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The Protein-tyrosine Phosphatase TCPTP Regulates Epidermal Growth Factor Receptor-mediated and Phosphatidylinositol 3-Kinase-dependent Signaling*

  • Tony Tiganis
    Correspondence
    National Health and Medical Research Council of Australia C. J. Martin Fellow. To whom correspondence should be addressed: St. Vincent's Inst. of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia. Tel.: 61-3-9288-2480; Fax: 61-3-9416-2676; E-mail: [email protected]
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  • Bruce E. Kemp
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  • Nicholas K. Tonks
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant CA53840 (to N. K. T.) and by grants from the National Health and Medical Research Council of Australia (to T. T. and B. E. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 24, 1999DOI:https://doi.org/10.1074/jbc.274.39.27768
      In this study we have investigated the down-regulation of epidermal growth factor (EGF) receptor signaling by protein-tyrosine phosphatases (PTPs) in COS1 cells. The 45-kDa variant of the PTP TCPTP (TC45) exits the nucleus upon EGF receptor activation and recognizes the EGF receptor as a cellular substrate. We report that TC45 inhibits the EGF-dependent activation of the c-Jun N-terminal kinase, but does not alter the activation of extracellular signal-regulated kinase 2. These data demonstrate that TC45 can regulate selectively mitogen-activated protein kinase signaling pathways emanating from the EGF receptor. In EGF receptor-mediated signaling, the protein kinase PKB/Akt and the mitogen-activated protein kinase c-Jun N-terminal kinase, but not extracellular signal-regulated kinase 2, function downstream of phosphatidylinositol 3-kinase (PI 3-kinase). We have found that TC45 and the TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, inhibit almost completely the EGF-dependent activation of PI 3-kinase and PKB/Akt. TC45 and TC45-D182A act upstream of PI 3-kinase, most likely by inhibiting the recruitment of the p85 regulatory subunit of PI 3-kinase by the EGF receptor. Recent studies have indicated that the EGF receptor can be activated in the absence of EGF following integrin ligation. We find that the integrin-mediated activation of PKB/Akt in COS1 cells is abrogated by the specific EGF receptor protein-tyrosine kinase inhibitor tyrphostin AG1478, and that TC45 and TC45-D182A can inhibit activation of PKB/Akt following the attachment of COS1 cells to fibronectin. Thus, TC45 may serve as a negative regulator of growth factor or integrin-induced, EGF receptor-mediated PI 3-kinase signaling.
      PTP
      protein-tyrosine phosphatase
      PTK
      protein-tyrosine kinase
      TCPTP
      T-cell PTP
      ER
      endoplasmic reticulum
      EGF
      epidermal growth factor
      HA
      hemagglutinin
      PAGE
      polyacrylamide gel electrophoresis
      FBS
      fetal bovine serum
      BSA
      bovine serum albumin
      PBS
      phosphate-buffered saline
      DMEM
      Dulbecco's modified Eagle's medium
      SH
      Src homology
      ERK
      extracellular signal-regulated kinase
      PKB
      protein kinase B
      JNK
      c-Jun N-terminal kinase
      PI
      phosphatidylinositol
      GST
      glutathioneS-transferase
      Protein-tyrosine phosphatases (PTPs)1 are a large and structurally diverse family of enzymes, characterized by the consensus sequence (I/V)HC XAGXX R. They are found in eukaryotes, prokaryotes and viruses and can either antagonize or potentiate protein-tyrosine kinase (PTK)-dependent signaling. PTPs have been shown to participate as either positive or negative regulators of signal transduction in a wide range of physiological processes, which include cellular growth and proliferation, migration, differentiation and survival (
      • Tonks N.K.
      • Neel B.G.
      ,
      • Tonks N.K.
      ,
      • Neel B.G.
      • Tonks N.K.
      ). Despite their important roles in such fundamental physiological processes, the mechanism by which PTPs exert their effects is often poorly understood.
      The human T-Cell PTP (TCPTP) is an intracellular non-transmembrane phosphatase that was originally cloned from a T-cell cDNA library but is now known to be expressed in many tissues. TCPTP contains a conserved catalytic domain and a non-catalytic C-terminal segment that varies in size and function as a result of alternative splicing. Two splice variants differing only in their extreme C termini are expressed. The 48-kDa form of human TCPTP (TC48) contains a 34-residue hydrophobic tail, which is replaced by a hydrophilic 6-residue sequence in the 45-kDa form (TC45). TC48 localizes to the endoplasmic reticulum (ER) (
      • Cool D.E.
      • Tonks N.K.
      • Charbonneau H.
      • Fischer E.H.
      • Krebs E.G.
      ,
      • Lorenzen J.A.
      • Dadabay C.Y.
      • E. H. F.
      ), whereas under basal conditions TC45 is localized in the nucleus due to the presence of a bipartite nuclear localization sequence (
      • Lorenzen J.A.
      • Dadabay C.Y.
      • E. H. F.
      ,
      • Mosinger Jr., B.
      • Tillmann U.
      • Westphal H.
      • Tremblay M.L.
      ,
      • Champion-Arnaud P.
      • Gesnel M.C.
      • Foulkes N.
      • Ronsin C.
      • Sassone-Corsi P.
      • Breathnach R.
      ,
      • Tillmann U.
      • Wagner J.
      • Boerboom D.
      • Westphal H.
      • Tremblay M.L.
      ,
      • Tiganis T.
      • Flint A.J.
      • Adam S.A.
      • Tonks N.K.
      ).
      All PTPs contain an aspartic acid that is essential for catalysis. Mutation of this residue, Asp-182 in TCPTP, to alanine reduces the catalytic activity but maintains a high affinity for substrates, thereby generating a “substrate trapping” mutant, which can form stable complexes with tyrosine-phosphorylated proteins in vitro (
      • Garton A.J.
      • Flint A.J.
      • Tonks N.K.
      ) and in vivo (
      • Flint A.J.
      • Tiganis T.
      • Barford D.
      • Tonks N.K.
      ,
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ). Using the TCPTP D182A substrate trapping mutants, we have shown previously that TCPTP displays a restricted specificity in a cellular context, and that the EGF receptor is one of its substrates (
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ). Both TC48 and TC45 recognize the tyrosine-phosphorylated EGF receptor as a substrate in a cellular context; TC48 recognizes the receptor as it proceeds through the ER and may function to prevent inappropriate signaling by the nascent receptor during synthesis, whereas TC45 can exit the nucleus in response to EGF and gain access to signaling complexes containing the EGF receptor at the plasma membrane (
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ).
      In the present study we have examined the effect of overexpression of TC45 on EGF receptor-induced signaling events. We show that TC45 can inhibit the EGF-induced activation of PKB/Akt and that this correlates with a reduced association of PI 3-kinase with the activated EGF receptor. In addition, we show that plating of COS1 cells on fibronectin leads to activation of PKB/Akt, in an EGF-independent but EGF receptor and PI 3-kinase-dependent manner, which is also inhibited by TC45. Thus, TC45 may serve as a negative regulator of specific signals from the EGF receptor that mediate PKB/Akt activation.

      EXPERIMENTAL PROCEDURES

      Materials

      Recombinant human EGF was purchased from Genzyme Diagnostics (Cambridge, MA), human plasma fibronectin from Life Technologies, Inc., wortmannin and crude brain lipids were from Sigma. Monoclonal EGF receptor Ab-1 antibody used for immunoprecipitation was purchased from Calbiochem Oncogene Research Products (Cambridge, MA), polyclonal EGF receptor (1005) antibody used for immunoblotting from Santa Cruz Biotechnology (Santa Cruz, CA), PI 3-kinase p85 (P13020) antibody from Transduction Laboratories (Lexington, KY), phospho-Akt (Ser-473) and Akt antibodies from New England BioLabs (Beverly, MA), and FLAG M2 antibody from Eastman Kodak Co. The following constructs and reagents were generously provided by colleagues: hemagglutinin-tagged (HA)-PKB/Akt pECE by B. Hemmings (Friedrich Miescher Institut, Basel, Swizerland), p110K227E pSG5 by J. Downward (Imperial Cancer Research Fund, London, United Kingdom), FLAG-JNK by L. Van Aelst (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), HA-Erk2 pJ3H by J. Chernoff (Temple University, Philadelphia, PA), monoclonal ERK2 1B3B9 antibody by M. Weber (University of Virginia, Charlottesville, VA), monoclonal anti-TCPTP antibody CF4 by D. Hill (Calbiochem Oncogene Research Products, Cambridge, MA), and tyrphostin AG1478 by E. Thompson (St. Vincent's Institute of Medical Research, Melbourne, Australia). Monoclonal anti-phosphotyrosine antibodies G98 (subtype IgM) and G104 (subtype IgG) have been described previously (
      • Garton A.J.
      • Flint A.J.
      • Tonks N.K.
      ,
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ).

      Cell Culture, Transfections, and Electroporations

      COS1 cells were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. Where indicated, COS1 cells were serum-starved for 24 h in DMEM containing 0.1% FBS, plus antibiotics.
      COS1 cells were transfected by the calcium phosphate precipitation method as described previously (
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ). Unless otherwise indicated cells were transfected using TCPTP pMT2 plasmid DNA at 20 μg/10-cm dish. Cells were washed three times with phosphate buffered saline (PBS) at 5–6 h after transfection and supplemented with fresh DMEM containing 10% FBS. Where indicated approximately 1–2 × 106COS1 cells were electroporated in 250 μl of medium with 20 μg of TCPTP pMT2 plasmid at 200 V and 950 microfarads in 0.4-cm cuvettes and seeded into a 10-cm dish. Transfected or electroporated cells were collected at 36–48 h after transfection, or washed once with PBS at 24 h after transfection, supplemented with DMEM containing 0.1% FBS and processed at 48 h after transfection. The efficiency of electroporation, as assessed by 5-bromo-4-chloro-3-indolyl β-d-galactosidase staining of pCMV-β-galactosidase-electroporated COS1 cells, was routinely 50–75%.

      Immune Complex Kinase Reactions

      ERK2 and JNK Assays

      COS1 cells, in 10-cm dishes, were transfected with 2 μg of HA-tagged ERK2 pJ3H plasmid or 5 μg of FLAG-tagged JNK pCMV plasmid and either 15 μg of pMT2 plasmid or 15 μg of the TC45 or TC45-D182A pMT2 plasmids. At 24 h after transfection, cells were washed once with PBS and serum-starved overnight for 24 h. Cells were then left unstimulated or stimulated with 100 ng/ml EGF for 15 min and processed for either ERK2 kinase assays as described previously (
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ) or JNK assays. For JNK assays, cells were lysed in 0.9 ml of immunoprecipitation (IP) lysis buffer (50 mm Tris, pH 7.5, 1% w/v Nonidet P-40, 150 mm NaCl, 50 mm NaF, 1 mm vanadate, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 mm benzamidine, and 2 mm phenylmethylsulfonyl fluoride), centrifuged (12,000 × g for 10 min at 4 °C) and FLAG-tagged JNK immunoprecipitated from the supernatant with 5 μg of anti-FLAG M2 antibody for 90 min at 4 °C. Immune complexes were collected on protein G-Sepharose for 30 min at 4 °C and JNK kinase activity measured using GST-Jun (GST fused to the N terminus of c-Jun) as substrate as described previously (
      • Joneson T.
      • McDonough M.
      • Bar-Sagi D.
      • Van Aelst L.
      ). Anti-FLAG immune complexes from these reactions were resolved by SDS-PAGE, immunoblotted with anti-FLAG antibodies, and quantitated by densitometry in order to normalize for FLAG-JNK activity.

      PI 3-Kinase Assays

      COS1 cells were electroporated with either pMT2 vector control, TC45 or TC45-D182A plasmids as indicated above. At 24 h after electroporation, cells were serum-starved for 24 h and then stimulated with EGF (100 ng/ml) for 15 min. Cells were lysed in 0.9 ml of IP lysis buffer, and lysates were precleared with 0.1 ml of Pansorbin (Cambridge, MA) for 60 min at 4 °C. Precleared lysates were subsequently centrifuged (12,000 ×g for 10 min at 4 °C), and the phosphotyrosine-containing proteins were immunoprecipitated overnight at 4 °C under constant mixing, using a mixture of the anti-phosphotyrosine antibodies G98 and G104 (20 μl of G98 and 20 μl of G104 ascites for every 10-cm dish of cells) (
      • Garton A.J.
      • Flint A.J.
      • Tonks N.K.
      ,
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ). Immune complexes were collected on protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Uppsala, Sweden) for 60 min at 4 °C, washed four times with IP lysis buffer; two times with 100 mm Tris, pH 7.5, buffer containing 100 mmNaCl and 1 mm EDTA; and three times with PI 3-kinase buffer (20 mm HEPES, pH 7.5, 5 mm MgCl2, 1 mm EGTA). PI 3-kinase assays were performed in 100 μl of PI 3-kinase buffer containing 100 μm ATP (plus 10 μCi of [γ-32P]ATP) and 50 μg of sonicated crude brain lipids (containing approximately 10% phosphatidylinositol) (
      • Shaw L.M.
      • Rabinovitz I.
      • Wang H.H.
      • Toker A.
      • Mercurio A.M.
      ) for 20 min at room temperature. Reactions were stopped with 100 μl of 1m HCl, and the phospholipids were extracted and separated by thin layer chromatography on silica plates coated with potassium oxalate. [32P]Phosphatidylinositol was quantitated on a PhosphorImager using ImageQuant software (Molecular Dynamics).

      PKB/Akt Assays

      COS1 cells, in 10-cm dishes, were transfected with 5 μg of HA-tagged PKB/Akt pECE plasmid, with or without 5 μg of p110K227E pSG5 plasmid, and either 15 μg of the pMT2 plasmid or 15 μg of the TC45 or TC45-D182A pMT2 plasmids. At 24 h after transfection, cells were serum-starved and either left unstimulated or stimulated with EGF (100 ng/ml) for 15 min. Cells were then lysed in 0.9 ml of IP lysis buffer, the lysates centrifuged (12,000 × g for 10 min at 4 °C) and HA-PKB/Akt immunoprecipitated from the supernatant with anti-HA antibody 12CA5 (2 μl of ascites/10-cm dish of cells) for 2 h at 4 °C. Immune complexes were collected on protein A-Sepharose CL-4B for 60 min at 4 °C, washed three times with IP lysis buffer, and washed two times with PKB/Akt kinase buffer (50 mm Tris, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol). PKB/Akt kinase activity was assayed using the peptide substrate RPRAATF-NH2 (
      • Alessi D.R.
      • Caudwell F.B.
      • Andjelkovic M.
      • Hemmings B.A.
      • Cohen P.
      ) by incubating the immunoprecipitated HA-PKB/Akt for 10 or 20 min at 30 °C in 30 μl of PKB/Akt kinase buffer containing 50 μm ATP (plus 0.25 μm[γ-32P]ATP) and 50 μm peptide substrate. The reaction was terminated by adding 15 μl of 0.5 m EDTA and after brief centrifugation 20 μl of the supernatant was spotted onto P-81 phosphocellulose paper. Unincorporated [γ-32P]ATP was eliminated by three 5-min washes in 75 mm orthophosphoric acid and phosphorylated peptide bound to the paper counted. Anti-HA immune complexes from the kinase reactions were resolved by SDS-PAGE, immunoblotted with anti-HA antibodies, and quantitated by densitometry in order to normalize for HA-PKB/Akt activity. Transfections for PKB/Akt assays were conducted either in duplicate or triplicate.

      Cell Stimulation with Fibronectin

      COS1 cells were electroporated with pMT2 vector control, TC45, or TC45-D182A expression plasmids as indicated above. At 24 h after electroporation, cells were serum-starved for 24 h, washed in PBS, and then harvested by limited trypsin-EDTA treatment (1 ml of 0.025% trypsin plus 5 mm EDTA in DMEM minus phenol red/10-cm dish of cells). After trypsin inhibition by soybean trypsin inhibitor (1 mg of chromatographically purified type I-S trypsin inhibitor (Sigma)/10-cm dish of cells) in DMEM minus phenol red containing 0.25% (w/v) bovine serum albumin (BSA) (radioimmunoassay grade, fraction V from Sigma), the cells were pelleted by centrifugation and washed twice with DMEM minus phenol red containing 0.25% (w/v) BSA and resuspended in DMEM minus phenol red containing 0.1% (w/v) BSA. The cells were held in suspension at 37 °C for 30 min prior to attachment for 1 h onto tissue culture dishes precoated with fibronectin (5 ml of 10 μg/ml human plasma fibronectin/10-cm dish incubated overnight at 4 °C and then washed once with DMEM minus phenol red and warmed to 37 °C for 30 min). Attached cells were rinsed twice in DMEM minus phenol red containing 0.1% (w/v) BSA, once in ice-cold PBS, and then collected in hot 3× Laemmli sample buffer containing 6% (v/v) β-mercaptoethanol. Proteins were resolved by SDS-PAGE and immunoblotted as indicated.

      DISCUSSION

      Activation of the EGF receptor PTK by ligand results in receptor dimerization and autophosphorylation on tyrosyl residues. The phosphotyrosyl residues that are produced serve as docking sites for SH2 domain-containing signaling molecules, leading to the assembly of multiprotein signaling complexes required for cell growth, proliferation, and survival. Although significant progress has been made in defining the tyrosine phosphorylation-dependent signaling pathways downstream of the EGF receptor, relatively little is known about which members of the PTP family serve to antagonize these signaling events. Through the use of substrate trapping mutants, we have demonstrated previously that the nuclear, 45-kDa form of TCPTP, TC45, can exit the nucleus in response to EGF and recognize tyrosine-phosphorylated substrates such as the EGF receptor and the 52-kDa isoform of Shc (
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ). In the case of Shc, TC45 can recognize preferentially Shc phosphorylated on tyrosine 239, compared to tyrosine 317, indicating that TC45 may be capable of regulating selectively Shc-dependent signaling events (
      • Tiganis T.
      • Bennett A.M.
      • Ravichandran K.S.
      • Tonks N.K.
      ).
      In this study we have characterized the ability of TC45 to regulate EGF receptor-induced signaling. TC45 inhibited the EGF receptor-mediated activation of PI 3-kinase and PKB/Akt and, to a lesser extent, JNK, but did not modulate the activation of ERK2. Thus, TC45 can regulate in a selective manner signaling processes which emanate from the EGF receptor. Our data indicate that TC45 may exert its effects on PI 3-kinase and PKB/Akt by inhibiting the recruitment of PI 3-kinase to the EGF receptor. Recruitment of the p85 regulatory subunit of PI 3-kinase to growth factor receptors is necessary for PI 3-kinase activation. The lipid products of PI 3-kinase then engage the pleckstrin homology domain of PKB/Akt and translocate it in the plasma membrane, where it is phosphorylated on Ser/Thr residues for complete activation (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Boudewijn M.
      • Burgering T.
      • Coffer P.J.
      ,
      • Downward J.
      ,
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ,
      • Hemmings B.A.
      ,
      • Anderson K.E.
      • Coadwell J.
      • Stephens L.R.
      • Hawkins P.T.
      ). EGF receptor-mediated activation of PI 3-kinase can also contribute to the activation of JNK, although the mechanism involved is not defined, but ERK2 activation is PI 3-kinase-independent.
      The p85 regulatory subunit of PI 3-kinase contains two Src homology 2 (SH2) domains that bind tyrosine-phosphorylated amino acids that have a consensus Tyr-X-X-Met motif (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ). Binding of p85 via its SH2 domain to tyrosine-phosphorylated receptors allows for the recruitment and activation of the PI 3-kinase p110 catalytic subunit. The five major autophosphorylation sites on the EGF receptor do not fit the Tyr-X-X-Met motif, but under certain circumstances Src can phosphorylate the EGF receptor on Tyr920 which has the motif for p85 binding (
      • Stover D.R.
      • Becker M.
      • Liebetanz J.
      • Lydon N.B.
      ). Also, others have reported that the SH2 domains of p85 can interact directly with the tyrosine-phosphorylated EGF receptor (
      • Moscatello D.K.
      • Holgado-Madruga M.
      • Emlet D.R.
      • Montgomery R.B.
      • Wong A.J.
      ). Alternatively, the EGF receptor can phosphorylate docking proteins such as p120cbl and Gab1 (
      • Meisner H.
      • Conway B.R.
      • Hartley D.
      • Czech M.P.
      ,
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      ), which in turn bind p85 and therefore recruit PI 3-kinase activity. Regardless of the precise mechanism by which PI 3-kinase associates with the EGF receptor, tyrosine phosphorylation of the EGF receptor is necessary for this event. By dephosphorylating the EGF receptor, TC45 can inhibit the association of p85 and concomitant activation of PI 3-kinase and PKB/Akt.
      The inhibition of p85 recruitment to the EGF receptor that we observed is consistent with the effects of TC45 and TC45-D182A on the activities of PI 3-kinase and PKB/Akt but not entirely consistent with their effects on JNK. Although both TC45 and TC45-D182A could equally inhibit the recruitment of p85 and the activation of PI 3-kinase and PKB/Akt, we observed that only wild type TC45 inhibited JNK. However, it is important to note that, unlike PKB/Akt, whose EGF-induced activation can be completely inhibited by the PI 3-kinase inhibitor wortmannin, JNK activity is only partially inhibited by this PI 3-kinase antagonist (this study and Ref.
      • Logan S.K.
      • Falasca M.
      • Hu P.
      • Schlessinger J.
      ). In addition, dominant negative forms of the p85 regulatory subunit only partially inhibit the EGF-induced activation of JNK (
      • Logan S.K.
      • Falasca M.
      • Hu P.
      • Schlessinger J.
      ). Therefore, other signaling events in addition to PI 3-kinase would seem to be necessary for EGF-mediated activation of JNK. Until the exact nature of the PI 3-kinase-mediated activation of JNK has been defined, it will be difficult to speculate as to the mechanism of differential regulation of JNK and PKB/Akt by TCPTP.
      Moro et al. (
      • Moro L.
      • Venturino M.
      • Bozzo C.
      • Silengo L.
      • Altruda F.
      • Beguinot L.
      • Tarone G.
      • Defilippi P.
      ) have recently reported that adhesion of human primary skin fibroblasts or ECV304 endothelial cells to fibronectin results in EGF receptor activation in the absence of EGF and that this is necessary for the integrin-mediated activation of the MAPK ERK1. We found that, in COS1 fibroblast cells, the integrin-mediated activation of ERK2 as well as PKB/Akt are also dependent on EGF receptor activation. Moreover, as in the case of EGF-induced signaling, the activation of PKB/Akt, but not ERK2, is PI 3-kinase-dependent. In light of these observations, we examined whether TC45 could also regulate EGF receptor signaling processes following transactivation by integrins. Consistent with the effect we observe of TC45 on the EGF-induced activation of PKB/Akt, we have also demonstrated that TC45 can inhibit the activation of PKB/Akt, but not ERK2, following attachment of COS1 cells to fibronectin. Thus, it would be appear that TC45 may regulate EGF receptor signaling irrespective of whether activation of the receptor PTK is initiated by growth factor or integrins.
      The EGF receptor is overexpressed or mutated in many human tumors including those derived from brain, lung, breast, and skin (
      • Schlegel J.
      • Merdes A.
      • Stumm G.
      • Albert F.K.
      • Forsting M.
      • Hynes N.
      • Kiessling M.
      ,
      • Garcia de Palazzo I.E.
      • Adams G.P.
      • Sundareshan P.
      • Wong A.J.
      • Testa J.R.
      • Bigner D.D.
      • Weiner L.M.
      ,
      • Harris A.L.
      • Nicholson S.
      • Sainsbury R.
      • Wright C.
      • Farndon J.
      ,
      • Wong A.J.
      • Ruppert J.M.
      • Bigner S.H.
      • Grzeschik C.H.
      • Humphrey P.A.
      • Bigner D.S.
      • Vogelstein B.
      ,
      • Wong A.J.
      • Bigner S.H.
      • Bigner D.D.
      • Kinzler K.W.
      • Hamilton S.R.
      • Vogelstein B.
      ,
      • Hunts J.
      • Ueda M.
      • Ozawa S.
      • Abe O.
      • Pastan I.
      • Shimizu N.
      ,
      • Libermann T.A.
      • Nusbaum H.R.
      • Razon N.
      • Kris R.
      • Lax I.
      • Soreq H.
      • Whittle N.
      • Waterfield M.D.
      • Ullrich A.
      • Schlessinger J.
      ). The high level of EGF receptor-mediated MAPK and PI 3-kinase signaling, which occurs as a consequence of this aberrant expression of the PTK, is believed to play an important role in the pathogenesis of these tumors (
      • Moscatello D.K.
      • Holgado-Madruga M.
      • Emlet D.R.
      • Montgomery R.B.
      • Wong A.J.
      ,
      • Di Fiore P.P.
      • Pierce J.H.
      • Fleming T.P.
      • Hazan R.
      • Ullrich A.
      • King C.R.
      • Schlessinger J.
      • Aaronson S.A.
      ). Certain PTPs such as SHP-2 and SHP-1 have been reported to exert positive or negative effects on the regulation of EGF receptor signaling to MAPK (
      • Bennett A.M.
      • Hausdorff S.F.
      • Oreilly A.M.
      • Freeman R.M.
      • Neel B.G.
      ,
      • Su L.
      • Zhao Z.
      • Bouchard P.
      • Banville D.
      • Fischer E.H.
      • Krebs E.G.
      • Shen S.-H.
      ,
      • You M.
      • Zhao Z.
      ,
      • Keilhack H.
      • Tenev T.
      • Nyakatura E.
      • Godovac-Zimmermann J.
      • Nielsen L.
      • Seedorf K.
      • Bohmer F.-D.
      ,
      • Tomic S.
      • Greiser U.
      • Lammers R.
      • Kharitonenkov A.
      • Imyanitov E.
      • Ullrich A.
      • Bohmer F.-D.
      ,
      • Deb T.B.
      • Wong L.
      • Salomon D.S.
      • Zhou G.
      • Dixon J.E.
      • Gutkind J.S.
      • Thompson S.A.
      • Johnson G.R.
      ). However, to our knowledge, our data represent the first occasion on which a PTP has been shown to act upstream of PI 3-kinase, most likely on the EGF receptor, to regulate negatively EGF receptor-mediated and PI 3-kinase-dependent signaling events. Thus, TC45 may serve as an important target for intervention in tumors where excessive EGF receptor-mediated PI 3-kinase signaling contributes to the disease.

      Acknowledgments

      We thank David Hill for the TCPTP CF4 antibody and Mike Weber for the ERK2 antibody. We also thank Jörg Heierhorst for critical reading of the manuscript and Richard Pearson for help with the PKB/Akt assays.

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