FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2030253887> ?p ?o ?g. }

• W2030253887 endingPage "25855" @default.
• W2030253887 startingPage "25848" @default.
• W2030253887 abstract "Previous biochemical evidence has yielded conflicting models for the role of protein tyrosine phosphatase-1B (PTP-1B) in the regulation of integrin signaling. Thus, to establish the physiological relevance for such a role, we employed a genetic approach by generating embryonic fibroblasts from PTP-1B knockout mice. Both primary fibroblasts and their derived cell lines were used in this study. Immortalization of wild-type primary cells with the SV40 Large T antigen resulted in a dramatic increase in the endogenous expression of PTP-1B, suggesting a role during transformation. Moreover, the absence of PTP-1B in the transformed cell lines led to a more pronounced effect on different pathways of fibronectin-mediated signaling compared with the untransformed state. Specifically, p130Cas phosphorylation, Erk activation as well as cell spreading were delayed in PTP-1B-deficient cells, compared with their wild-type counterparts. Interestingly, this attenuation in integrin-mediated events closely resembles that of Src-deficient fibroblasts. Indeed, PTP-1B deficient, transformed fibroblasts held in suspension do exhibit a hyperphosphorylation of the inhibitory site (Tyr-527) of Src, compared with their wild-type counterparts. These results establish PTP-1B as a positive physiological regulator of integrin signaling in transformed cells, acting upstream of Src Tyr-527 dephosphorylation that leads to several adhesion-dependent events. Previous biochemical evidence has yielded conflicting models for the role of protein tyrosine phosphatase-1B (PTP-1B) in the regulation of integrin signaling. Thus, to establish the physiological relevance for such a role, we employed a genetic approach by generating embryonic fibroblasts from PTP-1B knockout mice. Both primary fibroblasts and their derived cell lines were used in this study. Immortalization of wild-type primary cells with the SV40 Large T antigen resulted in a dramatic increase in the endogenous expression of PTP-1B, suggesting a role during transformation. Moreover, the absence of PTP-1B in the transformed cell lines led to a more pronounced effect on different pathways of fibronectin-mediated signaling compared with the untransformed state. Specifically, p130Cas phosphorylation, Erk activation as well as cell spreading were delayed in PTP-1B-deficient cells, compared with their wild-type counterparts. Interestingly, this attenuation in integrin-mediated events closely resembles that of Src-deficient fibroblasts. Indeed, PTP-1B deficient, transformed fibroblasts held in suspension do exhibit a hyperphosphorylation of the inhibitory site (Tyr-527) of Src, compared with their wild-type counterparts. These results establish PTP-1B as a positive physiological regulator of integrin signaling in transformed cells, acting upstream of Src Tyr-527 dephosphorylation that leads to several adhesion-dependent events. extracellular matrix mitogen-activated kinase Jun N-terminal kinase protein tyrosine phosphatase protein-tyrosine kinase Dulbecco's modified Eagle's medium phosphate-buffered saline fibronectin focal adhesion kinase Adhesion to an extracellular matrix (ECM)1 has long been known to be critical for normal adherent cells to function efficiently. Engagement of cell surface integrins with the ECM generates intracellular signals that promote a variety of processes such as gene expression, cell survival, and cell-cycle progression (1Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3807) Google Scholar). Furthermore, adhesion per se is insufficient to generate survival signals such that cells that attach onto a matrix but are not allowed to effectively spread may undergo apoptosis (2Chen C.S. Mrksich M. Huang S. Whitesides G.M. Ingber D.E. Science. 1997; 276: 1425-1428Crossref PubMed Scopus (4181) Google Scholar). It is now well established that adhesion-dependent signaling involves changes in tyrosyl phosphorylation of several proteins that is regulated by two large protein superfamilies: protein-tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Among the PTKs that have long been recognized as critical mediators of integrin signaling, the Src family of kinases (3Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2161) Google Scholar), as well as the FAK subgroup of kinases (4Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1031) Google Scholar), are well characterized. Indeed genetic ablation of either FAK or Src has resulted in multiple defects in integrin signaling that include alterations in cell motility, cell spreading, and focal adhesion formation (5Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1585) Google Scholar, 6Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (646) Google Scholar, 7Kaplan K.B. Swedlow J.R. Morgan D.O. Varmus H.E. Genes Dev. 1995; 9: 1505-1517Crossref PubMed Scopus (295) Google Scholar). Together and independently, Src and p125FAK phosphorylate many other proteins, leading to the activation of multiple adhesion-dependent pathways, most notably the Erk subgroup of MAP kinases (8Schlaepfer D.D. Broome M.A. Hunter T. Mol. Cell. Biol. 1997; 17: 1702-1713Crossref PubMed Scopus (399) Google Scholar, 9Schlaepfer D.D. Jones K.C. Hunter T. Mol. Cell. Biol. 1998; 18: 2571-2585Crossref PubMed Scopus (357) Google Scholar). Independent of Src/p125FAKsignaling, the Src-like kinase Fyn has been shown to be critical in adhesion-dependent Erk activation (10Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 11Wary K.K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-634Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar). Furthermore, recent genetic studies have suggested that the Src/p125FAKand Fyn pathways may act in parallel to obtain optimal activation of Erk (12Barberis L. Wary K.K. Fiucci G. Liu F. Brancaccio M. Altruda F. Tarone G. Giancotti F.G. J. Biol. Chem. 2000; 275: 36532-36540Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Upon integrin stimulation, the adapter protein p130Cas also becomes tyrosine phosphorylated by Src and/or p125FAK (8Schlaepfer D.D. Broome M.A. Hunter T. Mol. Cell. Biol. 1997; 17: 1702-1713Crossref PubMed Scopus (399) Google Scholar,13Tachibana K. Urano T. Fujita H. Ohashi Y. Kamiguchi K. Iwata S. Hirai H. Morimoto C. J. Biol. Chem. 1997; 272: 29083-29090Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 14Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar). Tyrosine-phosphorylated p130Cas acts as a large scaffolding molecule to recruit additional signaling complexes and has recently been shown to be involved in the activation of the JNK subgroup of MAP kinases (15Dolfi F. Garcia-Guzman M. Ojaniemi M. Nakamura H. Matsuda M. Vuori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15394-15399Crossref PubMed Scopus (157) Google Scholar, 16Oktay M. Wary K.K. Dans M. Birge R.B. Giancotti F.G. J. Cell Biol. 1999; 145: 1461-1469Crossref PubMed Scopus (248) Google Scholar). At the other end, the evidence for specific PTPs in the regulation of integrin signaling has been lagging from that of PTKs, although recent advances have been made in understanding the roles of several PTPs during adhesion (17Angers-Loustau A. Côté J.F. Tremblay M.L. Biochem. Cell Biol. 1999; 77: 493-505Crossref PubMed Scopus (64) Google Scholar). Indeed, using knockout fibroblasts, SHP-2, PTP-PEST, and RPTPα have definitively been shown to modulate integrin-mediated responses (18Angers-Loustau A. Côté J.F. Charest A. Dowbenko D. Spencer S. Lasky L.A. Tremblay M.L. J. Cell Biol. 1999; 144: 1019-1031Crossref PubMed Scopus (249) Google Scholar, 19Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 20Ponniah S. Wang D.Z. Lim K.L. Pallen C.J. Curr. Biol. 1999; 9: 535-538Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 21Su J. Muranjan M. Sap J. Curr. Biol. 1999; 9: 505-511Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Interestingly, each of these PTPs has been postulated to act through different mechanisms, either through dephosphorylation of p125FAK, the inhibitory tyrosine of Src kinases, or p130Cas, suggesting that each of these PTPs possess unique roles during adhesion signaling. Another PTP that has been implicated in integrin signaling is protein tyrosine phosphatase 1B (PTP-1B), the first PTP that was identified and cloned (22Brown-Shimer S. Johnson K.A. Lawrence J.B. Johnson C. Bruskin A. Green N.R. Hill D.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5148-5152Crossref PubMed Scopus (156) Google Scholar, 23Guan K.L. Haun R.S. Watson S.J. Geahlen R.L. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1501-1505Crossref PubMed Scopus (153) Google Scholar, 24Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6731-6737Abstract Full Text PDF PubMed Google Scholar, 25Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6722-6730Abstract Full Text PDF PubMed Google Scholar, 26Chernoff J. Schievella A.R. Jost C.A. Erikson R.L. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2735-2739Crossref PubMed Scopus (167) Google Scholar). Its structure consists of an N-terminal catalytic domain followed by two tandem proline-rich motifs that possess a consensus for binding to proteins with SH3 domains (27Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). At the extreme C terminus, a small hydrophobic stretch has been shown to be sufficient to localize the enzyme to the endoplasmic reticulum (28Frangioni J.V. Beahm P.H. Shifrin V. Jost C.A. Neel B.G. Cell. 1992; 68: 545-560Abstract Full Text PDF PubMed Scopus (504) Google Scholar). Biochemical analyses and PTP “substrate-trapping methods” have suggested that PTP-1B may dephosphorylate the insulin, IGF-I, and EGF receptors, as well as antagonize bcr-abl signaling (29Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (680) Google Scholar, 30LaMontagne Jr., K.R. Flint A.J. Franza Jr., B.R. Pandergast A.M. Tonks N.K. Mol. Cell. Biol. 1998; 18: 2965-2975Crossref PubMed Scopus (102) Google Scholar, 31LaMontagne Jr., K.R. Hannon G. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14094-14099Crossref PubMed Scopus (96) Google Scholar, 32Kenner K.A. Anyanwu E. Olefsky J.M. Kusari J. J. Biol. Chem. 1996; 271: 19810-19816Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 33Liu F. Chernoff J. Biochem. J. 1997; 327: 139-145Crossref PubMed Scopus (161) Google Scholar). In contrast, the role for PTP-1B in integrin signaling is less clear. Originally, overexpression studies have shown that PTP-1B negatively regulates integrin signaling, which was thought to depend on its association with and dephosphorylation of p130Cas (27Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 34Liu F. Sells M.A. Chernoff J. Curr. Biol. 1998; 8: 173-176Abstract Full Text Full Text PDF PubMed Google Scholar). Overexpression of wild-type PTP-1B in Rat-1 fibroblasts impaired adhesion-dependent Erk activation as well as tyrosine phosphorylation of p130Cas and p125FAK. Furthermore, overexpression of PTP-1B readily reduced fibronectin (FN)-mediated cell spreading and cell migration. Importantly, these effects were abrogated when a proline mutant that did not bind p130Cas was used. Independently, experiments using a catalytically inactive mutant indicated that PTP-1B had a positive role in integrin signaling (35Arregui C.O. Balsamo J. Lilien J. J. Cell Biol. 1998; 143: 861-873Crossref PubMed Scopus (129) Google Scholar). In comparison with the above study, expression of this mutant in L cells decreased FN-mediated cell spreading and p125FAKphosphorylation, whereas wild-type PTP-1B had no effect. In this study, PTP-1B is suggested to dephosphorylate the inhibitory site on Src kinases and thus activate them, thereby promoting integrin-mediated events. Consistent with this notion, L cells expressing the catalytically inactive mutant displayed decreased Src activity. It has been argued that the opposite roles of PTP-1B on integrin signaling may reflect the different cell systems employed, although in both cases a fibroblast cell type was used. Alternatively, the promiscuous nature of overexpressed PTP-1B in cells may reflect its dephosphorylation of non-physiological substrates and affect signaling in an artificial manner. Herein we have used a genetic loss-of-function approach to ascertain the physiological role of PTP-1B in adhesion-dependent signaling. We provide evidence that PTP-1B is required for the efficient transduction of integrin-mediated signals and that loss of PTP-1B delays fibronectin-induced cell signaling and cell spreading. In PTP-1B-deficient fibroblasts, a transient hyperphosphorylation of Tyr-527 on Src was observed, suggesting that PTP-1B is involved in Src activation during adhesion. The p130Cas rabbit antiserum was generously given by Jean François Côtéand has been previously described (18Angers-Loustau A. Côté J.F. Charest A. Dowbenko D. Spencer S. Lasky L.A. Tremblay M.L. J. Cell Biol. 1999; 144: 1019-1031Crossref PubMed Scopus (249) Google Scholar). TC-PTP monoclonal antibodies (36You-Ten K.E. Muise E.S. Itié A. Michaliszyn E. Wagner J. Jothy S. Lapp W.S. Tremblay M.L. J. Exp. Med. 1997; 186: 683-693Crossref PubMed Scopus (316) Google Scholar) were kindly provided by Maria de Jesus Ibarra-Sanchez. Polyclonal rabbit antibodies against PTP-1B have been previously described (37Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramachandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1906) Google Scholar). Pan Src and phosphospecific (Tyr-527) Src antibodies were purchased from BIOSOURCE. Antibodies against Erk and phospho-Erk were obtained from New England BioLabs. Anti-β1 integrin and monoclonal p130Casantibodies were purchased from Transduction Laboratories. To detect phosphotyrosine-containing proteins, mAb 4G10 (Upstate Biotechnology) conjugated to horseradish peroxidase was used (kind gift of J. F. Côté). Fibronectin was purchased from BD Biosciences. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (BIOSOURCE) and 5 mg/ml penicillin/streptomycin (Life Technologies). To obtain primary embryonic fibroblasts, wild-type (+/+) and mutant (−/−) E14 embryos were isolated. The head and organs were removed, and the remaining carcasses were minced and dispersed into single cell suspensions in trypsin at 37 °C for 30 min. Afterward, cells were resuspended in medium and experiments were performed within three passages. Immortalized cells were derived by infection of primary cultures with a recombinant retrovirus expressing the SV40 Large T antigen. Independent “cell lines” were derived from immortalized cells obtained from separate embryos. Prior to the start of experiments, near confluent cultures were starved overnight in 0.1% fetal bovine serum in DMEM. Cells were washed with PBS, harvested by limited trypsin treatment (0.01% trypsin, 0.1 mm EDTA, 5 min at 37 °C) and then inactivated by two washes with 0.1% fetal bovine serum, 0.2% bovine serum albumin in DMEM. Cells were kept in suspension at 37 °C for 30 min, then lysed immediately or replated onto tissue culture dishes that had been precoated overnight with 10 µg/ml FN in PBS. Prior to lysis, cells were washed twice in ice-cold PBS and then scraped into TNE buffer (10 mm Tris, pH 7.5, 1 mm EDTA, 150 mm NaCl, 1% Nonidet P-40) supplemented with 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and CompleteTM EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals). Crude lysates were rotated end-over-end at 4 °C for 20 min and then cleared by centrifugation at 4 °C for 15 min. In the analyses of Src phosphorylation, cells were lysed in RIPA buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 0.1% SDS, 1% sodium deoxycholate, 1 mm EDTA, 1% Triton X-100) supplemented with the same phosphatase and protease inhibitors. An equivalent amount of SDS sample buffer was added to the TNE or RIPA lysates and boiled for 5 min at 95 °C. For immunoprecipitations, equal amounts of (TNE) protein lysates (0.5–1.0 mg) were incubated with the appropriate antibodies and 25 µl of protein A-agarose (Life Technologies). The mixtures were then incubated end-over-end at 4 °C for 2 h. The beads were washed three times in TNE and then resuspended in SDS sample buffer, followed by boiling at 95 °C for 5 min. Samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Blots were blocked in 1% bovine serum albumin, 1% goat serum in TBST (10 mm Tris, pH 8.0, 150 mm NaCl, 0.05% Tween 20) for 2 h. Incubation with the primary antibodies took place at room temperature for 1.5 h (or overnight at 4 °C for Src and Erk antibodies), washed five times in TBST, and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 30 min. After five washes with TBST, proteins were detected using an enhanced chemiluminescence kit (PerkinElmer Life Sciences). Cells were treated similarly to that for the cell signaling studies. Approximately 2 × 105cells in a volume of 1 ml were added to 35-mm FN-coated dishes for the indicated times at 37 °C. Afterward the dishes were chilled on ice for 10 min, placed on an eclipse TE300 (inverted) Nikon microscope, and photographed with a CCD camera using Metamorph software (version 4.1). Single cells that were phase-bright with rounded morphology were scored as non-spread, whereas those that possessed a flattened shape and were phase-dark were scored as spread. Three different fields for each sample were averaged. At least 100 cells were counted in each field. Error bars represent S.E. from these counts. Cells were seeded at a density of 5000 cells/cm2 onto Falcon chamber slides and allowed to grow for 21 h. Cells were washed once with PBS, fixed in 4% paraformaldehyde in PBS for 20 min at 4 °C, and then permeabilized with 0.1% Triton X-100. Slides were blocked overnight with 1% bovine serum albumin in PBS at 4 °C. To detect filamentous/polymerized actin, the cells were incubated with rhodamine-conjugated phalloidin (1:500 dilution) for 2 h at room temperature, washed three times with PBS, and then mounted in a 1:1 mixture of 2.5% 1,4-diazabicyclo [2.2.2.] octane (DABCO; Sigma). Cells were then visualized with the eclipse TE300 (inverted) Nikon microscope, and photographed with a CCD camera using Metamorph software (version 4.1). The details of the targeted inactivation of PTP-1B and its role in insulin signaling have been previously described (37Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramachandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1906) Google Scholar). However, to clarify the role of PTP-1B in integrin signaling, we have generated embryonic fibroblasts and cell lines to use as a model. Primary embryonic fibroblasts were derived from E14 wild-type (+/+) or mutant (−/−) embryos, and experiments were performed within three passages. Cell lines were also obtained by immortalization of primary cells by the SV40 Large T antigen (Tag). Western blotting analysis of PTP-1B protein confirmed the genotype of each cell line (Fig. 1 A). Interestingly, the levels of PTP-1B in SV40 Tag immortalized cell lines were dramatically increased compared with levels found in primary embryonic fibroblasts (Fig. 1 A, lanes 1, 3, 4,and 5), suggesting a role for PTP-1B during immortalization or transformation. Consistent with this notion, PTP-1B protein levels has been shown to be increased by the expression of the bcr-abl oncogene (30LaMontagne Jr., K.R. Flint A.J. Franza Jr., B.R. Pandergast A.M. Tonks N.K. Mol. Cell. Biol. 1998; 18: 2965-2975Crossref PubMed Scopus (102) Google Scholar). Furthermore, this effect seems to be specific for PTP-1B, as the expression level of T-cell protein tyrosine phosphatase (TC-PTP), a closely related PTP, was not significantly altered by SV40 Tag (Fig. 1 B). In embryonic fibroblasts, the α5β1 receptor is the major integrin involved in the recognition of the fibronectin ligand. Immunoblotting with antibodies against the β1chain suggests that this was the case in all our cells tested (Fig.1 C), validating their use for subsequent cell adhesion studies. One of the earliest events following engagement of integrins by the extracellular matrix includes the tyrosine phosphorylation of several proteins, most noticeably within the 120–130 kDa range that includes p130Cas and p125FAK (38Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y.,. et al.J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 39Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). In addition, previous biochemical studies suggested that PTP-1B negatively regulates integrin signaling through the binding and dephosphorylation of the adapter protein p130Cas (27Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 34Liu F. Sells M.A. Chernoff J. Curr. Biol. 1998; 8: 173-176Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, to determine the physiological importance of PTP-1B toward integrin induced tyrosine phosphorylation of p130Cas, serum-starved PTP-1B wild-type and mutant cells were either kept in suspension or replated onto fibronectin-coated dishes for the indicated period of times. The levels of protein tyrosine phosphorylation were then analyzed by immunoblotting of cell lysates with anti-phosphotyrosine (anti-pY) antibodies. Cells held in suspension exhibited almost a complete absence of phosphotyrosine (pY)-containing proteins of around 120–130 kDa as expected (Fig. 2). Upon re-attachment to FN, the pY levels within the 120–130-kDa range were rapidly elevated. In primary fibroblasts, these pY levels were not considerably different between wild-type and mutant cells when normalized against p130Cas protein levels (Fig. 2 A). Interestingly, the pY levels for the 120–130 kDa proteins were somewhat decreased in PTP-1B mutant Tag cell lines after 20 min of replating onto FN (Fig.2 B, lanes 6–10) but were restored by 40 min (Fig. 2 B, lanes 11–20). In contrast, constitutive hyperphosphorylation of proteins around 180 and 80 kDa were observed in PTP-1B mutant cells compared with wild-type controls and was more pronounced in the transformed mutant cell lines. One probable explanation for the magnified differences seen in the cell lines (as opposed to primary cells) is the increased expression of PTP-1B in the transformed state. Previous experiments demonstrated that overexpression of PTP-1B in Rat 3Y1 fibroblasts reduced adhesion-dependent p130Cas phosphorylation (34Liu F. Sells M.A. Chernoff J. Curr. Biol. 1998; 8: 173-176Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, to further determine whether FN-induced p130Cas phosphorylation is altered in the absence of PTP-1B, the cell lysates were then immunoprecipitated with antibodies toward p130Cas and analyzed by immunoblotting with anti-pY antibodies. Equal immunoprecipitation of p130Cas protein was monitored in all experiments by subsequent reprobing of stripped blots with anti-p130Casantibodies. In primary fibroblasts, cells in suspension lacked detectable p130Cas phosphorylation, which was readily induced upon replating onto FN for 20 min (Fig.3 A). However, no discernible change in p130Cas phosphorylation was observed between wild-type and mutant primary cells, consistent with the observations of FN-induced pY levels (Fig. 2 A). In comparison, this was not the case for the Tag-transformed cell lines. After 20 min of replating onto FN, PTP-1B mutant Tag cell lines exhibited a decrease in p130Cas phosphorylation compared with wild-type cells, which once again paralleled the observations of FN-induced pY levels (Fig. 2 B, lanes 6–10). However, this effect was not significant after 40 min of replating onto FN (Fig. 2 B,lanes 11–20). Nevertheless, these results suggest that p130Cas is not a robust physiological substrate of PTP-1B under these conditions. In fact, it appears that PTP-1B is required for efficient FN-induced p130Cas phosphorylation, especially in the transformed state. Our results demonstrating that PTP-1B is required for efficient FN-induced p130Cas phosphorylation raises the possibility that PTP-1B could be required for other FN-mediated events. One particular adhesion-dependent signaling pathway includes the activation of the Erk subgroup of the MAP kinase cascade, presumably through activation of one or more Src kinases. To determine the ability of FN to induce Erk activation in PTP-1B wild-type and mutant cells, we performed immunoblot analysis with phosphospecific antibodies that recognize activated Erk protein. In suspension, only modest amounts of activated Erk were detected in all cell lines (Fig.4). In primary cells, adhesion onto FN for 20 min activated Erk (Fig. 4 A), similar to that previously observed for NIH 3T3 cells (40Aplin A.E. Juliano R.L. J. Cell Sci. 1999; 112: 695-706Crossref PubMed Google Scholar). No appreciable differences were observed for PTP-1B wild-type or mutant primary cells. However, in the case of Tag cell lines, after 20 min of replating onto fibronectin, wild-type cell lines readily displayed an increase in phospho-Erk levels (Fig. 4 B). In contrast, PTP-1B mutant Tag cell lines were impaired in their ability to induce adhesion-dependent Erk activation. Interestingly, PTP-1B mutant cells that were fully adherent in culture, displayed normal Erk activation in response to serum suggesting that the FN-dependent impairment is transient or rescued by other signaling pathways activated by components in serum (data not shown). Regardless, these results suggest that PTP-1B positively regulates FN-mediated Erk activation as well as p130Cas phosphorylation in transformed but not primary cells. It is well established that MAP kinase activation depends on an intact cytoskeleton and is associated with the formation of actin structures (40Aplin A.E. Juliano R.L. J. Cell Sci. 1999; 112: 695-706Crossref PubMed Google Scholar, 41Renshaw M.W. Ren X.D. Schwartz M.A. EMBO J. 1997; 16: 5592-5599Crossref PubMed Scopus (270) Google Scholar). Because FN-mediated cell spreading is dependent on actin bundling and correlates with efficient MAP kinase activation in NIH 3T3 cells, it is possible that PTP-1B mutant cells might exhibit a defect in cell spreading. To explore this possibility, we plated PTP-1B wild-type and mutant primary cells onto FN for either 10, 20, or 60 min. Afterward, cells were chilled on ice for 10 min and then photographed. Single cells that were phase-bright with rounded morphology were scored as non-spread, whereas those that possessed a flattened shape and were phase-dark were scored as spread. Fig.5 A shows representative photographs obtained from PTP-1B wild-type and mutant primary cells. Typically, the PTP-1B wild-type cells displayed around 30% spreading efficiency after 10 min on FN, whereas PTP-1B mutant cells displayed only 15% spreading efficiency (Fig. 5 B). By 20 min, PTP-1B mutant cells were still lagging behind wild-type cells in terms of spreading, although the difference became less apparent as time continued. In addition, similar results were obtained using the Tag cell lines (data not shown). Thus, these results suggest that PTP-1B is required for efficient spreading on FN in mouse embryonic fibroblasts. Cell morphology is known to be dependent on the actin cytoskeleton. Subsequently, we analyzed the actin staining in both PTP-1B wild-type and mutant cells using Rhodamine-conjugated Phalloidin, that specifically recognizes F-actin (polymerized actin). At low densities (5000 cells/cm2), most PTP-1B wild-type transformed cells exhibit a round, spread morphology (Fig. 5 C, aand c); in contrast a relatively high proportion of PTP-1B mutant transformed cells displayed an elongated, spindle like shape (Fig. 5 C, b and d). These results suggest that PTP-1B regulates an aspect of actin dynamics that affects cell morphology in transformed cells. Previous gene-targeting studies have clearly established the physiological importance of Src during adhesion-mediated cell spreading (7Kaplan K.B. Swedlow J.R. Morgan D.O. Varmus H.E. Genes Dev. 1995; 9: 1505-1517Crossref PubMed Scopus (295) Google Scholar). During adhesion onto FN, it has previously been shown that Tyr-527 of Src transiently becomes dephosphorylated and then quickly becomes rephosphorylated (42Kaplan K.B. Bibbins K.B. Swedlow J.R. Arnaud M. Morgan D.O. Varmus H.E. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar). In addition, Src is primarily regulated through this site (43MacAuley A. Cooper J.A. Mol. Cell. Biol. 1989; 9: 2648-2656Crossref PubMed Scopus (47) Google Scholar, 44Courtneidge S.A. EMBO J. 1985; 4: 1471-1477Cr" @default.
• W2030253887 created "2016-06-24" @default.
• W2030253887 creator A5007653418 @default.
• W2030253887 creator A5019628978 @default.
• W2030253887 creator A5036028850 @default.
• W2030253887 creator A5072673780 @default.
• W2030253887 date "2001-07-01" @default.
• W2030253887 modified "2023-09-27" @default.
• W2030253887 title "Attenuation of Adhesion-dependent Signaling and Cell Spreading in Transformed Fibroblasts Lacking Protein Tyrosine Phosphatase-1B" @default.
• W2030253887 cites W1497274503 @default.
• W2030253887 cites W1499420960 @default.
• W2030253887 cites W1749869160 @default.
• W2030253887 cites W180669350 @default.
• W2030253887 cites W1836212818 @default.
• W2030253887 cites W1849793110 @default.
• W2030253887 cites W1974517994 @default.
• W2030253887 cites W1980855212 @default.
• W2030253887 cites W1981043027 @default.
• W2030253887 cites W1982849870 @default.
• W2030253887 cites W1989899772 @default.
• W2030253887 cites W1999673185 @default.
• W2030253887 cites W2001386699 @default.
• W2030253887 cites W2004755427 @default.
• W2030253887 cites W2005076200 @default.
• W2030253887 cites W2008725579 @default.
• W2030253887 cites W2013355944 @default.
• W2030253887 cites W2014280822 @default.
• W2030253887 cites W2020537787 @default.
• W2030253887 cites W2021515559 @default.
• W2030253887 cites W2022506262 @default.
• W2030253887 cites W2028105686 @default.
• W2030253887 cites W2028691987 @default.
• W2030253887 cites W2029723738 @default.
• W2030253887 cites W2034842897 @default.
• W2030253887 cites W2041182684 @default.
• W2030253887 cites W2053544216 @default.
• W2030253887 cites W2054192585 @default.
• W2030253887 cites W2054575721 @default.
• W2030253887 cites W2055354269 @default.
• W2030253887 cites W2055910783 @default.
• W2030253887 cites W2056327110 @default.
• W2030253887 cites W2058096164 @default.
• W2030253887 cites W2069526456 @default.
• W2030253887 cites W2083358105 @default.
• W2030253887 cites W2084434864 @default.
• W2030253887 cites W2094173028 @default.
• W2030253887 cites W2098573045 @default.
• W2030253887 cites W2101257013 @default.
• W2030253887 cites W2115681043 @default.
• W2030253887 cites W2116045343 @default.
• W2030253887 cites W2123229219 @default.
• W2030253887 cites W2128576963 @default.
• W2030253887 cites W2139144434 @default.
• W2030253887 cites W2155723800 @default.
• W2030253887 cites W2158501218 @default.
• W2030253887 cites W2159228923 @default.
• W2030253887 cites W2160771960 @default.
• W2030253887 cites W2161873278 @default.
• W2030253887 cites W2313875470 @default.
• W2030253887 cites W4213141530 @default.
• W2030253887 cites W4250684467 @default.
• W2030253887 doi "https://doi.org/10.1074/jbc.m009734200" @default.
• W2030253887 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11346638" @default.
• W2030253887 hasPublicationYear "2001" @default.
• W2030253887 type Work @default.
• W2030253887 sameAs 2030253887 @default.
• W2030253887 citedByCount "101" @default.
• W2030253887 countsByYear W20302538872012 @default.
• W2030253887 countsByYear W20302538872013 @default.
• W2030253887 countsByYear W20302538872015 @default.
• W2030253887 countsByYear W20302538872016 @default.
• W2030253887 countsByYear W20302538872017 @default.
• W2030253887 countsByYear W20302538872018 @default.
• W2030253887 countsByYear W20302538872020 @default.
• W2030253887 countsByYear W20302538872021 @default.
• W2030253887 crossrefType "journal-article" @default.
• W2030253887 hasAuthorship W2030253887A5007653418 @default.
• W2030253887 hasAuthorship W2030253887A5019628978 @default.
• W2030253887 hasAuthorship W2030253887A5036028850 @default.
• W2030253887 hasAuthorship W2030253887A5072673780 @default.
• W2030253887 hasBestOaLocation W20302538871 @default.
• W2030253887 hasConcept C11960822 @default.
• W2030253887 hasConcept C1491633281 @default.
• W2030253887 hasConcept C178666793 @default.
• W2030253887 hasConcept C178790620 @default.
• W2030253887 hasConcept C185592680 @default.
• W2030253887 hasConcept C2776165026 @default.
• W2030253887 hasConcept C55493867 @default.
• W2030253887 hasConcept C84416704 @default.
• W2030253887 hasConcept C85528070 @default.
• W2030253887 hasConcept C85789140 @default.
• W2030253887 hasConcept C86803240 @default.
• W2030253887 hasConcept C95444343 @default.
• W2030253887 hasConceptScore W2030253887C11960822 @default.
• W2030253887 hasConceptScore W2030253887C1491633281 @default.
• W2030253887 hasConceptScore W2030253887C178666793 @default.
• W2030253887 hasConceptScore W2030253887C178790620 @default.
• W2030253887 hasConceptScore W2030253887C185592680 @default.

• next