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• W2051201358 abstract "Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro andex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B−/− mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B−/− fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice. Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro andex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B−/− mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B−/− fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice. receptor tyrosine kinase protein tyrosine phosphatase insulin receptor epidermal growth factor epidermal growth factor receptor platelet-derived growth factor platelet-derived growth factor receptor wild type mouse embryo fibroblast wild-type human PTP1B substrate-trapping mutant PTP1B-D181A knock-out phosphatidyl inositol-3 kinase Regulation of cellular proliferation, adhesion, and migration is pivotal for maintaining homeostasis. Multiple peptide growth factors direct these processes to differing extents in primary fibroblasts and fibroblast cell lines. These growth factors signal via receptors with intrinsic protein-tyrosine kinase activity, termed receptor tyrosine kinases (RTKs).1 Ligand binding activates the intrinsic kinase activity of these receptors, resulting in the phosphorylation of multiple receptor tyrosyl residues. These serve as docking sites to recruit signal relay molecules containing src homology-2 and/or phosphotyrosine binding domains, most of which also are RTK substrates. The resultant complexes lead to activation of downstream signaling, including the Ras-Erk and PI-3K/Akt pathways. Ultimately, downstream signaling pathways stimulate new transcription and changes in cellular behavior and/or state (reviewed in Ref. 1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1891) Google Scholar). Because of their pleiotropic actions, RTKs must be regulated carefully. Abnormally increased RTK activity can have dire consequences, including developmental abnormalities (reviewed in Ref. 2Robertson S.C. Tynan J.A. Donoghue D.J. Trends Genet. 2000; 16: 265-271Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), cancer (reviewed in Ref. 3Hunter T. Cell. 1997; 88: 333-346Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar), or fibrosis (reviewed in Ref. 4Ostman A. Heldin C.H. Adv. Cancer Res. 2001; 80: 801-838Google Scholar). Classic PTPs comprise a large family of receptor-like and non-receptor enzymes that share a highly conserved catalytic (PTP) domain that is absolutely specific for phosphotyrosine hydrolysis (reviewed in Ref. 5Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (462) Google Scholar). Because tyrosyl phosphorylation is reversible, PTPs probably play important roles in the regulation of RTKs and/or their substrates (reviewed in Refs. 5Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (462) Google Scholar,6Ostman A. Bohmer F.D. Trends Cell Biol. 2001; 11: 258-266Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Based largely on experiments in which wild-type PTPs or their catalytically impaired (dominant-negative) mutants were overexpressed, multiple PTPs, including LAR (7Kulas D.T. Goldstein B.J. Mooney R.A. J. Biol. Chem. 1996; 271: 748-754Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), DEP-1 (8Kovalenko M. Denner K. Sandstrom J. Persson C. Gross S. Jandt E. Vilella R. Bohmer F. Ostman A. J. Biol. Chem. 2000; 275: 16219-16226Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), TC-PTP (9Tiganis T. Bennett A.M. Ravichandran K.S. Tonks N.K. Mol. Cell. Biol. 1998; 18: 1622-1634Crossref PubMed Google Scholar, 10Tiganis T. Kemp B.E. Tonks N.K. J. Biol. Chem. 1999; 274: 27768-27775Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), Shp-1 (11Keilhack H. Tenev T. Nyakatura E. Godovac-Zimmermann J. Nielsen L. Seedorf K. Bohmer F.D. J. Biol. Chem. 1998; 273: 24839-24846Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar,12Keilhack H. Muller M. Bohmer S.A. Frank C. Weidner K.M. Birchmeier W. Ligensa T. Berndt A. Kosmehl H. Gunther B. Muller T. Birchmeier C. Bohmer F.D. J. Cell Biol. 2001; 152: 325-334Crossref PubMed Scopus (71) Google Scholar), Shp-2 (13Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (208) Google Scholar), and PTP1B (14Ahmad F., Li, P.M. Meyerovitch J. Goldstein B.J. J. Biol. Chem. 1995; 270: 20503-20508Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 15Seely B.L. Staubs P.A. Reichart D.R. Berhanu P. Milarski K.L. Saltiel A.R. Kusari J. Olefsky J.M. Diabetes. 1996; 45: 1379-1385Crossref PubMed Google Scholar, 16Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar, 17Liu F. Chernoff J. Biochem. J. 1997; 327: 139-145Crossref PubMed Scopus (161) Google Scholar) have been implicated in the dephosphorylation of various RTKs. Which, if any, of these PTPs regulate RTK signaling under physiologically relevant conditions of expression has remained largely unclear. Also unknown is the extent to which RTK dephosphorylation, as opposed to other down-regulatory mechanisms such as RTK degradation and/or inhibitory seryl phosphorylation, plays the (a) key role in receptor inactivation. PTP1B is a widely expressed non-receptor PTP that is localized on intracellular membranes via a hydrophobic C-terminal targeting sequence (18Frangioni J.V. Beahm P.H. Shifrin V. Jost C.A. Neel B.G. Cell. 1992; 68: 545-560Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 19Woodford-Thomas T.A. Rhodes J.D. Dixon J.E. J. Cell Biol. 1992; 117: 401-414Crossref PubMed Scopus (152) Google Scholar). A role for PTP1B in the regulation of many cellular functions has been suggested, including integrin (20Arregui C.O. Balsamo J. Lilien J. J. Cell Biol. 1998; 143: 861-873Crossref PubMed Scopus (129) Google Scholar, 21Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 22Liu F. Sells M.A. Chernoff J. Curr. Biol. 1998; 8: 173-176Abstract Full Text Full Text PDF PubMed Google Scholar, 23Cheng A. Bal G.S. Kennedy B.P. Tremblay M.L. J. Biol. Chem. 2001; 276: 25848-25855Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), cadherin (24Balsamo J. Arregui C. Leung T. Lilien J. J. Cell Biol. 1998; 143: 523-532Crossref PubMed Scopus (143) Google Scholar, 25Rhee J. Lilien J. Balsamo J. J. Biol. Chem. 2001; 276: 6640-6644Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and cytokine receptor signaling (26Myers M.P. Andersen J.N. Cheng A. Tremblay M.L. Horvath C.M. Parisien J.P. Salmeen A. Barford D. Tonks N.K. J. Biol. Chem. 2001; 276: 47771-47774Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 27Zabolotny J.M. Bence-Hanulec K.K. Stricker-Krongrad A. Haj F. Wang Y. Minokoshi Y. Kim Y.B. Elmquist J.K. Tartaglia L.A. Kahn B.B. Neel B.G. Dev. Cell. 2002; 2: 489-495Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 28Cheng A. Uetani N. Simoncic P.D. Chaubey V.P. Lee-Loy A. McGlade C.J. Kennedy B.P. Tremblay M.L. Dev. Cell. 2002; 2: 497-503Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar), cell cycle regulation (29Flint A.J. Gebbink M.F. Franza B.R., Jr. Hill D.E. Tonks N.K. EMBO J. 1993; 12: 1937-1946Crossref PubMed Scopus (124) Google Scholar, 30Schievella A.R. Paige L.A. Johnson K.A. Hill D.E. Erikson R.L. Cell Growth & Differ. 1993; 4: 239-246PubMed Google Scholar, 31Shifrin V.I. Neel B.G. J. Biol. Chem. 1993; 268: 25376-25384Abstract Full Text PDF PubMed Google Scholar), and the response to cellular stress (32Shifrin V.I. Davis R.J. Neel B.G. J. Biol. Chem. 1997; 272: 2957-2962Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Multiple studies indicated that PTP1B dephosphorylates the EGFR (16Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar, 17Liu F. Chernoff J. Biochem. J. 1997; 327: 139-145Crossref PubMed Scopus (161) Google Scholar) and the insulin receptor (IR) (14Ahmad F., Li, P.M. Meyerovitch J. Goldstein B.J. J. Biol. Chem. 1995; 270: 20503-20508Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 33Kenner K.A. Anyanwu E. Olefsky J.M. Kusari J. J. Biol. Chem. 1996; 271: 19810-19816Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 34Dadke S. Kusari J. Chernoff J. J. Biol. Chem. 2000; 275: 23642-23647Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 35Goldstein B.J. Ahmad F. Ding W., Li, P.M. Zhang W.R. Mol. Cell. Biochem. 1998; 182: 91-99Crossref PubMed Scopus (173) Google Scholar). Analysis of PTP1B−/− mice confirmed that the IR is a key physiological target of PTP1B (36Elchebly 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 (1897) Google Scholar, 37Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1121) Google Scholar). However, these mice lack any obvious signs of increased activity of the EGFR or PDGFR, such as increased tumor incidence or fibrosis. We used fibroblasts derived from wild-type (WT) and PTP1B−/− mice to address the role of PTP1B in the regulation of RTK signaling. We find that PTP1B−/− cells exhibit enhanced and sustained tyrosyl phosphorylation of both RTKs. Despite increased RTK phosphorylation, EGF- and PDGF-evoked Akt activation is not enhanced, whereas Erk activation is only minimally increased. Our results show that PTP1B is a bona fideregulator of EGF and PDGF signaling in vivo, but other cellular regulatory mechanisms, including other PTPs, can largely compensate for loss of PTP1B regulation of these RTKs. Affinity-purified polyclonal antibodies to murine PTP1B were described previously (37Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1121) Google Scholar). Antibodies against murine PDGFRβ were provided by Dr. D. DeMaio (Yale Medical School, New Haven, CT). Monoclonal antibodies against phosphotyrosine (4G10) were from Upstate Biotechnology, Inc (Lake Placid, NY) and anti-PTP1B monoclonal antibodies (FG6) were from Calbiochem. Rabbit polyclonal antibodies against total Akt, phosphorylated (Ser-473) Akt, and phosphorylated (Thr-202/Tyr-204) Erk were from Cell Signaling (Beverly, MA). Anti-Erk2, -Shp2, and -EGFR antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Primary mouse embryonic fibroblasts (MEFs) were generated from embryonic day 14 embryos from WT or PTP1B−/− (exon 1) mice (C57/Bl6J X SV129 background) (37Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1121) Google Scholar). Embryos were incubated in trypsin/EDTA (Invitrogen) for 30 min at 37 °C, and dissociated cells were collected by centrifugation and cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum,100 units/ml penicillin, and 10 mg/ml streptomycin at 37 °C in a 5% humidified CO2atmosphere. Four independent WT and PTP1B−/− MEF strains were used for experiments, with similar results. For immortalization with simian virus 40 large T antigen, PTP1B−/− MEFs were infected with pZipTex (38Jat P.S. Cepko C.L. Mulligan R.C. Sharp P.A. Mol. Cell. Biol. 1986; 6: 1204-1217Crossref PubMed Scopus (132) Google Scholar), and these cells were maintained as a pool and used for subsequent experiments. WT human PTP1B (hPTP1B-WT) or the substrate-trapping mutant PTP1B-D181A (hPTP1B-D/A) (16Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar) cloned into the retroviral vector pWZL (39LaMontagne K.R., Jr. Flint A.J. Franza B.R., Jr. Pandergast A.M. Tonks N.K. Mol. Cell. Biol. 1998; 18: 2965-2975Crossref PubMed Scopus (102) Google Scholar, 40LaMontagne K.R., Jr. Hannon G. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14094-14099Crossref PubMed Scopus (96) Google Scholar) were the generous gifts of Dr. N. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). To generate viral stocks, these vectors were transiently transfected into the Phoenix-Eco retroviral packaging line (http://www.stanford.edu/group/nolan/index.html), and supernatants were collected 48 h later. Immortalized PTP1B−/− cells were infected in the presence of polybrene (1 μg/ml) and selected in Dulbecco's modified Eagle's medium, 10% fetal calf serum plus hygromycin (700 μg/ml). Pools of hygromycin-resistant cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf serum, 100 units/ml penicillin, 10 mg/ml streptomycin, and 200 μg/ml hygromycin. MEFs were seeded at 2.5 × 105cells per 60-mm tissue culture plate for 24 h, starved in serum-free Dulbecco's modified Eagle's medium plus antibiotics for 48 h, and stimulated with EGF (50 ng/ml) or PDGF-BB (50 ng/ml). At the indicated times after stimulation, cells were lysed in Nonidet P-40 buffer (1% Nonidet P-40, 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm sodium orthovanadate, and protease inhibitor mixture (final concentrations, 20 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml of pepstatin, and 1 μg/ml of antipain)). Lysates were clarified by centrifugation at 13,000 rpm for 10 min and protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Chemical). For immunoprecipitations, lysates were incubated with the appropriate antibodies for 3 h overnight at 4 °C. Immune complexes were collected onto protein A-Sepharose beads, washed extensively, resolved by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). Immunoblots were blocked with 5% bovine serum albumin or Carnation non-fat dry milk in 10 mmTris-HCl, pH 7.4, 150 mm NaCl, and 0.05% Tween 20. After incubation with appropriate primary and secondary antibodies, blots were visualized using enhanced chemiluminescence reagents (ECL;Amersham Biosciences). Quantification was carried out using NIH Image Pro Software Version 1.62; data are expressed as relative units of phosphorylated protein per total protein for each band. Anti-PTP1B and anti-PDGFRβ antisera were used at 1/1000 for immunoblotting. Anti-human PTP1B monoclonal antibodies (FG6) were used at 1 μg/ml for immunoprecipitations and immunoblotting. All other antibodies were used at concentrations as recommended by the supplier. Cells were starved overnight in 0.5% fetal calf serum, treated with 2 mmH2O2 at 37 °C for 30 min, and stimulated with 50 ng/ml PDGF at room temperature for 10 min or left untreated. Anti-PDGFR immunoprecipitates were prepared from lysates of one 75-cm2 tissue culture flask. Control immunoprecipitations were carried out using normal rabbit IgG (Santa Cruz). In-gel phosphatase assays were performed as described by Burridge and Nelson (41Burridge K. Nelson A. Anal. Biochem. 1995; 232: 56-64Crossref PubMed Scopus (85) Google Scholar) with minor modifications. For substrate, poly(Glu4Tyr1)n was radiolabeled with [γ32P]ATP (PerkinElmer Life Sciences) using recombinant human p60c-Src, expressed as a glutathione S-transferase-fusion protein inEscherichia coli. The substrate was cast in a 10% SDS-polyacrylamide gel (acrylamide/bisacrylamide, 30:0.8). Instead of Tween 40, Tween 20 was used throughout, and the final renaturation step was carried out overnight in a buffer containing 50 mmTris-HCl, pH 7.4, 0.3% 2-mercaptoethanol, 1 mm EDTA, and 0.04% Tween 20. Previous reports indicated that PTP1B dephosphorylates the EGFR, at least when one or both of these proteins are overexpressed. To investigate whether the EGFR is a PTP1B target under physiological conditions, primary MEFs were generated from PTP1B (exon 1) −/− mice and WT control mice. Starved MEFs were stimulated with EGF (50 ng/ml) for various times or left unstimulated. Total cell lysates or EGFR immunoprecipitates were resolved by SDS-PAGE and subjected to anti-phosphotyrosine immunoblotting (Fig. 1 A). As expected, in starved cells, there was no detectable tyrosyl phosphorylation of the EGFR in total cell lysates or EGFR immunoprecipitates from either WT or PTP1B−/− MEFs. However, after stimulation, EGFR tyrosyl phosphorylation was enhanced and sustained in PTP1B−/−MEFs compared with control MEFs (KO/WT ratio ± S.E., 1.73 ± 0.22, 1.46 ± 0.18, 1.83 ± 0.13, and 1.55 ± 0.21 at 1, 5, 15, and 30 min, respectively, as quantified by scanning densitometry). Reprobing these blots with anti-EGFR antibodies revealed that the difference in EGFR tyrosyl phosphorylation was not caused by increased EGFR expression in PTP1B−/− MEFs, but instead reflected increased specific tyrosyl phosphorylation. Together with the earlier PTP1B overexpression/dominant-negative mutant studies, these data show that PTP1B plays a role in dephosphorylating the EGFR, at least in MEFs. Although to our knowledge PTP1B has not been shown to regulate the PDGFR, PDGF-stimulated PDGFR tyrosyl phosphorylation also was increased in the absence of PTP1B (Fig. 1 B), (KO/WT ratio ± S.E., 1.53 ± 0.22, 1.72 ± 0.12, 1.94 ± 0.35, and 2.1 ± 0.44 for 1, 5, 15, and 30 min, respectively), suggesting that PTP1B also regulates the PDGFR. Anti-PTP1B immunoblots confirmed the absence of PTP1B protein in the PTP1B−/−MEFs (Fig. 1 C). Although EGFR and PDGFR tyrosyl phosphorylation are increased in the absence of PTP1B, mice lacking PTP1B exhibit no obvious hypermorphism of EGFR or PDGFR pathways. To begin to understand this apparent paradox, we examined signaling pathways downstream of these RTKs in WT and PTP1B−/− MEFs. Activation of the Erk pathway, as assessed by immunoblotting with phospho-specific anti-Erk antibodies, was enhanced slightly in response to EGF stimulation of PTP1B−/−, compared with WT MEFs (KO/WT ratio ± S.E., 1.02 ± 0.08, 1.31 ± 0.1, 1.36 ± 0.25, and 1.39 ± 0.04 at 1, 5, 15, and 30 min, respectively). Erk activation was enhanced to an even greater extent in PDGF-stimulated PTP1B−/− MEFs compared with WT (Fig. 2 A) (KO/WT ratio ± S.E., 1.13 ± 0.15, 1.2 ± 0.07, 1.58 ± 0.14, and 1.76 ± 0.36 at 1, 5, 15, and 30 min, respectively). In contrast, growth factor-stimulated Akt activation (as assayed by pAkt immunoblotting) was not increased in PTP1B−/− MEFs (compared with WT MEFs) in response to either growth factor, in fact there was a tendency for decreased activation (Fig. 2 B) (e.g. KO/WT ratio ± S.E., 0.93 ± 0.21, 0.85 ± 0.04, 0.95 ± 0.11, and 0.99 ± 0.12 for 1, 5, 15, and 30 min of PDGF stimulation, respectively; 0.73 ± 0.28 and 0.98 ± 0.15 for 1 and 5 min post-EGF stimulation, respectively). These results suggested that MEFs compensate for the increased EGFR or PDGFR activation caused by PTP1B deficiency somewhere between the hyperphosphorylated RTK and Akt activation. The small enhancement of Erk activation in PTP1B−/− cells suggests compensatory mechanisms for the effects of increased RTK activation on this signaling pathway as well. To begin to understand the molecular basis for these effects, we focused on more immediate events after PDGFR activation. PDGF-stimulated tyrosyl phosphorylation of Shp2 (Fig. 3 A) and Shc (Fig. 3 B), two signal relay molecules implicated in Erk activation (reviewed in Refs. 42Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 43Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (732) Google Scholar), was enhanced significantly in PTP1B−/− MEFs compared with WT. Association of Shp2 with the PDGFR is probably increased as well (Fig. 4). Activation of the Akt pathway is mediated, at least in part, by recruitment of phosphatidyl inositol-3 kinase (PI-3K) to the PDGFR (reviewed in Ref. 44Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1221) Google Scholar). Consistent with the lack of enhanced Akt activation in PDGF-stimulated PTP1B−/− MEFs, association of the p85 (targeting) subunit of PI-3K with the PDGFR was not increased in the absence of PTP1B (Fig. 3 C). In addition, PDGFR-associated PI-3K activity was not altered significantly in PTP1B−/−, compared with WT cells (data not shown).Figure 4Reconstitution of PTP1B in immortalized murine PTP1B−/− fibroblasts. Immortalized PTP1B−/− cells were generated and then reconstituted with hPTP1B-WT, hPTP1B-D/A, or the parental pWZL vector, as described under “Experimental Procedures.” A, lysates from randomly growing cells were subjected to SDS-PAGE and then immunoblotted for hPTP1B and Shp2. B, lysates of PDGF-stimulated PTP1B−/− and hPTP1B-WT fibroblasts were subjected to SDS-PAGE and then immunoblotted with either anti-phosphotyrosine (Ptyr), anti-phospho-Erk (pMAPK), anti-total Erk-2 (ERK 2), anti-phospho-Akt (pAKT) and anti-total Akt (AKT) antibodies, respectively. C, lysates of unstimulated and PDGF-stimulated (15 min) PTP1B−/−, hPTP1B-WT-, and hPTP1B-D/A-reconstituted cells were immunoprecipitated with anti-hPTP1B antibodies and then subjected to anti-phosphotyrosine (Ptyr) immunoblotting. Right panel, multiple PTPs, including PTP1B, associate with the PDGFR in murine fibroblasts. Lysates of immortalized PTP1B−/−fibroblasts, reconstituted with hPTP1B or the parental vector, were subjected to immunoprecipitation with anti-PDGFR antibodies or normal rabbit IgG as control, as indicated. The immunoprecipitates were separated by SDS-PAGE in 10% gels containing32P-tyrosine-phosphorylated poly(Glu4Tyr1), and PTPs were visualized as activity bands (white) after renaturation. Identification of PTPs is based on immunodepletion experiments and comparison of wild type and PTP1B−/− cells. Note that several fragments of PTP1B are detectable. A component of about 40 kDa is present in the PTP1B−/− cells and is therefore not a PTP1B fragment. Different parts of the same gel have been exposed for different lengths of time to optimize PTP detection. Lower panels show controls for PDGFR immunoprecipitation (PDGFR-IP) and PDGFR tyrosine phosphorylation (pTyr).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conceivably, increased RTK activation in PTP1B−/− MEFs could reflect an indirect effect of PTP1B on fibroblast differentiation, rather than an effect of PTP1B on RTK signaling per se. To exclude this possibility, we restored PTP1B expression to PTP1B−/− cells (Fig. 4 A). A large T Ag-immortalized PTP1B−/−fibroblast cell line was generated and reconstituted with human PTP1B (hPTP1B-WT), the PTP1B substrate-trapping mutant D181A (hPTP1B-D/A), or the parental pWZL expression vector (see “Experimental Procedures”). Immunostaining revealed that >90% of hPTP1B-WT- and hPTP1B-D/A-infected, hygromycin-resistant cells expressed hPTP1B protein (data not shown); thus, these pools can be studied directly, avoiding potential clone-to-clone variation. Notably, PDGF-stimulated (but not basal) PDGFR tyrosyl phosphorylation was greater in the vector control-infected PTP1B−/− cell pool, compared with the cells reconstituted with hPTP1B-WT (Fig. 4 B). Restoration of hPTP1B-WT expression in immortalized PTP1B−/− fibroblasts diminished PDGF-evoked Erk activation but left PDGF-evoked Akt activation unaffected (Fig. 4 B). The effects of restoring hPTP1B expression on PDGFR tyrosyl phosphorylation were observed only at submaximal doses of PDGF. The reason for this is not clear, but may be caused, at least in part, by the increased level of PDGFR expression that accompanied immortalization (data not shown). The resultant increase in the concentration of activated PDGFR may allow access to a secondary PDGFR phosphatase with a higher Kmvalue (see below). These data indicate that PTP1B regulates PDGFR tyrosyl phosphorylation in a cell-autonomous manner. To seek further evidence that PTP1B directly regulates PDGFR tyrosyl phosphorylation, we asked whether the PDGFR could form a stable complex with PTP1B-D/A, which is impaired catalytically, but retains substrate-binding ability (16Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar, 43Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (732) Google Scholar, 45Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar). PTP1B immunoprecipitates prepared from starved or PDGFR-stimulated, immortalized PTP1B−/− cells reconstituted with hPTP1B-WT, hPTP1B-D/A, or the vector controls were immunoblotted with anti-phosphotyrosine antibodies. An ∼180-kDa band corresponding in size to the PDGFR coimmunoprecipitated with hPTP1B-D/A but not hPTP1B-WT (Fig. 4 C); this interaction was detected only upon PDGF stimulation. Unfortunately, available anti-PDGFR antibodies were insufficiently sensitive to unambiguously identify this band as the PDGFR. However, this is highly likely; using a highly sensitive fluorescence resonance energy transfer approach, we recently demonstrated complex formation between the PDGFR and PTP1B in hPTP1B-D/A-expressing cells (46Haj F.G. Verveer P.J. Squire A. Neel B.G. Bastiaens P.I. Science. 2002; 295: 1708-1711Crossref PubMed Scopus (367) Google Scholar). Previous studies reported associations between the C215S and/or D181A substrate trapping mutants of PTP1B and the PDGFR (17Liu F. Chernoff J. Biochem. J. 1997; 327: 139-145Crossref PubMed Scopus (161) Google Scholar) as well as the EGFR (16Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar) and IR (15Seely B.L. Staubs P.A. Reichart D.R. Berhanu P. Milarski K.L. Saltiel A.R. Kusari J. Olefsky J.M. Diabetes. 1996; 45: 1379-1385Crossref PubMed Google Scholar), respectively. Unlike the inducible interaction we observed, in these earlier studies, constitutive complex formation was observed. This most probably reflects the high level of overexpression of PTP1B trapping mutant and/or the RTK used in those studies. Although coimmunoprecipitation of the PDGFR and WT-PTP1B was not observed by immunoblotting, PDGFR immunoprecipitates contained an ∼50-kDa PTP activity detectable by a highly sensitive in-gel phosphatase assay (Fig. 4 D). This activity is almost certainly PTP1B, because it is absent in PDGFR immunoprecipitates prepared from PTP1B−/− cells. Interestingly, PTP1B/PDGFR association was detected only when cells were pretreated with hydrogen peroxide (data not shown). Much recent work suggests that specific PTPs, including PTP1B, undergo physiological oxidation (and thus transient inactivation) by peroxide (reviewed in Ref. 47Xu D. Rovira I.I. Finkel T. Dev. Cell. 2002; 2: 251-252Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Conceivably, PDGFR/PTP1B association requires a conformational change in PTP1B that is induced upon oxidation. Alternatively, transient PDGFR/PTP1B interactions may be “trapped” by the peroxide-induced generation of one or more intermolecular disulfide bonds. A peroxide-dependent a" @default.
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• W2051201358 date "2003-01-01" @default.
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• W2051201358 title "Regulation of Receptor Tyrosine Kinase Signaling by Protein Tyrosine Phosphatase-1B" @default.
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• W2051201358 doi "https://doi.org/10.1074/jbc.m210194200" @default.
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