luria broth cultures (500 ml) of UT5600 bacteria (New England Biolabs) containing the various pGex plasmids were grown to absorbance 0.6–0.9 at 37 °C. Cells were then shifted to 26 °C and induced overnight with 100 μmisopropyl-1-thio-β-d-galactopyranoside. Bacteria were sedimented and lysed by sonication in buffer composed of 50 mm Tris, pH 7.5, 150 mm NaCl, 5 mmβ-mercaptoethanol and 1 mm phenylmethylsulfonyl fluoride. Triton X-100 was then added to 1% final concentration, and cellular debris were removed by ultracentrifugation at 30,000 ×g. The supernatant was removed and incubated with 1 ml of a 50% slurry of glutathione-Sepharose (Amersham Pharmacia Biotech) for 1 h. The beads were thoroughly washed before cleavage in 1 ml of buffer containing 50 mm Tris, pH 8.0, 2.5 mmCaCl2, 150 mm NaCl, 10 mmβ-mercaptoethanol, and 50 μl of thrombin (400 μg/ml). Glycerol was added to a final concentration of 15% before storage at −80 °C. Phosphatase activity was determined using the malachite green microtiter plate (MGMP) phosphatase assay to detect the release of phosphate from phosphotyrosine-containing synthetic peptides as described previously (38Harder K.W. Owen P. Wong L.K.H. Aebersold R. Clark-Lewis I. Jirik F.R. Biochem. J. 1994; 298: 395-401Crossref PubMed Scopus (182) Google Scholar). Briefly, recombinant PTPα or PTPα immunoprecipitated directly from A431 cell lysates was incubated with phosphopeptides in buffer containing 25 mm MES, pH 6.0, and 0.1 mmβ-mercaptoethanol. Enzyme reactions were carried out in half-volume microtiter plates (Costar) in a final volume of 25 μl for the indicated times. Phosphatase reactions were terminated and free phosphate detected by addition of 100 μl of malachite green solution to each well. Changes in absorbance at 620 nm of each well were measured in an enzyme-linked immunosorbent assay plate reader and phosphate release was determined by comparison to a standard curve (38Harder K.W. Owen P. Wong L.K.H. Aebersold R. Clark-Lewis I. Jirik F.R. Biochem. J. 1994; 298: 395-401Crossref PubMed Scopus (182) Google Scholar). Phosphopeptides Src-527YTSTEPQpYQPGENL and CSF-1 receptor 708YIHLEKKpYVRRDSG were synthesized as described previously (38Harder K.W. Owen P. Wong L.K.H. Aebersold R. Clark-Lewis I. Jirik F.R. Biochem. J. 1994; 298: 395-401Crossref PubMed Scopus (182) Google Scholar, 39Clark-Lewis I. Moser B. Walz A. Baggiolini M. Scott G.J. Aebersold R. Biochemistry. 1991; 30: 3128-3135Crossref PubMed Scopus (134) Google Scholar). The activity of PTPα-D2 towardpara-nitrophenylphosphate (40Wang Y. Pallen C.J. EMBO J. 1991; 10: 3231-3237Crossref PubMed Scopus (102) Google Scholar) was detected using the MGMP assay as above. Passage number 10 or less control vector alone-transfected and PTPα-overexpressing A431 cell lines were plated at 104 cells/well in quadruplicate in 96-well plates. EGF was added, and cell proliferation was determined 4 days later by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Boehringer Mannheim). The effect of PTPα expression on the growth characteristics of BHK-IR cells was analyzed by comparing growth curves obtained with and without insulin as described previously (36Moller N.P.H. Moller K.B. Lammers R. Kharitonenkov A. Hoppe E. Wiberg F.C. Sures I. Ullrich A. J. Biol. Chem. 1995; 270: 23126-23131Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In short, five 6-well plates were plated at 2 × 104 cells/well. After 24 h, the number of adherent cells per well was determined in one plate (day 0). Three wells in each of the remaining four plates then received insulin at a final concentration of 100 nm (+ insulin) with the other three wells serving as controls (− insulin). The number of adherent cells was determined in one plate after a further 24 h of incubation (day 1), whereas the remaining plates were washed thoroughly three times to remove nonadherent cells. Fresh medium with and without insulin was added. This procedure was repeated every 24 h for the next three days. Similar results were obtained in two independent experiments. During routine passaging of PTPα-overexpressing A431 clones, we observed that these cells were resistant to removal from the substratum by low concentrations of trypsin (0.05%). To quantitate this characteristic, an adhesion assay was developed to assess cell-substratum adhesion. Cells containing vector alone, PTPα, or PTPα (D1 C433A) were plated in 96-well flat-bottom plates in quadruplicate at 104 cells/well. Cells were allowed to adhere and spread in serum-containing medium to 75–85% confluency (1–2 days). Medium was then carefully removed so as not to disturb the cell monolayer, and the cells were gently washed 3–5 times with 100 μl of Ca2+- and Mg2+-free PBS/well/wash over a period of 20–30 min. During this treatment, control cells rounded and lifted off the substratum in a manner that was dependent on the number and duration of the PBS washes. To be able to discern the lower adherence of control and PTPα (D1 C433A)-transfected A431 cells, the number and duration of PBS washes was reduced. Wash solutions were then discarded, and 100 μl of culture medium was added to each well. One hour later, 25 μl of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (5 mg/ml) was added, and the cells remaining in each well were stained for 2.5 h. Medium was then carefully removed, 100 μl of Me2SO was added to each well to dissolve the precipitated 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide crystals, and the absorbance at 550 nm was determined using an enzyme-linked immunosorbent assay plate reader. The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay allowed linear detection of between 1000 and 70,000 cells/well. The absorbance of each well was compared with untreated wells to determine the percentage of cells removed by each washing protocol. For each cell line, the percentage difference in absorbance was found to be equivalent to the percentage of cells removed. To analyze adherence changes in response to agents known to alter cell-substratum adhesion, EGF (100 ng/ml), pervanadate (100 μm vanadate, 2 mmH2O2), and EDTA (10 mm) were added to the PBS washes, and the cells were treated as above. Lysates from A431 cells containing vector alone or from PTPα-expressing or PTPα (D1 C433A)-expressing cells were incubated for 1 h with Src Tyr527phosphopeptide immobilized on CNBr-activated Sepharose. Beads were thoroughly washed in cell lysis buffer before resuspension in Laemmli buffer. Src family kinases precipitated by the beads were separated by 10% SDS-PAGE, transferred to Duralose membrane (Stratagene) and immunoblotted with SRC2 antisera or the Src-specific antibody (GD11). Immobilized nonphosphorylated Src Tyr527 peptide was used as a control. Blots were developed using horseradish peroxidase-linked goat anti-rabbit or goat anti-mouse antiserum and the ECL system (Amersham). To investigate the effects of PTPα expression, A431 cells were transfected with cDNAs encoding the 123-residue extracellular domain-containing isoform of PTPα, as well as a catalytically inactive mutant form of PTPα (PTPα D1 C433A), using the expression vector pBCMGNeo (34Karasuyama H. Melchers F. Eur. J. Immunol. 1988; 18: 97-101Crossref PubMed Scopus (1076) Google Scholar). G418-resistant clones were obtained with similar frequency in all three instances. Three vector alone transfected control A431 lines and 10 clones of PTPα-transfected and PTPα (D1 C433A)-transfected cells were examined for PTPα expression. Three clones of each, in the case of PTPα and PTPα (D1 C433A) transfectants, were then selected based on their having similar levels of PTPα expression. Results presented are representative examples of each group of clones. A431 cells normally express relatively low levels of endogenous PTPα (Fig. 1 A). PTPα expression was determined by immunoblot analysis of A431 total cell lysates with polyclonal anti-peptide antibodies (anti-PTPα-1) (Fig. 1 A) or with anti-recombinant PTPα-specific antibodies (anti-PTPα-2) (data not shown). Both of these antibodies recognized proteins of approximately 130–150, 100, 85, and 68 kDa. N- and O-glycosylation of the predicted 85-kDa PTPα polypeptide chain results in a mature protein of between 130 and 150 kDa (11Daum G. Regenass S. Sap J. Schlessinger J. Fischer E.H. J. Biol. Chem. 1994; 269: 10524-10528Abstract Full Text PDF PubMed Google Scholar). A 100-kDa form of PTPα, observed in immunoblots of lysates derived from PTPα-expressing cells (Fig. 1 A), was also immunoprecipitated from these lysates with antibodies specific for the extracellular region of PTPα (Fig. 1 B) and likely corresponds to an N-glycosylated precursor of the larger form. This is in agreement with Daum et al. (11Daum G. Regenass S. Sap J. Schlessinger J. Fischer E.H. J. Biol. Chem. 1994; 269: 10524-10528Abstract Full Text PDF PubMed Google Scholar), who reported that antibodies against baculovirus-expressed PTPα recognized a glycosylation-dependent epitope in the extracellular domain of PTPα. The anti-PTPα-extracellular domain antibodies used in our study were generated against residues 20–60 of the extracellular domain, a region containing multiple potentialN- and O-glycosylated residues. Thus, the PTP-extracellular domain antiserum likely recognized an incompletely glycosylated PTPα species. This antibody, however, bound to the surface of PTPα-overexpressing cells with little or no binding observed to vector alone transfected control cells, demonstrating PTPα cell-surface expression in the transfected clones (data not shown). To assess whether PTPα expression would alter the EGF-induced growth inhibition response of A431 cells, we treated vector alone control cells and PTPα-transfected clones with various concentrations of EGF. Cell numbers were then determined by WST-1 assay (as described under “Experimental Procedures”). Maximal inhibition of vector control transfected cell growth was observed at EGF concentrations between 5–10 ng/ml (Fig. 2 A). The response of two PTPα-expressing A431 clones (PTPα-1 and PTPα-2) to EGF are shown in Fig. 2 A. The expression of PTPα in A431 cells was unable to rescue these cells from the growth inhibitory effects of EGF. However, a dramatic change in cell morphology was evident following exposure of A431 cells to EGF in serum-free conditions. Whereas EGF caused control A431 cells to round-up and lift-off the substratum within 5–10 min (Fig. 2, B and C, rounded phase-bright cells in A431-Ctr 1 and A431-Ctr 2 EGF-treated panels) clones expressing PTPα remained adherent and spread following exposure to EGF (Fig. 2, B and C, clones PTPα-1 and PTPα-3). This phenotype was observed in all of the PTPα-expressing clones and was dependent on the catalytic activity of PTPα D1 (data not shown). A previously established BHK-IR cell line (35Anderson A.S. Kjeldsen T. Wiberg F.C. Vissing H. Schaffer L. Rasmussen J.S. De Meyts P. Moller N.P.H. J. Biol. Chem. 1992; 267: 13681-13686Abstract Full Text PDF PubMed Google Scholar) responds to insulin stimulation with growth inhibition, cell rounding, and detachment from the substratum of cell culture dishes (36Moller N.P.H. Moller K.B. Lammers R. Kharitonenkov A. Hoppe E. Wiberg F.C. Sures I. Ullrich A. J. Biol. Chem. 1995; 270: 23126-23131Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). These cells round up quickly after addition of insulin, with detachment being detectable as early as 1 h after the addition of insulin. The optimal insulin concentration required to bring about this effect was approximately 100 nm, with the half-maximal effect being observed at a concentration of approximately 1 nm. In contrast, stable BHK-IR cell lines overexpressing PTPα failed to round up and remained attac" @default.
• W2037291219 created "2016-06-24" @default.
• W2037291219 creator A5025721279 @default.
• W2037291219 creator A5037158345 @default.
• W2037291219 creator A5061429410 @default.
• W2037291219 creator A5064798252 @default.
• W2037291219 date "1998-11-01" @default.
• W2037291219 modified "2023-10-14" @default.
• W2037291219 title "Protein-tyrosine Phosphatase α Regulates Src Family Kinases and Alters Cell-Substratum Adhesion" @default.
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