FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1999791629> ?p ?o ?g. }

• W1999791629 endingPage "16989" @default.
• W1999791629 startingPage "16979" @default.
• W1999791629 abstract "Epidermal growth factor receptor (EGFR) gene amplification, mutations, and/or aberrant activation are frequent abnormalities in malignant gliomas and other human cancers and have been associated with an aggressive clinical course and a poor therapeutic outcome. Elevated glutathione S-transferase P1 (GSTP1), a major drug-metabolizing and stress response signaling protein, is also associated with drug resistance and poor clinical outcome in gliomas and other cancers. Here, we provide evidence that GSTP1 is a downstream EGFR target and that EGFR binds to and phosphorylates tyrosine residues in the GSTP1 protein in vitro and in vivo. Mass spectrometry and mutagenesis analyses in a cell-free system and in gliomas cells identified Tyr-7 and Tyr-198 as major EGFR-specific phospho-acceptor residues in the GSTP1 protein. The phosphorylation increased GSTP1 enzymatic activity significantly, and computer-based modeling showed a corresponding increase in electronegativity of the GSTP1 active site. In human glioma and breast cancer cells, epidermal growth factor stimulation rapidly increased GSTP1 tyrosine phosphorylation and decreased cisplatin sensitivity. Lapatinib, a clinically active EGFR inhibitor, significantly reversed the epidermal growth factor-induced cisplatin resistance. These data define phosphorylation and activation of GSTP1 by EGFR as a novel, heretofore unrecognized component of the EGFR signaling network and a novel mechanism of tumor drug resistance, particularly in tumors with elevated GSTP1 and/or activated EGFR. Epidermal growth factor receptor (EGFR) gene amplification, mutations, and/or aberrant activation are frequent abnormalities in malignant gliomas and other human cancers and have been associated with an aggressive clinical course and a poor therapeutic outcome. Elevated glutathione S-transferase P1 (GSTP1), a major drug-metabolizing and stress response signaling protein, is also associated with drug resistance and poor clinical outcome in gliomas and other cancers. Here, we provide evidence that GSTP1 is a downstream EGFR target and that EGFR binds to and phosphorylates tyrosine residues in the GSTP1 protein in vitro and in vivo. Mass spectrometry and mutagenesis analyses in a cell-free system and in gliomas cells identified Tyr-7 and Tyr-198 as major EGFR-specific phospho-acceptor residues in the GSTP1 protein. The phosphorylation increased GSTP1 enzymatic activity significantly, and computer-based modeling showed a corresponding increase in electronegativity of the GSTP1 active site. In human glioma and breast cancer cells, epidermal growth factor stimulation rapidly increased GSTP1 tyrosine phosphorylation and decreased cisplatin sensitivity. Lapatinib, a clinically active EGFR inhibitor, significantly reversed the epidermal growth factor-induced cisplatin resistance. These data define phosphorylation and activation of GSTP1 by EGFR as a novel, heretofore unrecognized component of the EGFR signaling network and a novel mechanism of tumor drug resistance, particularly in tumors with elevated GSTP1 and/or activated EGFR. Epidermal growth factor receptor (EGFR), 2The abbreviations used are: EGFRepidermal growth factor (EGF) receptorGBMglioblastoma multiformeGSTP1glutathione S-transferase P1GSHglutathioneEAethacrynic acidMAPKmitogen-activated protein kinaseIPimmunoprecipitationMSmass spectrometryLCliquid chromatographysiRNAsmall interfering RNA. 2The abbreviations used are: EGFRepidermal growth factor (EGF) receptorGBMglioblastoma multiformeGSTP1glutathione S-transferase P1GSHglutathioneEAethacrynic acidMAPKmitogen-activated protein kinaseIPimmunoprecipitationMSmass spectrometryLCliquid chromatographysiRNAsmall interfering RNA. a 170-kDa receptor-type tyrosine kinase, mediates diverse signaling pathways and cellular processes, including, proliferation, differentiation, motility, and survival (1Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (542) Google Scholar, 2Ali-Osman F. Friedman H.S. Antoun G.R. Readon D. Bigner D.D. Buolamwini J.K. Brain Tumors. Humana Press, Totowa, NJ2005: 359-381Crossref Google Scholar, 3Arteaga C.L. J. Clin. Oncol. 2001; 19: 32-40PubMed Google Scholar, 4Shinojima N. Tada K. Shiraishi S. Kamiryo T. Kochi M. Nakamura H. Makino K. Saya H. Hirano H. Kuratsu J. Oka K. Ishimaru Y. Ushio Y. Cancer Res. 2003; 63: 6962-6970PubMed Google Scholar). Ligand binding and activation of EGFR result in receptor dimerization, autophosphorylation, and activation of downstream effector pathways, such as phosphatidylinositol 3-kinase/AKT, Janus kinases/signal transducers and activators of transcription (STAT), and Ras/Raf/mitogen-activated protein kinase (MAPK) (1Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (542) Google Scholar, 2Ali-Osman F. Friedman H.S. Antoun G.R. Readon D. Bigner D.D. Buolamwini J.K. Brain Tumors. Humana Press, Totowa, NJ2005: 359-381Crossref Google Scholar). The cellular signaling cascades initiated and transduced by EGFR have been implicated in oncogenesis and functional dysregulation of EGFR, and its downstream pathways are frequently observed in malignant gliomas and other human cancers and have been shown to regulate features of the malignant phenotype, such as tumor progression, adhesion, invasion, angiogenesis, and apoptosis (3Arteaga C.L. J. Clin. Oncol. 2001; 19: 32-40PubMed Google Scholar, 5Yang Z. Bagheri-Yarmand R. Wang R.A. Adam L. Papadimitrakopoulou V.V. Clayman G.L. El-Naggar A. Lotan R. Barnes C.J. Hong W.K. Kumar R. Clin. Cancer Res. 2004; 10: 658-667Crossref PubMed Scopus (74) Google Scholar). epidermal growth factor (EGF) receptor glioblastoma multiforme glutathione S-transferase P1 glutathione ethacrynic acid mitogen-activated protein kinase immunoprecipitation mass spectrometry liquid chromatography small interfering RNA. epidermal growth factor (EGF) receptor glioblastoma multiforme glutathione S-transferase P1 glutathione ethacrynic acid mitogen-activated protein kinase immunoprecipitation mass spectrometry liquid chromatography small interfering RNA. EGFR gene amplification is a hallmark of glioblastoma multiforme (GBM), the most aggressive and most common intrinsic malignant brain tumor (6Gurney J.G. Kadan-Lottick N. Curr. Opin. Oncol. 2001; 13: 160-166Crossref PubMed Scopus (155) Google Scholar). In primary (de novo) GBM, which accounts for ∼95% of all GBM (7Kleihues P. Cavenee W.K. WHO Classification of Tumors: Pathology and Genetics of Tumours of the Nervous System. IARC Press, Lyon, France2000Google Scholar, 8Ohgaki H. Dessen P. Jourde B. Horstmann S. Nishikawa T. Di Patre P.L. Burkhard C. Schüler D. Probst-Hensch N.M. Maiorka P.C. Baeza N. Pisani P. Yonekawa Y. Yasargil M.G. Lütolf U.M. Kleihues P. Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1034) Google Scholar), EGFR amplification follows Loss of heterozygosity of chromosome 10q as the most frequently observed genetic alteration (8Ohgaki H. Dessen P. Jourde B. Horstmann S. Nishikawa T. Di Patre P.L. Burkhard C. Schüler D. Probst-Hensch N.M. Maiorka P.C. Baeza N. Pisani P. Yonekawa Y. Yasargil M.G. Lütolf U.M. Kleihues P. Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1034) Google Scholar). In preclinical studies, a strong correlation has been reported between high aberrant EGFR signaling and ligand-dependent GBM cell proliferation (9Halatsch M.E. Gehrke E. Borhani F.A. Efferth T. Werner C. Nomikos P. Schmidt U. Buchfelder M. Anticancer Res. 2003; 23: 2315-2320PubMed Google Scholar) and the resistance of GBM to chemotherapy and radiation therapy (10Chakravarti A. Chakladar A. Delaney M.A. Latham D.E. Loeffler J.S. Cancer Res. 2002; 62: 4307-4315PubMed Google Scholar). Consistent with this, clinical studies of GBM have shown EGFR gene amplification to be a significant negative predictor of patient survival (4Shinojima N. Tada K. Shiraishi S. Kamiryo T. Kochi M. Nakamura H. Makino K. Saya H. Hirano H. Kuratsu J. Oka K. Ishimaru Y. Ushio Y. Cancer Res. 2003; 63: 6962-6970PubMed Google Scholar), and EGFR overexpression has been associated with failure to respond to radiation therapy (11Barker Jr., F.G. Simmons M.L. Chang S.M. Prados M.D. Larson D.A. Sneed P.K. Wara W.M. Berger M.S. Chen P. Israel M.A. Aldape K.D. Int. J. Radiat. Oncol. Biol. Phys. 2001; 51: 410-418Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). In addition to its amplification and overexpression, a mutant EGFR, EGFRvIII, characterized by deletion of exons 2–7, is present in almost half of GBMs with amplified EGFR (4Shinojima N. Tada K. Shiraishi S. Kamiryo T. Kochi M. Nakamura H. Makino K. Saya H. Hirano H. Kuratsu J. Oka K. Ishimaru Y. Ushio Y. Cancer Res. 2003; 63: 6962-6970PubMed Google Scholar, 12Aldape K.D. Ballman K. Furth A. Buckner J.C. Giannini C. Burger P.C. Scheithauer B.W. Jenkins R.B. James C.D. J. Neuropathol. Exp. Neurol. 2004; 63: 700-707Crossref PubMed Scopus (213) Google Scholar). EGFRvIII is unique in that the loss of a large portion of the extracellular ligand binding domain leads to its constitutive and ligand-independent activation (13Batra S.K. Castelino-Prabhu S. Wikstrand C.J. Zhu X. Humphrey P.A. Friedman H.S. Bigner D.D. Cell Growth Differ. 1995; 6: 1251-1259PubMed Google Scholar). In GBM, EGFRvIII has been associated with increased tumor growth, cell proliferation, and drug resistance, which, similar to wild-type EGFR, occurs via constitutive activation of downstream EGFR pathways (14Raizer J.J. J. Neuro-Oncol. 2005; 74: 77-86Crossref PubMed Scopus (68) Google Scholar, 15Narita Y. Nagane M. Mishima K. Huang H.J. Furnari F.B. Cavenee W.K. Cancer Res. 2002; 62: 6764-6769PubMed Google Scholar). Glutathione S-transferase P1 (GSTP1), a major phase II-metabolizing enzyme, encoded in a polymorphic gene locus (16Goto S. Iida T. Cho S. Oka M. Kohno S. Kondo T. Free Radic. Res. 1999; 31: 549-558Crossref PubMed Scopus (156) Google Scholar), catalyzes the S-conjugation of endogenous and exogenous electrophiles, including many genotoxins, carcinogens, and anticancer agents, to the nucleophilic thiol group of reduced glutathione, GSH (17Chasseaud L.F. Adv. Cancer Res. 1979; 29: 175-274Crossref PubMed Scopus (1086) Google Scholar). In addition, GSTP1 is a major regulator of cell signaling in response to stress, hypoxia, growth factors, and other stimuli. This results in part from its ability to inhibit downstream mitogen-activated protein kinase signaling, notably that mediated by c-Jun N-terminal kinase (18Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (948) Google Scholar, 19Wang T. Arifoglu P. Ronai Z. Tew K.D. J. Biol. Chem. 2001; 276: 20999-21003Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 20Ranganathan P.N. Whalen R. Boyer T.D. Biochem. J. 2005; 386: 525-533Crossref PubMed Scopus (14) Google Scholar). GSTP1 also regulates important normal cellular functions through interaction with a number of critical cellular proteins, including transglutaminase 2, apoptosis signal-regulating kinase 1, and Fanconi anemia group C protein (21Lo H.W. Ali-Osman F. Curr. Opin. Pharmacol. 2007; 7: 367-374Crossref PubMed Scopus (190) Google Scholar). Recently, we reported that GSTP1 is a substrate for two Ser/Thr protein kinases, viz. cAMP-dependent protein kinase and protein kinase C (22Lo H.W. Antoun G. Ali-Osman F. Cancer Res. 2004; 64: 9131-9138Crossref PubMed Scopus (51) Google Scholar). In many human cancers, including gliomas, leukemias, lymphomas, melanoma, and carcinomas of the breast, ovary, colorectum, lung, liver, etc. (23Ali-Osman F. Brunner J.M. Kutluk T.M. Hess K. Clin. Cancer Res. 1997; 3: 2253-2261PubMed Google Scholar, 24Okamura T. Kurisu K. Yamamoto W. Takano H. Nishiyama M. Int. J. Oncol. 2000; 16: 295-303PubMed Google Scholar, 25Nishiyama M. Suzuki K. Kumazaki T. Yamamoto W. Toge T. Okamura T. Kurisu K. Int. J. Cancer. 1997; 72: 649-656Crossref PubMed Scopus (32) Google Scholar), GSTP1 is frequently overexpressed, and the high expression is associated with a more aggressive tumor biology and poor patient survival. Given the roles that both EGFR and GSTP1 play in cell signaling and in both normal and neoplastic biology, we investigated using GBM and inflammatory breast cancer cell lines the possibility and consequences of the interaction between the two proteins in vitro and in GBM xenografts growing in vivo. The nature of the interaction was characterized structurally and functionally by a combination of mass spectrometry and other biochemical analyses, and its effects on the response of the tumor cells to chemotherapy were investigated. Our findings support EGFR-mediated tyrosine phosphorylation of GSTP1 to be a heretofore unrecognized component of the EGFR cellular network and constitutes an important mechanism of cellular protection and drug resistance, particularly in tumors and/or tissues with activated EGFR and/or elevated GSTP1 expression. Recombinant human GSTP1-1 protein was purchased from Invitrogen, and recombinant human EGFR active kinase domain and normal mouse IgG was from Upstate Biotechnology Inc., (Lake Placid, NY). [γ-32P]ATP and Protein A-Sepharose were from Amersham Biosciences. Anti-human GSTP1-1 rabbit polyclonal antibody was from Oxford Biomedical Research (Oxford, MI), and anti-human GSTP1 mouse monoclonal antibody was from BIODESIGN International (Saco, ME). Anti-phosphotyrosine (phospho-Tyr-100) and anti-phospho-EGFR (Tyr-1068) monoclonal antibodies were from Cell Signaling Technology (Danvers, MA). Anti-GRB2 and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The EGFR inhibitor, lapatinib, was purchased from LC Laboratories (Woburn, MA) and prepared in DMSO stock solution. All other chemicals and biochemicals were purchased from Sigma-Aldrich unless otherwise stated. The human cell lines MGR1 (anaplastic astrocytoma) and MGR3 (GBM) were established in our laboratory from primary specimens (22Lo H.W. Antoun G. Ali-Osman F. Cancer Res. 2004; 64: 9131-9138Crossref PubMed Scopus (51) Google Scholar). The high EGFR expressing human GBM U87MG.wtEGFR was derived by stable transfection of the parental U87MG cells with wild-type EGFR (26Huang H.S. Nagane M. Klingbeil C.K. Lin H. Nishikawa R. Ji X.D. Huang C.M. Gill G.N. Wiley H.S. Cavenee W.K. J. Biol. Chem. 1997; 272: 2927-2935Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). SUM 149 (Asterand PLC, Detroit, MI) is a human inflammatory breast cancer cell line. Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum (FCS) (MGR1, MGR3), Improved MEM Zinc Option with 10% FCS (U87MG, U87MG.wtEGFR), or Ham's F-12 with 5% FCS (SUM 149). To mimic intracellular conditions in which the GSTP1 protein exists in equilibrium with GSH bound to its GSH binding site (22Lo H.W. Antoun G. Ali-Osman F. Cancer Res. 2004; 64: 9131-9138Crossref PubMed Scopus (51) Google Scholar, 27Caccuri A.M. Antonini G. Ascenzi P. Nicotra M. Nuccetelli M. Mazzetti A.P. Federici G. Lo Bello M. Ricci G. J. Biol. Chem. 1999; 274: 19276-19280Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), 1 μg of human recombinant GSTP1 was preincubated with 5 mm GSH for 20 min at 37 °C and then added to a reaction mixture containing EGFR active kinase domain (25 ng) and [γ-32P]ATP in Mn2+, Mg2+-containing kinase buffer. After 1 h of incubation at 30 °C, the reaction was terminated by boiling and resolved by SDS-PAGE followed by Coomassie Blue staining and autoradiography. For stoichiometry of the GSTP1 phosphorylation by EGFR, the phosphorylation reaction was set up containing 1 μg of GSTP1, 0.05 μm EGFR, and a saturating (100 μm) ATP concentration. Over 0–4 h, aliquots were removed and subjected to SDS-PAGE, and the phosphorylated GSTP1 bands were excised and solubilized, and the radioactivity counted by β-scintillation. The incorporated 32P phosphate was computed from the specific activity of the [γ-32P]ATP, expressed per mol of the dimeric GSTP1 protein, and plotted against time. GSTP1 was 32P-phosphorylated by EGFR, subjected to SDS-PAGE, and transferred to polyvinylidene difluoride membranes. After autoradiography, the phospho-GSTP1 bands were excised, hydrolyzed at 110 °C in 5 m HCl for 1 h, vacuum-dried, and resuspended in a loading buffer of acetic acid:pyridine:water (10:1:189) containing phosphoserine, phosphothreonine, and phosphotyrosine standards, and electrophoresed at pH 3.5 on a cellulose TLC plate. The plate was dried, stained with ninhydrin, and autoradiographed. A mixture of GSTP1 preincubated with or without 5 mm GSH was applied to the GSTP1 phosphorylation assay with 50 nm EGFR and 200 μm ATP followed by SDS-PAGE/Western blotting with anti-phosphotyrosine (Tyr(P)) antibody. After stripping, the membrane was reprobed with anti-GSTP1 antibody to control for loading. To inhibit the EGFR activation, a 50 nm EGFR preincubated for 30 min with 0–1 mm lapatinib, an EGFR inhibitor, was used for the GSTP1 phosphorylation assay. Tyrosine-phosphorylated GSTP1 was normalized against total GSTP1 protein. Tumor cells grown in serum-free medium for 24 h were treated with 100 ng/ml EGF for 10 min, rinsed with ice-cold phosphate-buffered saline, and lysed in 50 mm Tris-HCl (pH 7.5) containing 1% Triton X-100, protease and phosphatase inhibitors mixture (Pierce). The lysates were centrifuged at 20,000 rpm for 15 min. Supernatants were subjected to Western blotting with anti-Tyr(P), phospho-EGFR, and GSTP1 antibodies. For a combined immunoprecipitation (IP)-Western blotting, supernatants (1 mg total protein) were incubated (4 °C; overnight) with anti-GSTP1, phospho-EGFR antibodies, or normal mouse IgG (as a negative control). The protein A-Sepharose beads were incubated with immunocomplexes for 1 h and washed 4 times with the lysis buffer, and the immunoprecipitates were subjected to Western blotting. To examine the effect of EGFR inhibition, tumor cells with activated EGFR were treated with 2.5 μm lapatinib for 30 min, as per experimental protocol before lysis and Western blotting. These were performed as we had previously described (22Lo H.W. Antoun G. Ali-Osman F. Cancer Res. 2004; 64: 9131-9138Crossref PubMed Scopus (51) Google Scholar). Briefly, 0.1 unit each of unphosphorylated recombinant GSTP1 and GSTP1 phosphorylated by EGFR as described earlier were used to set up reactions containing 0.05–0.5 mm GSTP1-specific substrate, ethacrynic acid (EA), and 0.25 mm GSH in a 0.1 mm potassium phosphate buffer (pH 6.8). The rate of formation of the reaction product between EA and GSH was monitored at 270 nm. Reaction rates, normalized against that of the nonenzymatic reaction, were computed and used to generate double reciprocal plots from which the enzyme kinetic constants, Km, Vmax, Kcat, and Kcat/Km, were computed for phosphorylated and unphosphorylated GSTP1 (28Segel I.H. Biochemical Calculations. John Wiley and Sons, New York1976: 208-318Google Scholar), and the results are presented as the mean ± 1 S.D. of triplicate experiments. Xenografts (approximately, 200 mm2 in diameter) of U87MG and U87MG.wtEGFR growing subcutaneously in the flanks of 4-week-old male athymic BALB/c nu/nu mice were excised, minced, and sonicated on ice in the lysis buffer. Supernatants were subjected to the IP (anti-GSTP1)-Western (anti-Tyr(P)) procedure as described earlier. Recombinant GSTP1 was EGFR-phosphorylated GSTP1, reduced, and alkylated, and second dimension acrylamide gel electrophoresis was performed. SYPRO Ruby-stained protein spots were robotically excised, reduced with dithiothreitol, alkylated with iodoacetamide, digested with trypsin, and subjected to LC-MS/MS. MS/MS data were analyzed using the MASCOT MS/MS Ions Search, and de novo sequence analysis performed with the Scaffold Software (Proteome Software Inc., Portland, OR). Details of the protocols used are available online at Proteome Software Inc. and in supplemental material. Peptides containing each of the 12 tyrosine residues in the GSTP1 protein, namely, Tyr-3, Tyr-7, Tyr-49, Tyr-63, Tyr-79, Tyr-103, Tyr-108, Tyr-111, Tyr-118, Tyr-153, Tyr-179, and Tyr-198 as well as human angiotensin II peptide (DRVYIHPF as a positive control; Calbiochem) and Crosstide (GRPRTSSFAEG as a negative control; AnaSpec Inc., San Jose, CA), were EGFR-phosphorylated using [32P]ATP, spotted on Whatman P81 cellulose phosphate filters, acetone-washed, and air-dried. The radioactivity was quantitated by β-scintillation counting and used to compute the incorporated phosphate in each peptide. To better ascertain the GSTP1 phospho-acceptor residues, Tyr-3/Tyr-7, Tyr-63, Tyr-118, and Tyr-198 in six peptides selected from the LC-MS/MS analysis were mutated to aspartic acid (BioSynthesis, Louisville, TX), and the level of EGFR phosphorylation was determined as described above. Peptide information is available in supplemental Table S1. Mutant GSTP1 cDNAs were created by PCR on a template plasmid vector pBK-CMV/GSTP1A (16Goto S. Iida T. Cho S. Oka M. Kohno S. Kondo T. Free Radic. Res. 1999; 31: 549-558Crossref PubMed Scopus (156) Google Scholar) using GSTP1-specific primers containing tyrosine to phenylalanine mutations at Tyr-3, -7, and -198. All mutations were verified by DNA sequencing. Cloning was performed using the Gateway technology (Invitrogen) with the pcDNA-DEST40 destination vector to allow C-terminal fusions with a six-histidine tag. Transient transfections were performed with FuGENE HD (Roche Applied Science) according to the manufacturer's instructions. Briefly, 5 × 105 U87MG.wtEGFR cells were plated in 6-well plates and transfected with 2 μg of pcDNA-DEST40 expression vector carrying the wild-type GSTP1, the single tyrosine to phenylalanine mutants Y3F, Y7F, and Y198F, the double mutants, Y3F/Y7F, Y3F/Y198F and Y7F/Y198F, the triple mutant Y3F/Y7F/Y198F, and the empty vector (negative control). After 48 h the cells were treated with 100 ng/ml EGF for 10 min and lysed. The histidine-tagged GSTP1 proteins were immunoprecipitated with TALON cobalt Dynabeads (Invitrogen) according to the manufacturer's instructions followed by SDS-PAGE and Western blotting with anti-Tyr(P) antibody as described earlier. The relative levels of tyrosine phosphorylation of the mutant GSTP1 proteins relative to the wild-type GSTP1 were quantified by densitometry using ImageJ Version 1.34s software. Primer information is available in supplemental Table S2. X-ray crystallographic data were imported from the Brookhaven Protein Data Bank, and using the Insight II modeling program (Accelerys Software, San Diego), the three-dimensional structures of the GSH-bound GSTP1 monomer with and without the hydroxyl group of Tyr-7 phosphorylated were created. The modeled structures were soaked in a cubic box of water molecules and subjected to energy minimization and long-duration molecular dynamics simulation using the NAMD program 2.5 running on a 5 node Scyld Beowulf linux cluster. The coordinate and parameter files for input were generated using the “psfgen” utility in the CHARMM PARAM 22 topology file, whereas the all atom CHARMM PARAM 22 force field was used to describe the potential energy. The results of the analyses of energies and structure frames of the simulated system were extracted using the VMD software and illustrations produced with both the VMD and SYBYL software. Details of the simulation procedure are provided in the supplemental material. EGFR was activated in exponentially growing tumor cells by a 10-min treatment with 100 ng/ml EGF. Extracts from cells with and without subsequent treatment with 2.5 μm EGFR inhibitor, lapatinib, for 30 min were assayed for specific GSTP1 activity as we described earlier (22Lo H.W. Antoun G. Ali-Osman F. Cancer Res. 2004; 64: 9131-9138Crossref PubMed Scopus (51) Google Scholar). These studies were performed with the GSTP1- and EGF-overexpressing human inflammatory breast cancer cell line, SUM 149. Approximately 5 × 106 cells in exponential growth were pretreated with 100 ng/ml EGF for 10 min in triplicate, after which the medium was replaced with fresh medium containing 100 μm cisplatin. After an additional 2 h at 37 °C, the cells were washed twice, harvested, and homogenized in 500 μl of phosphate-buffered saline. Supernatants after centrifugation at 15,000 × g for 20 min were removed, and protein was precipitated by adding trichloroacetic acid to 10% (final concentration) and incubating at 4 °C for 3 h. After final centrifugation, the supernatants (normalized for equal protein content) were used for glutathionylplatinum metabolite quantitation, as we previously described (30Ishikawa T. Ali-Osman F. J. Biol. Chem. 1993; 268: 20116-20125Abstract Full Text PDF PubMed Google Scholar). Briefly, aliquots of the supernatant were diluted 1:10 with 10% trichloroacetic acid, and the absorbance was measured by scanning spectrophotometry over a wavelength range of 240–400 nm (Beckman DU-70 spectrophotometer). The absorbance at 265 nm, A265 (peak absorbance of the glutathionylplatinum conjugate), of the supernatants with and without EGF pretreatment were normalized against that of control without cisplatin treatment, and the resulting ΔA265 was used as a measure of the level of glutathionylcisplatin in the cells. Tumor cells with activated EGFR were treated with and without 2.5 μm lapatinib for 30 min followed by 0–50 μm cisplatin for 3 h. The cells were washed, and cell survival was examined after 48 h using the CellTiter-Blue Assay (Promega, Madison, WI) according to the manufacturer's instructions. MGR3 and SUM 149 cells were plated at 1 × 103–104 cells in 100 μl of Dulbecco's modified Eagle's medium containing 10% fetal calf serum in a flat-bottomed microtiter plate. After 24 h at 37 °C, the cells were transfected with siRNA with the sequence (5′-ACCAGAUCUCCUUCGCUGACUACAA-3′) targeting the N-terminal region of GSTP1 mRNA using LipofectamineTM (Invitrogen) according to the manufacturer's instructions. After 6 h the cultures were refed with fresh medium and incubated at 37 °C for a further 24 h. Untransfected cells and cells transfected with scrambled siRNA served as controls. After 24 h the cells were treated with and without 100 ng/ml EGF for 10 min followed by 0–50 μm cisplatin. Cell survival was determined as described earlier. Cisplatin sensitivity (surviving fraction) after each treatment relative to controls (normalized against scrambled siRNA) was determined, and IC50 values were computed. Replicate cells after siRNA treatment were lysed and subjected to Western blotting for GSTP1 expression to monitor the level of GSTP1 knockdown. Figs. 1, A–C, summarizes the results of the cell-free analysis of the phosphorylation of GSTP1 by EGFR, performed with recombinant GSTP1 and EGFR proteins. The 32P-labeling results (Fig. 1A) show that, after its pre-equilibration with GSH, GSTP1 undergoes dose-dependent phosphorylation by EGFR. The Western blots with an anti-phosphotyrosine (Tyr(P))-specific antibody (Fig. 1B) show that, while for GSTP1, tyrosine phosphorylation required GSH and was significantly reduced in its absence, the presence of GSH resulted in a slight reduction in the level of EGFR autophosphorylation, consistent with previous reports that EGFR is a redox-regulated protein and that its intracellular activation is suppressed by reducing agents (31Kamata H. Shibukawa Y. Oka S.I. Hirata H. Eur. J. Biochem.. 2000; 267: 1933-1944Crossref PubMed Scopus (72) Google Scholar). Equal loading of GSTP1 in the lanes is shown by the Western blots for GSTP1 (lowest panel in Fig. 1B). To determine which amino acids in the GSTP1 protein undergo phosphorylation by EGFR, recombinant GSTP1 was 32P-phosphorylated by EGFR, acid-hydrolyzed, and subjected to thin layer electrophoresis and autoradiography. The results (Fig. 1C) show tyrosine to be the only amino acid residue phosphorylated by EGFR in the GSTP1 protein. No phosphoserines or phosphothreonines were detected. In the cell-free system and at saturating ATP concentrations EGFR phosphorylated the dimeric GSTP1 protein with a stoichiometry of 0.0436 ± 0.0003 mol of phosphate/mol of GSTP1. Intracellular EGFR-dependent GSTP1 phosphorylation was examined using the isogenic pair of human GBM cell lines, U87MG and U87MG.wtEGFR, with low and high constitutive wild-type EGFR expression, respectively. Protein extracts prepared from tumor cells that had been treated with and without 100 ng/ml EGF for 10 min were subjected to Western blotting with anti-Tyr(P) antibody. The results, Fig. 2A, show no detectable tyrosine-phosphorylated proteins of around 23 kDa in either EGF-treated and untreated U87MG cells. Similarly, phospho-EGFR was undetectable in control untreated U87MG cells and increased only modestly after EGF treatment. In contrast, in untreated U87MG.wtEGFR cells moderate levels of phospho-EGFR were observed even without EGF treatment, and after EGF treatment these levels increased by more than 10-fold. Western blotting with Tyr(P) antibody showed the level of 23-kDa tyrosine-phosphorylated protein, absent in the non-EGF treated cells, to be significantly increased after EGF treatment. After stripping and reprobing with anti-GSTP1 antibody, this 23-kDa band was confirmed to be GSTP1. The results of GSTP1 tyrosine phosphorylation in GBM cells examined by immunoprecipitation with an anti-GSTP1 antibody followed by Western blotting for phosphotyrosine (Fig. 2B) were similar to those of the direct Western blotting and showed that, in U87MG.wtEGFR, levels of tyrosine-phosphorylated GSTP1 were low in non-EGF-treated cells but increased significantly after EGF treatment. Western blotting with anti-GSTP1 confirmed the amount of GSTP1 immunoprecipitated to be e" @default.
• W1999791629 created "2016-06-24" @default.
• W1999791629 creator A5000972766 @default.
• W1999791629 creator A5004785534 @default.
• W1999791629 creator A5044465742 @default.
• W1999791629 creator A5050348822 @default.
• W1999791629 creator A5051836172 @default.
• W1999791629 creator A5057371623 @default.
• W1999791629 creator A5058728556 @default.
• W1999791629 date "2009-06-01" @default.
• W1999791629 modified "2023-10-15" @default.
• W1999791629 title "Tyrosine Phosphorylation of the Human Glutathione S-Transferase P1 by Epidermal Growth Factor Receptor" @default.
• W1999791629 cites W129370812 @default.
• W1999791629 cites W1504784308 @default.
• W1999791629 cites W1536437858 @default.
• W1999791629 cites W1980827500 @default.
• W1999791629 cites W1987413233 @default.
• W1999791629 cites W1995584424 @default.
• W1999791629 cites W1999805805 @default.
• W1999791629 cites W2001598945 @default.
• W1999791629 cites W2002695473 @default.
• W1999791629 cites W2007041998 @default.
• W1999791629 cites W2010338582 @default.
• W1999791629 cites W2012907539 @default.
• W1999791629 cites W2015848531 @default.
• W1999791629 cites W2020455451 @default.
• W1999791629 cites W2020947886 @default.
• W1999791629 cites W2035536974 @default.
• W1999791629 cites W2038163496 @default.
• W1999791629 cites W2061701210 @default.
• W1999791629 cites W2080559885 @default.
• W1999791629 cites W2085037886 @default.
• W1999791629 cites W2088089215 @default.
• W1999791629 cites W2095267500 @default.
• W1999791629 cites W2134672568 @default.
• W1999791629 cites W2140578136 @default.
• W1999791629 cites W2143373549 @default.
• W1999791629 cites W2150081412 @default.
• W1999791629 cites W2150638747 @default.
• W1999791629 cites W2167805508 @default.
• W1999791629 cites W54894650 @default.
• W1999791629 doi "https://doi.org/10.1074/jbc.m808153200" @default.
• W1999791629 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2719335" @default.
• W1999791629 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19254954" @default.
• W1999791629 hasPublicationYear "2009" @default.
• W1999791629 type Work @default.
• W1999791629 sameAs 1999791629 @default.
• W1999791629 citedByCount "39" @default.
• W1999791629 countsByYear W19997916292012 @default.
• W1999791629 countsByYear W19997916292013 @default.
• W1999791629 countsByYear W19997916292014 @default.
• W1999791629 countsByYear W19997916292015 @default.
• W1999791629 countsByYear W19997916292016 @default.
• W1999791629 countsByYear W19997916292018 @default.
• W1999791629 countsByYear W19997916292020 @default.
• W1999791629 countsByYear W19997916292021 @default.
• W1999791629 countsByYear W19997916292022 @default.
• W1999791629 countsByYear W19997916292023 @default.
• W1999791629 crossrefType "journal-article" @default.
• W1999791629 hasAuthorship W1999791629A5000972766 @default.
• W1999791629 hasAuthorship W1999791629A5004785534 @default.
• W1999791629 hasAuthorship W1999791629A5044465742 @default.
• W1999791629 hasAuthorship W1999791629A5050348822 @default.
• W1999791629 hasAuthorship W1999791629A5051836172 @default.
• W1999791629 hasAuthorship W1999791629A5057371623 @default.
• W1999791629 hasAuthorship W1999791629A5058728556 @default.
• W1999791629 hasBestOaLocation W19997916291 @default.
• W1999791629 hasConcept C11960822 @default.
• W1999791629 hasConcept C170493617 @default.
• W1999791629 hasConcept C181199279 @default.
• W1999791629 hasConcept C185592680 @default.
• W1999791629 hasConcept C2776165026 @default.
• W1999791629 hasConcept C2776362946 @default.
• W1999791629 hasConcept C2776907368 @default.
• W1999791629 hasConcept C2777553839 @default.
• W1999791629 hasConcept C2779438470 @default.
• W1999791629 hasConcept C538909803 @default.
• W1999791629 hasConcept C55493867 @default.
• W1999791629 hasConcept C86803240 @default.
• W1999791629 hasConcept C95444343 @default.
• W1999791629 hasConceptScore W1999791629C11960822 @default.
• W1999791629 hasConceptScore W1999791629C170493617 @default.
• W1999791629 hasConceptScore W1999791629C181199279 @default.
• W1999791629 hasConceptScore W1999791629C185592680 @default.
• W1999791629 hasConceptScore W1999791629C2776165026 @default.
• W1999791629 hasConceptScore W1999791629C2776362946 @default.
• W1999791629 hasConceptScore W1999791629C2776907368 @default.
• W1999791629 hasConceptScore W1999791629C2777553839 @default.
• W1999791629 hasConceptScore W1999791629C2779438470 @default.
• W1999791629 hasConceptScore W1999791629C538909803 @default.
• W1999791629 hasConceptScore W1999791629C55493867 @default.
• W1999791629 hasConceptScore W1999791629C86803240 @default.
• W1999791629 hasConceptScore W1999791629C95444343 @default.
• W1999791629 hasIssue "25" @default.
• W1999791629 hasLocation W19997916291 @default.
• W1999791629 hasLocation W19997916292 @default.
• W1999791629 hasLocation W19997916293 @default.
• W1999791629 hasLocation W19997916294 @default.

• next