Chemical Knockdown of Protein-tyrosine Phosphatase 1B by 1,2-Naphthoquinone through Covalent Modification Causes Persistent Transactivation of Epidermal Growth Factor Receptor

Abstract

1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells. 1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells. Most intracellular signaling cascades are mediated by receptor tyrosine kinases, and the activated signals are transmitted to downstream proteins by sequential phosphorylation. Because protein tyrosine phosphorylation is negatively regulated by protein-tyrosine phosphatases (PTPs), 2The abbreviations used are: PTP, protein-tyrosine phosphatase; 1,2-NQ, 1,2-naphthoquinone; PTP1B, protein-tyrosine phosphatase 1B; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ROS, reactive oxygen species; DTT, dithiothreitol; HRP, horseradish peroxidase; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); TBS, Tris-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; pNP, p-nitrophenol; pNPP, p-nitrophenol phosphate; KLH, keyhole limpet hemocyanin. 2The abbreviations used are: PTP, protein-tyrosine phosphatase; 1,2-NQ, 1,2-naphthoquinone; PTP1B, protein-tyrosine phosphatase 1B; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ROS, reactive oxygen species; DTT, dithiothreitol; HRP, horseradish peroxidase; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); TBS, Tris-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; pNP, p-nitrophenol; pNPP, p-nitrophenol phosphate; KLH, keyhole limpet hemocyanin. these dephosphorylation enzymes play important roles in the signal transduction controlled by receptor tyrosine kinases. PTPs are a diverse family of enzymes that can be divided into several subgroups, including receptor PTPs and non-receptor PTPs. All PTPs contain an essential cysteine residue in the active site motif, HCX5R(S/T) (1Zhang Z.Y. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 209-234Crossref PubMed Scopus (374) Google Scholar). This sulfhydryl group, as thiolate anion, contributes to the formation of a thiol-phosphate intermediate in the dephosphorylation reaction and is therefore essential for enzyme activity (2Barford D. Jia Z. Tonks N.K. Nat. Struct. Biol. 1995; 2: 1043-1053Crossref PubMed Scopus (181) Google Scholar). Because of the low pKa of the catalytic cysteine (e.g. ∼5.4 for mammalian PTPs) (3Lohse D.L. Denu J.M. Santoro N. Dixon J.E. Biochemistry. 1997; 36: 4568-4575Crossref PubMed Scopus (158) Google Scholar), PTP thiols are readily oxidized to their sulfenic acid (-SOH) state by reactive oxygen species (ROS) (4Claiborne A. Yeh J.I. Mallett T.C. Luba J. Crane 3rd, E.J. Charrier V. Parsonage D. Biochemistry. 1999; 38: 15407-15416Crossref PubMed Scopus (460) Google Scholar, 5Barrett W.C. DeGnore J.P. Keng Y.F. Zhang Z.Y. Yim M.B. Chock P.B. J. Biol. Chem. 1999; 274: 34543-34546Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), thereby decreasing dephosphorylation activity. Current consensus is that PTP activity is regulated by endogenous oxidant such as hydrogen peroxide (H2O2) in cells (6Caselli A. Marzocchini R. Camici G. Manao G. Moneti G. Pieraccini G. Ramponi G. J. Biol. Chem. 1998; 273: 32554-32560Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 7Lee S.R. Kwon K.S. Kim S.R. Rhee S.G. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). The decreased PTP activity associated with epidermal growth factor receptor (EGFR) activation caused by H2O2 is transient and restored by cellular reduction systems (8Tonks N.K. FEBS Lett. 2003; 546: 140-148Crossref PubMed Scopus (318) Google Scholar). However, if reactive thiols of PTPs undergo covalent modification by exogenous electrophiles, irreversible inactivation of PTPs, resulting in prolonged phosphorylation of EGFR, would occur. We have been examining the reactivity and toxicology of selected quinones (9Kumagai Y. Koide S. Taguchi K. Endo A. Nakai Y. Yoshikawa T. Shimojo N. Chem. Res. Toxicol. 2002; 15: 483-489Crossref PubMed Scopus (274) Google Scholar, 10Kumagai Y. Hayashi T. Miyauchi T. Endo A. Iguchi A. Kiriya-Sakai M. Sakai S. Yuki K. Kikushima M. Shimojo N. Am. J. Physiol. 2001; 281: R25-R30Google Scholar, 11Sugimoto R. Kumagai Y. Nakai Y. Ishii T. Free Radic. Biol. Med. 2005; 38: 388-395Crossref PubMed Scopus (68) Google Scholar, 12Inoue K. Takano H. Hiyoshi K. Ichinose T. Sadakane K. Yanagisawa R. Tomura S. Kumagai Y. Eur. Respir. J. 2007; 29: 259-267Crossref PubMed Scopus (49) Google Scholar, 13Taguchi K. Fujii S. Yamano S. Cho A.K. Kamisuki S. Nakai Y. Sugawara F. Froines J.R. Kumagai Y. Free Radic. Biol. Med. 2007; 43: 789-799Crossref PubMed Scopus (63) Google Scholar) in an effort to establish mechanistic pathways that may account for the adverse health effects associated with exposure to mixtures in which they are present. Quinones, including 1,2-NQ of this study, have been found at significant concentrations in vehicle exhaust particles and in ambient air samples (14Chung M.Y. Lazaro R.A. Lim D. Jackson J. Lyon J. Rendulic D. Hasson A.S. Environ. Sci. Technol. 2006; 40: 4880-4886Crossref PubMed Scopus (171) Google Scholar, 15Cho A.K. Di Stefano E. Ying Y. Rodriguez C.E. Schmitz D.A. Kumagai Y. Miguel A.H. Eiguren-Fernandez A. Kobayashi T. Avol E. Froines J.R. Aerosol Sci. Technol. 2004; 38: 68-81Crossref Scopus (220) Google Scholar, 16Valavanidis A. Fiotakis K. Vlahogianni T. Papadimitriou V. Pantikaki V. Environ. Chem. 2006; 3: 118-123Crossref Scopus (59) Google Scholar), so their toxic actions have considerable relevance to understanding air pollution toxicity. Quinones are a class of organic compounds that undergo two toxicologically relevant reactions (9Kumagai Y. Koide S. Taguchi K. Endo A. Nakai Y. Yoshikawa T. Shimojo N. Chem. Res. Toxicol. 2002; 15: 483-489Crossref PubMed Scopus (274) Google Scholar, 17Bolton J.L. Trush M.A. Penning T.M. Dryhurst G. Monks T.J. Chem. Res. Toxicol. 2000; 13: 135-160Crossref PubMed Scopus (1385) Google Scholar, 18Brunmark A. Cadenas E. Free Radic. Biol. Med. 1989; 7: 435-477Crossref PubMed Scopus (363) Google Scholar). One is as an electrophile, forming covalent bonds with tissue nucleophiles to irreversibly alter key cellular proteins. In the second reaction the quinone acts as an electron transfer agent, transferring electrons from a reducing source such as NADPH to oxygen, generating ROS such as H2O2. We recently reported that 1,2-NQ elicits a contraction of smooth muscle in guinea pig tracheal preparations under conditions that indicated transactivation of EGFR was involved in the contraction process (19Kikuno S. Taguchi K. Iwamoto N. Yamano S. Cho A.K. Froines J.R. Kumagai Y. Toxicol. Appl. Pharmacol. 2006; 210: 47-54Crossref PubMed Scopus (43) Google Scholar). These studies also suggested that 1,2-NQ-induced EGFR activation was caused, at least partially, by covalent modification and/or thiol oxidation of PTP1B, leading to the reduction of its dephosphorylation activity. Of the multiple PTPs, PTP1B is ubiquitously expressed (20Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6722-6730Abstract Full Text PDF PubMed Google Scholar) and negatively regulates receptor tyrosine kinases such as EGFR (21Haj F.G. Markova B. Klaman L.D. Bohmer F.D. Neel B.G. J. Biol. Chem. 2003; 278: 739-744Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), so that inhibition of this PTP would lead to EGFR activation. Although 1,2- and 1,4-NQ and their derivatives are reported to be potent inhibitors for PTP1B (22Urbanek R.A. Suchard S.J. Steelman G.B. Knappenberger K.S. Sygowski L.A. Veale C.A. Chapdelaine M.J. J. Med. Chem. 2001; 44: 1777-1793Crossref PubMed Scopus (78) Google Scholar, 23Kar S. Lefterov I.M. Wang M. Lazo J.S. Scott C.N. Wilcox C.S. Carr B.I. Biochemistry. 2003; 42: 10490-10497Crossref PubMed Scopus (43) Google Scholar), their detailed mechanism of action still remains unclear. This report describes results of a study of 1,2-NQ-PTP1B interaction, which demonstrates that 1,2-NQ-mediated covalent modification of PTP1B, accompanied by reduction of its enzyme activity, results in the transactivation of EGFR in human epithelial A431 cells. The results also show that arylation of PTP1B through Cys-121 is the predominant event in the loss of enzyme activity caused by was from p-nitrophenol and acid from was by S. Yamano and from was from cells and from and from was from acid was from was from was from All used of the of 1,2-NQ was by a modification of the of and J. Chem. Res. Toxicol. PubMed Scopus Google Scholar). keyhole limpet was in of by of of to oxidized thiol of the The was under at in the for The proteins to an which been with to the to was with 1,2-NQ in under at for and the was the described The binding of 1,2-NQ to was a of the reaction was to an at a of was used as the and was at these the of 1,2-NQ was of was of 1,2-NQ of 1,2-NQ in The was from the to described Y. A. S. T. H. K. H. Cho A.K. Pharmacol. Google Scholar). and A431 cells at in a in with and A431 cells to on was with to A431 cells with PTP1B for with of and was to a protein of with the of of and The samples at for and by PubMed Scopus Google Scholar). proteins for with in and with the indicated EGFR that and used as that EGFR, and used as with with PTP1B of of which was to the The at for and with of and the PTP1B from the was with of at by analysis as described on the by and to the and on J. J. PubMed Scopus Google Scholar). for are of was with the PubMed Scopus Google Scholar) the protein with as a and to the form of PTP1B, was by downstream of the at the site of the catalytic of human PTP1B in a was used as a for and a R. K. in Scholar). The resulting was by on an The resulting was into E. cells for protein The in at with at to at The by the of and for an The cells by and the by and the of PTP1B and at E. cells with the in of and The was at for of the and to a which been with The was with the and PTP1B and its with the The of the PTP1B as by was oxidized by with for and the was by an enzyme was in The PTP1B was by the used for cells with concentrations of 1,2-NQ for the was and was to the The from the and to from at for used for phosphatase was with and The reaction was to for at and by of of The was at a was to the of 1,2-NQ on PTP1B activity. The for the of PTP1B concentrations of 1,2-NQ and by of at for to the second to PTP1B activity. The second reaction of the pNPP, and at for and by of to a of these dephosphorylation of was with and protein of Free was at with 1,2-NQ in a of the reaction was by of The reaction and enzymes with of DTNB, of and of the of acid was a of at B. PubMed Scopus Google Scholar). Matrix-assisted was with 1,2-NQ for at in a and into an to The PTP1B with and the ionization of mass samples purified with and with acid and acid and on at and The an mass with a All in the and the was to of expressed as from at least was at by analysis to a Phosphorylation of EGFR to of PTP by 1,2-NQ in the of A431 cells to 1,2-NQ for caused a concentration-dependent phosphorylation of EGFR these 1,2-NQ also cellular PTP activity in a concentration-dependent but the reduction of PTP activity by 1,2-NQ a at not 1,2-NQ-induced EGFR phosphorylation is the cells with a for EGFR to exposure of A431 cells to in A431 cells to the a inhibition of EGFR a of 1,2-NQ-induced EGFR phosphorylation was These results suggested that a EGFR phosphorylation through the inhibition of PTP activity exposure of A431 cells to In experiments EGFR phosphorylation by in A431 cells was to after which In EGFR activation caused by 1,2-NQ was at but with a transactivation of EGFR phosphorylation Because PTP inactivation by 1,2-NQ be due to thiolate oxidation the of on the phosphorylation of EGFR was by of A431 cells with with a for ROS, EGFR activation caused by 1,2-NQ was not These results that such a prolonged phosphorylation of EGFR exposure of A431 cells to 1,2-NQ was not due to thiol oxidation and be due to covalent binding of this quinone to cellular PTPs. PTP1B is responsible for the transactivation of EGFR caused by 1,2-NQ, of PTP1B in A431 cells EGFR phosphorylation by the with this 1,2-NQ activation of EGFR was by PTP1B in the cells not PTP1B is of the for irreversible modification by 1,2-NQ, a 1,2-NQ as described under in with by analysis with the revealed that 1,2-NQ was to PTP1B in A431 cells. the mechanistic details of the covalent modification of PTP1B by 1,2-NQ with purified PTP1B in of of 1,2-NQ in PTP1B in PTP1B activity was by 1,2-NQ in a and concentration-dependent The with with a of concentrations of 1,2-NQ with PTP1B for the reduction of PTP1B activity caused by 1,2-NQ the in covalent modification of this quinone to PTP1B H2O2 is reported to PTP1B activity through the oxidation of Cys-215 to the sulfenic acid S.R. Kwon K.S. Kim S.R. Rhee S.G. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar), but the decreased PTP1B activity was readily restored by of through reduction of the oxidized thiol However, on the loss of PTP1B activity caused by 1,2-NQ the that an irreversible covalent inactivation thiol oxidation of 1,2-NQ is the covalent modification of PTP1B by 1,2-NQ, 1,2-NQ with the derivatives in In studies guinea pig tracheal contraction caused by 1,2-NQ by EGFR and on the action and activation of EGFR (19Kikuno S. Taguchi K. Iwamoto N. Yamano S. Cho A.K. Froines J.R. Kumagai Y. Toxicol. Appl. Pharmacol. 2006; 210: 47-54Crossref PubMed Scopus (43) Google Scholar). This an of an in the contraction of guinea pig in the of covalent binding of 1,2-NQ and with a reduction of PTP1B activity. However, and which are of binding to PTP1B, not action on PTP1B These results that the decreased PTP1B activity was on the of of the thiol of PTP1B after reaction with Although are of cysteine in PTP1B and the thiol of the PTP1B in the study was of thiol of the of the of 1,2-NQ with PTP1B, as by the of reactive thiols and 1,2-NQ a reaction of the protein with 1,2-NQ at was suggested that two of thiols in PTP1B modification by of the modification in 1,2-NQ, the and PTP1B with and by mass by analysis of the from the PTP1B for of the protein and with the mass of the to showed an mass of to the of a of The their and are: and and These also in the analysis of from the PTP1B with 1,2-NQ by by from in a the 1,2-NQ binding the by 1,2-NQ and by analysis The of the at from the is in In the the and and loss and and to have by that the site in the is The of the at from the is in The and and the in the and to have by and and to by These results that the 1,2-NQ is with was by analysis In this the two as His-25 and the covalent modification of Cys-121 and His-25 of PTP1B is involved in the reduction of its dephosphorylation activity, of these nucleophilic with that are of with 1,2-NQ the loss of catalytic activity caused by this this of Cys-121 and Cys-215 to and and His-25 to of and of PTP1B and of that these acid not PTP1B activity. However, of for but not of for the in PTP1B activity caused by 1,2-NQ with this covalent binding of 1,2-NQ to the PTP1B was after mutation of Cys-121 to but after mutation of His-25 to also be that was a reduction of covalent binding of 1,2-NQ to PTP1B with of for that not Cys-121 and His-25 but also Cys-215 undergo irreversible modification by 1,2-NQ under the Quinones with cellular systems by two as and as electron transfer to ROS, with quinones these to J.L. Trush M.A. Penning T.M. Dryhurst G. Monks T.J. Chem. Res. Toxicol. 2000; 13: 135-160Crossref PubMed Scopus (1385) Google Scholar). We reported recently that 1,2-NQ potent contraction of guinea pig tracheal through transactivation of receptor tyrosine kinases, including EGFR (19Kikuno S. Taguchi K. Iwamoto N. Yamano S. Cho A.K. Froines J.R. Kumagai Y. Toxicol. Appl. Pharmacol. 2006; 210: 47-54Crossref PubMed Scopus (43) Google Scholar). In the study, exposure of A431 cells to 1,2-NQ in a phosphorylation of EGFR coupled to decreased PTP activity in a concentration-dependent EGF, 1,2-NQ caused a prolonged activation which at least to with for and These that 1,2-NQ irreversibly PTPs that as for EGFR thereby its This inactivation be due to a reaction by the quinone to the protein thiol to its oxidation by H2O2 by electron these a of the effects of on phosphorylation of EGFR in A431 cells by H2O2 and 1,2-NQ The of but not that of 1,2-NQ, was by with with that a reaction was for 1,2-NQ be that of cellular PTP activity was by 1,2-NQ at that not of the PTPs in A431 cells are to Because PTP1B is to EGFR negatively and a reactive thiol at the active D. Tonks N.K. 263: PubMed Scopus Google Scholar), used PTP1B as a for PTP modification by EGFR phosphorylation caused by 1,2-NQ was in cells with human PTP1B of PTP1B the of a of 1,2-NQ to cellular with and analysis with 1,2-NQ revealed that 1,2-NQ to endogenous PTP1B These that PTP1B a in the 1,2-NQ-mediated activation of EGFR in the cells. K. H. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) reported that and EGFR in epithelial cells and that the activation of EGFR caused by is associated with inhibition of PTP that which is also and 1,2-NQ are to PTP1B, thereby transactivation of on the cellular on the reaction PTP1B and with purified PTP1B showed that a inactivation of the PTP1B by 1,2-NQ second with a of These results that is a reaction 1,2-NQ and PTP1B with a of The would that a modification is responsible for enzyme activation residue is Although Rhee (6Caselli A. Marzocchini R. Camici G. Manao G. Moneti G. Pieraccini G. Ramponi G. J. Biol. Chem. 1998; 273: 32554-32560Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) showed that oxidation of the catalytic site thiol to sulfenic acid of PTP1B decreased its enzyme activity, the was restored by S.R. Kwon K.S. Kim S.R. Rhee S.G. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). However, as in this reducing was on the in PTP1B activity caused by a study with and showed that the quinone is for binding to PTP1B and for reduction of the enzyme activity. Collectively, these that 1,2-NQ PTP1B by covalent attachment by a reaction with second reaction The PTP is by a active site to as HCX5R(S/T) (1Zhang Z.Y. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 209-234Crossref PubMed Scopus (374) Google Scholar) in which the the thiol the pKa by several of The pKa of the active thiol in PTP1B is reported to be (3Lohse D.L. Denu J.M. Santoro N. Dixon J.E. Biochemistry. 1997; 36: 4568-4575Crossref PubMed Scopus (158) Google Scholar), that the cysteine thiol is at and of through a nucleophilic attack to form a The reactivity resulting from the low pKa also the active site cysteine a for covalent this reactivity in the of 1,2-NQ to PTP1B from the thiol 1,2-NQ The results showed that two of thiolate in the protein covalent modification not The arylation of PTP1B, by the Cys-121 and His-25 studies indicated that the arylation at Cys-121 was important to the catalytic activity that at of Cys-121 to showed of PTP1B inhibition with reduction in 1,2-NQ binding 1,2-NQ of decreased PTP1B activity caused by 1,2-NQ was in of of for We therefore that but not in PTP1B is a nucleophilic for covalent attachment by 1,2-NQ, thereby reduction of dephosphorylation activity. J. T. D.A. Biochemistry. 2005; 44: PubMed Scopus Google Scholar) reported that a site cysteine is the site of inhibition of PTP1B activity. This cysteine residue is from the catalytic Cys-215 Tonks N.K. Biol. 2001; PubMed Scopus Google Scholar) and the the of which in forms a hydrogen with the residue the active site of the 1,2-NQ to Cys-121 would this thereby the active Although not covalent modification of Cys-215 by 1,2-NQ binding in analysis in the study, such a was also by and not of the that the of PTP1B with 1,2-NQ Cys-215 and that mutation of covalent binding of 1,2-NQ to PTP1B, that Cys-215 is also a for for to Cys-215 attachment is that the used in analysis may have the of the Cys-215 are a of PTPs in which Cys-121 and Cys-215 are Tonks N.K. Biol. 2001; PubMed Scopus Google Scholar). In this study, also found that transactivation of EGFR coupled to reduction of PTP activity exposure of A431 cells to 1,2-NQ was 1,2-NQ exposure EGFR activation a at to a at by an the several We that not PTP1B but PTPs also undergo covalent modification at Cys-121 and/or Cys-215 by 1,2-NQ, leading to the of EGFR at after 1,2-NQ in of for Cys-121 caused a of decreased PTP1B activity caused by 1,2-NQ at 1,2-NQ, of the loss of PTP1B caused by 1,2-NQ was restored by mutation of that the of thiol modification in PTP1B reaction with 1,2-NQ a such at Cys-121 arylation is that of at the In 1,2-NQ been to the contraction of tracheal smooth muscle in the through activation of the EGFR (19Kikuno S. Taguchi K. Iwamoto N. Yamano S. Cho A.K. Froines J.R. Kumagai Y. Toxicol. Appl. Pharmacol. 2006; 210: 47-54Crossref PubMed Scopus (43) Google Scholar) leading to of the receptor to smooth muscle In experiments in which 1,2-NQ was to in and two actions that may that is associated with K. Takano H. Hiyoshi K. Ichinose T. Sadakane K. Yanagisawa R. Tomura S. Kumagai Y. Eur. Respir. J. 2007; 29: 259-267Crossref PubMed Scopus (49) Google Scholar). Because these tissue and are at that the in effects are mediated by inactivation of The study for the that of PTP1B by of 1,2-NQ to the and Cys-215 This reaction is irreversible so that of PTP1B in dephosphorylation of EGFR PTP1B the of on the of the the is exposure to low concentrations of compounds 1,2-NQ have a on PTP activity in the The EGFR been to play a key in epithelial and of Am. J. Physiol. 2002; Scholar), and effects on this by be relevant to their effects on J.M. Am. J. Physiol. 2001; 281: Google Scholar) reported that exposure of epithelial cells to the ambient a significant transactivation of showed that are of EGFR through a The reported that such as 1,2-NQ, which are in ambient can effects on the EGFR and may to the adverse health effects associated with We the of from Yamano with