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• W2009476974 abstract "Despite its toxicity, H2O2 is produced as a signaling molecule that oxidizes critical cysteine residues of effectors such as protein tyrosine phosphatases in response to activation of cell surface receptors. It has remained unclear, however, how H2O2 concentrations above the threshold required to modify effectors are achieved in the presence of the abundant detoxification enzymes peroxiredoxin (Prx) I and II. We now show that PrxI associated with membranes is transiently phosphorylated on tyrosine-194 and thereby inactivated both in cells stimulated via growth factor or immune receptors in vitro and in those at the margin of healing cutaneous wounds in mice. The localized inactivation of PrxI allows for the transient accumulation of H2O2 around membranes, where signaling components are concentrated, while preventing the toxic accumulation of H2O2 elsewhere. In contrast, PrxII was inactivated not by phosphorylation but rather by hyperoxidation of its catalytic cysteine during sustained oxidative stress. Despite its toxicity, H2O2 is produced as a signaling molecule that oxidizes critical cysteine residues of effectors such as protein tyrosine phosphatases in response to activation of cell surface receptors. It has remained unclear, however, how H2O2 concentrations above the threshold required to modify effectors are achieved in the presence of the abundant detoxification enzymes peroxiredoxin (Prx) I and II. We now show that PrxI associated with membranes is transiently phosphorylated on tyrosine-194 and thereby inactivated both in cells stimulated via growth factor or immune receptors in vitro and in those at the margin of healing cutaneous wounds in mice. The localized inactivation of PrxI allows for the transient accumulation of H2O2 around membranes, where signaling components are concentrated, while preventing the toxic accumulation of H2O2 elsewhere. In contrast, PrxII was inactivated not by phosphorylation but rather by hyperoxidation of its catalytic cysteine during sustained oxidative stress. Phosphorylation inactivates peroxiredoxin I (PrxI), an enzyme that reduces H2O2 Ligation of growth factor or immune receptors induces PrxI tyrosine phosphorylation Phosphorylation occurs on membrane-associated but not cytosolic PrxI This localized inactivation of PrxI allows accumulation of H2O2 needed for signaling Hydrogen peroxide (H2O2) is generated in all aerobic organisms as a byproduct of normal cellular processes. Given that H2O2 is toxic to cells, however, such organisms are equipped with detoxifying enzymes such as catalase, glutathione peroxidases (GPxs), and peroxiredoxins (Prxs). Many mammalian cell types also produce H2O2 in response to a variety of extracellular stimuli, with the H2O2 so produced serving as a signaling molecule that regulates various biological processes. Stimulation of cells with various agonists thus induces H2O2 production, and blockage of H2O2 accumulation results in inhibition of signaling by such stimulants (D'Autréaux and Toledano, 2007D'Autréaux B. Toledano M.B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis.Nat. Rev. Mol. Cell Biol. 2007; 8: 813-824Crossref PubMed Scopus (2136) Google Scholar, Oakley et al., 2009Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Signaling components of redox active endosomes: the redoxosomes.Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (145) Google Scholar, Rhee, 2006Rhee S.G. Cell signaling. H2O2, a necessary evil for cell signaling.Science. 2006; 312: 1882-1883Crossref PubMed Scopus (1539) Google Scholar, Xu et al., 2002Xu D. Rovira I.I. Finkel T. Oxidants painting the cysteine chapel: redox regulation of PTPs.Dev. Cell. 2002; 2: 251-252Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). For example, in cells stimulated with growth factors such as platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), production of H2O2 is required for propagation of growth factor signaling (Bae et al., 1997Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation.J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar, Sundaresan et al., 1995Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.Science. 1995; 270: 296-299Crossref PubMed Scopus (2257) Google Scholar). Among the best characterized H2O2 effectors are protein tyrosine phosphatases (PTPs): H2O2 specifically oxidizes the catalytic cysteine residue of these enzymes and thereby inhibits their activity (Chiarugi and Cirri, 2003Chiarugi P. Cirri P. Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction.Trends Biochem. Sci. 2003; 28: 509-514Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, Rhee et al., 2005Rhee S.G. Kang S.W. Jeong W. Chang T.S. Yang K.S. Woo H.A. Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins.Curr. Opin. Cell Biol. 2005; 17: 183-189Crossref PubMed Scopus (581) Google Scholar, Tonks, 2005Tonks N.K. Redox redux: revisiting PTPs and the control of cell signaling.Cell. 2005; 121: 667-670Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). Indeed, the activation of protein tyrosine kinases (PTKs) is not sufficient to increase the steady-state level of protein tyrosine phosphorylation in cells; the concurrent inhibition of PTPs by H2O2 is also required. Many other H2O2 effector molecules also contain an H2O2-sensitive cysteine residue. Evidence indicates that NADPH oxidase (Nox) is largely responsible for receptor-dependent H2O2 production (Clempus and Griendling, 2006Clempus R.E. Griendling K.K. Reactive oxygen species signaling in vascular smooth muscle cells.Cardiovasc. Res. 2006; 71: 216-225Crossref PubMed Scopus (271) Google Scholar, Lambeth, 2004Lambeth J.D. NOX enzymes and the biology of reactive oxygen.Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2301) Google Scholar, Oakley et al., 2009Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Signaling components of redox active endosomes: the redoxosomes.Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (145) Google Scholar). Seven distinct catalytic subunits of Nox (Nox1 to Nox5, Duox1, and Duox2) have been identified (Lambeth, 2004Lambeth J.D. NOX enzymes and the biology of reactive oxygen.Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2301) Google Scholar). These catalytic subunits are transmembrane proteins and form multisubunit complexes with other membrane as well as cytosolic proteins. Nox generates superoxide by transferring one electron from NADPH to O2. Superoxide then undergoes dismutation to H2O2. Activated Nox complexes appear to assemble within discrete subcellular compartments, including lipid rafts and endosomes, thereby making it possible to produce toxic H2O2 molecules within a restricted region of the cell (Li et al., 2006Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Nox2 and Rac1 regulate H2O2-dependent recruitment of TRAF6 to endosomal interleukin-1 receptor complexes.Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (187) Google Scholar, Oakley et al., 2009Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Signaling components of redox active endosomes: the redoxosomes.Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (145) Google Scholar, Ushio-Fukai, 2006Ushio-Fukai, M. (2006). Localizing NADPH Oxidase-derived ROS. Science's Stke, http://www.stke.org/cgi/content/full/2006/349/re8.Google Scholar). For H2O2 to serve as a signaling molecule, its concentration must increase rapidly above a certain threshold (10 to 100 μM) and remain elevated long enough for it to oxidize effector molecules (Stone and Yang, 2006Stone J.R. Yang S. Hydrogen peroxide: a signaling messenger.Antioxid. Redox Signal. 2006; 8: 243-270Crossref PubMed Scopus (848) Google Scholar). However, in most cells, H2O2-eliminating enzymes are present in large concentrations in the cytosol in order to ensure that the toxic molecule remains at low intracellular levels (<0.1 μM) (Stone and Yang, 2006Stone J.R. Yang S. Hydrogen peroxide: a signaling messenger.Antioxid. Redox Signal. 2006; 8: 243-270Crossref PubMed Scopus (848) Google Scholar). PrxI to PrxIV belong to the 2-Cys Prx subfamily of Prx enzymes, which exist as homodimers and reduce H2O2 with the use of reducing equivalents provided by thioredoxin (Trx) (Rhee et al., 2005Rhee S.G. Kang S.W. Jeong W. Chang T.S. Yang K.S. Woo H.A. Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins.Curr. Opin. Cell Biol. 2005; 17: 183-189Crossref PubMed Scopus (581) Google Scholar). PrxI and PrxII are localized to the cytosol, whereas PrxIII and PrxIV are restricted to mitochondria and the endoplasmic reticulum, respectively. PrxI and PrxII are also abundant, constituting a total of 0.2% to 1% of soluble protein in cultured mammalian cells (Chae et al., 1999Chae H.Z. Kim H.J. Kang S.W. Rhee S.G. Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin.Diabetes Res. Clin. Pract. 1999; 45: 101-112Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Catalase is exclusively localized in peroxisomes. GPx1 is present predominantly in the cytosol, but its concentration is smaller than that of PrxI or PrxII in most tissues. Prx enzymes are efficient in eliminating low concentrations of H2O2 because of their low Km values for this substrate (Chae et al., 1999Chae H.Z. Kim H.J. Kang S.W. Rhee S.G. Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin.Diabetes Res. Clin. Pract. 1999; 45: 101-112Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). It has therefore remained unclear how, in the presence of PrxI and II, H2O2 can accumulate in the cytosol to a concentration sufficient for it to modify target proteins. The catalytic site of 2-Cys Prxs comprises the NH2-terminal region of one subunit and the COOH-terminal region of the other subunit in the homodimer (Rhee et al., 2005Rhee S.G. Kang S.W. Jeong W. Chang T.S. Yang K.S. Woo H.A. Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins.Curr. Opin. Cell Biol. 2005; 17: 183-189Crossref PubMed Scopus (581) Google Scholar). The peroxidatic Cys-SH (CP-SH) is located in the NH2-terminal region and is selectively oxidized by H2O2 to CP-SOH, which then reacts with the resolving Cys-SH (CR-SH) in the COOH-terminal region of the other subunit to form an intermolecular disulfide. The disulfide is reduced by Trx. During catalysis, the CP-SOH intermediate occasionally undergoes further oxidation by H2O2 to CP-SO2H before the relatively slow formation of a disulfide with CR-SH can occur. This hyperoxidation, which results in inactivation of peroxidase activity, is reversed by sulfiredoxin (Biteau et al., 2003Biteau B. Labarre J. Toledano M.B. ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.Nature. 2003; 425: 980-984Crossref PubMed Scopus (765) Google Scholar, Woo et al., 2003aWoo H.A. Chae H.Z. Hwang S.C. Yang K.S. Kang S.W. Kim K. Rhee S.G. Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation.Science. 2003; 300: 653-656Crossref PubMed Scopus (459) Google Scholar). Given that bacterial Prxs are resistant to hyperoxidation and that prokaryotes do not express sulfiredoxin, reversible inactivation through hyperoxidation has been proposed to be a eukaryotic adaptation that allows H2O2 to accumulate to substantial levels under certain circumstances, thereby facilitating H2O2-dependent signaling. This concept has been termed the “floodgate” hypothesis (Wood et al., 2003Wood Z.A. Poole L.B. Karplus P.A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling.Science. 2003; 300: 650-653Crossref PubMed Scopus (1083) Google Scholar). However, no hyperoxidized PrxI or II was observed in cells stimulated with PDGF (Choi et al., 2005Choi M.H. Lee I.K. Kim G.W. Kim B.U. Han Y.H. Yu D.Y. Park H.S. Kim K.Y. Lee J.S. Choi C. et al.Regulation of PDGF signalling and vascular remodelling by peroxiredoxin II.Nature. 2005; 435: 347-353Crossref PubMed Scopus (325) Google Scholar). We have now found that PrxI is phosphorylated on Tyr194 and is thereby inactivated in cells stimulated via receptors for growth factors such as PDGF or EGF or via immune receptors such as T cell (TCR) and B cell (BCR) receptors. This phosphorylation is confined to PrxI molecules associated with cell membranes; it was not observed with PrxI present in the cytosol. The spatially confined inactivation of PrxI thus provides a means for generating favorable H2O2 gradients around a submembrane compartment, where signaling proteins are concentrated, while minimizing the general accumulation of H2O2 to toxic levels and disturbance of global redox potential. To determine whether Prxs are phosphorylated, we subjected recombinant human PrxI and II to an in vitro kinase assay with two nonreceptor PTKs, Lck and Abl, in the presence of [γ-32P]ATP. Both PTKs phosphorylated PrxI and PrxII, with the extent of phosphorylation for the former being markedly greater than that for the latter (Figure 1A , Figure S1A available online). Tyrosine-194 of PrxI was one of 600 phosphorylation sites revealed by a large-scale proteomic analysis in a cancer cell line treated with pervanadate (Rush et al., 2005Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (920) Google Scholar). We therefore generated antibodies to Tyr194-phosphorylated PrxI by injection with a phosphopeptide (KSKEpYFSKQK) corresponding to residues 190 to 199 of PrxI. The sequence SKEYFSK is conserved between PrxI and II. The extents of PrxI and PrxII phosphorylation detected by immunoblot analysis with the phospho-specific antibodies was similar to that revealed by measurement of 32P radioactivity, indicative of similar reactivities of the antibodies for the phosphorylated isozymes. To verify the phosphorylation site of PrxI, we expressed wild-type PrxI or a Tyr194 to Phe mutant (Y194F) in the PTK-expressing Escherichia coli strain TKB1. Phosphorylation of the wild-type protein was detected, whereas that of the Y194F mutant was not (Figure 1B), indicating that Tyr194 is the only site of tyrosine phosphorylation.Figure S1Phosphorylation of PrxI and PrxII In Vitro and Its Effect on the Catalytic Activity of PrxI, Related to Figure 1Show full caption(A) Phosphorylation of PrxI and PrxII in vitro by Abl. Purified recombinant PrxI or II (1 μg) was incubated for 1 hr at 30°C in the absence or presence of purified recombinant Abl (15 ng) in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 100 μM ATP, and 10 μCi of [γ-32P]ATP. Each reaction mixture was then subjected to SDS-PAGE on a 14% gel followed by autoradiography, immunoblot analysis with antibodies to phosphorylated PrxI, or staining with Ponceau S.(B) Peroxidase activity of wild-type or mutant forms of PrxI in the presence of 500 μM H2O2. The peroxidase activity of nonphosphorylated (open circles) or Tyr194- phosphorylated (closed squares) forms of wild-type PrxI or of Y194D (open triangles) or Y194F (closed triangles) mutants of PrxI was monitored for 600 s as a decrease in A340 by coupling of the reaction to the oxidation of NADPH (left panel). The initial decrease in A340 between 20 and 100 s is expanded in the right panel. NADPH oxidation in the absence of PrxI (closed circles) was also monitored. The assay conditions are identical to those in Figure 1C with the exception that the H2O2 concentration was increased to 500 μM.(C) Ribbon diagrams comparing the active sites and COOH-terminal regions of PrxI (green) and PrxII (gold). The homology model was built as described in the Expanded Legend for Figure 1. His197 of PrxII (human erythrocytes, 1QMV) appears to form a salt bridge network with Asp181 and Lys196 in the same subunit, and this interaction appears to hinder access of a PTK to Tyr193. The salt bridge network of PrxII might stabilize the structure of the COOH-terminal domain, in which the salt bridge distance between Asp181 and Lys196 and that between Asp181 and His197 are 2.64 and 2.69 Å, respectively. Asp182 and Lys197 are conserved in PrxI, but PrxI contains Gln198 instead of His197 of PrxII. The salt bridge network is displayed in cyan in the enlarged image (right), which was obtained after a rotation of 180° around the z-axis followed by a rotation of 45° around the x-axis of the image on the upper left. Primed residue numbers correspond to the subunit whose catalytic Cys is not shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Phosphorylation of PrxI and PrxII in vitro by Abl. Purified recombinant PrxI or II (1 μg) was incubated for 1 hr at 30°C in the absence or presence of purified recombinant Abl (15 ng) in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 100 μM ATP, and 10 μCi of [γ-32P]ATP. Each reaction mixture was then subjected to SDS-PAGE on a 14% gel followed by autoradiography, immunoblot analysis with antibodies to phosphorylated PrxI, or staining with Ponceau S. (B) Peroxidase activity of wild-type or mutant forms of PrxI in the presence of 500 μM H2O2. The peroxidase activity of nonphosphorylated (open circles) or Tyr194- phosphorylated (closed squares) forms of wild-type PrxI or of Y194D (open triangles) or Y194F (closed triangles) mutants of PrxI was monitored for 600 s as a decrease in A340 by coupling of the reaction to the oxidation of NADPH (left panel). The initial decrease in A340 between 20 and 100 s is expanded in the right panel. NADPH oxidation in the absence of PrxI (closed circles) was also monitored. The assay conditions are identical to those in Figure 1C with the exception that the H2O2 concentration was increased to 500 μM. (C) Ribbon diagrams comparing the active sites and COOH-terminal regions of PrxI (green) and PrxII (gold). The homology model was built as described in the Expanded Legend for Figure 1. His197 of PrxII (human erythrocytes, 1QMV) appears to form a salt bridge network with Asp181 and Lys196 in the same subunit, and this interaction appears to hinder access of a PTK to Tyr193. The salt bridge network of PrxII might stabilize the structure of the COOH-terminal domain, in which the salt bridge distance between Asp181 and Lys196 and that between Asp181 and His197 are 2.64 and 2.69 Å, respectively. Asp182 and Lys197 are conserved in PrxI, but PrxI contains Gln198 instead of His197 of PrxII. The salt bridge network is displayed in cyan in the enlarged image (right), which was obtained after a rotation of 180° around the z-axis followed by a rotation of 45° around the x-axis of the image on the upper left. Primed residue numbers correspond to the subunit whose catalytic Cys is not shown. We next investigated the effect of phosphorylation of PrxI on its peroxidase activity by subjecting PrxI produced in TKB1 bacteria to affinity chromatography with an immobilized monoclonal antibody to phosphotyrosine (4G10) in order to separate phosphorylated and nonphosphorylated proteins. The peroxidase activity of phosphorylated and nonphosphorylated forms of PrxI was then monitored in the presence of 50 μM H2O2 by coupling the peroxidase reaction to NADPH oxidation. After a lag time of ∼50 s, the reaction rate for phosphorylated PrxI achieved a steady state that was approximately one-seventh of that for nonphosphorylated PrxI (Figure 1C). No lag time was observed for nonphosphorylated PrxI. The reaction rate for nonphosphorylated PrxI decreased slightly with time because CP-SH becomes hyperoxidized during catalysis (see below), and the rate became zero when A340 reached 0.9 because of exhaustion of H2O2 (Figure 1C). A similar lag time (∼20 s) and reduced activity (approximately one-fourth of that for nonphosphorylated PrxI) were observed for phosphorylated PrxI when the H2O2 concentration was increased to 500 μM (Figure S1B). These results suggest that, at H2O2 concentrations in the low micromolar range, the lag time for phosphorylated PrxI would be substantially greater than 50 s and the steady-state reaction rate would be much less than one-seventh of that for the nonphosphorylated enzyme. The catalytic CP-SH of PrxI is highly reactive with H2O2, because it is surrounded by Arg128 and Arg151 that stabilize the thiolate anion (CP-S−), which is more readily oxidized by H2O2 than is its protonated thiol counterpart. Structural modeling predicted that the phosphate moiety of phospho-Tyr194 of one subunit of the PrxI homodimer is positioned within 9 Å of the sulfur atom of CP-SH (Cys52) of the other subunit (Figure 1D). The negatively charged phosphate group might thus be expected to impair the deprotonation of Cp-SH and thereby to decrease its reactivity with H2O2. However, after CP-SH has entered the catalytic cycle, reduction by Trx may return the CP residue to a state that is not affected to the same extent by the phosphate moiety and that is slightly more reactive with H2O2, resulting in a catalytic activity for the phosphorylated enzyme that is reduced but measurable. In support of this notion, a PrxI mutant in which Tyr194 is replaced by Asp (Y194D), which therefore contains a negatively charged carboxyl group at position 194, exhibited an activity time course similar to that for phosphorylated PrxI, whereas neutral replacement of the same residue by Phe did not substantially affect enzyme activity (Figure 1C). Comparison of the structures of PrxI and PrxII might also suggest an explanation for the observed slower phosphorylation of PrxII. His197 of PrxII appears to form a hydrogen bond with Asp181, with this interaction possibly hindering access of a PTK to Tyr193 (the residue corresponding to Tyr194 of PrxI) (Figure S1C). No analogous interaction is apparent in PrxI, in which Gln is present at the position corresponding to His197 of PrxII. With the antibodies specific for Tyr194-phosphorylated PrxI, we next examined whether PrxI or PrxII is phosphorylated in cells. PrxI and PrxII, which differ by one amino acid residue in size, cannot be separated by SDS-PAGE. Immunoblot analysis revealed that stimulation of rat smooth muscle cells with PDGF resulted in the transient phosphorylation of PrxI or II (Figure 2A ). Similar time-dependent phosphorylation of PrxI or II was observed in NIH 3T3 cells stimulated with PDGF (Figure S2A), HER (NIH 3T3 cells that stably express EGF receptor) stimulated with PDGF or EGF (Figure 3, Figure S2B), A431 cells stimulated with EGF (see Figure 4), Ramos B cells stimulated with antibodies to IgM (see Figure 4), and Jurkat T cells stimulated with antibodies to CD3 (see Figure 4). Stimulation of mouse embryonic fibroblasts (MEFs) derived from wild-type or PrxI knockout mice with PDGF yielded a phosphorylated Prx band with the former cells but not with the latter (Figure 2B), suggesting that PrxI is the primary target for growth factor-induced phosphorylation.Figure S2Tyrosine Phosphorylation of PrxI in Growth Factor-Stimulated Cells and Its Role in Receptor Signaling, Related to Figure 2Show full caption(A) Serum-deprived NIH 3T3 cells were stimulated for the indicated times with PDGF (25 ng/ml). Cell lysates (25 μg) were then subjected to immunoblot analysis with antibodies to phosphorylated or total PrxI.(B) Serum-deprived HER cells were stimulated with PDGF (25 ng/ml) or EGF (50 ng/ml) for the indicated times, after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated or total PrxI.(C) Lysates (250 μg of protein) of NIH 3T3 cells that had been stimulated with PDGF (25 ng/ml) in the presence of 100 μM sodium pervanadate for 10 min were subjected to 2D-PAGE followed by immunoblot analysis with antibodies to phosphorylated or total forms of PrxI or PrxII. The positions of phosphorylated and unphosphorylated PrxI are indicated. Tyr194-phosphorylated PrxI is detected at two spots, probably because PrxI undergoes an additional unknown modification when stimulated with PDGF in the presence of pervanadate. The amount of Tyr194-phosphorylated was estimated to be ∼5% of total PrxI from the immunoblot intensities.(D) The indicated amounts of lysates from (A) and (C) were subjected to immunoblot analysis with antibodies to phosphorylated or total PrxI.(E) MEFs from PrxI knockout mouse were transiently transfected with plasmids encoding wild-type PrxI (W) or Y194F mutant PrxI (M) using MEF 2 Nucleofector Kit (Amaxa) according to the manufacturer's instructions. The transfection efficiency was ∼30%. The cells were deprived of serum and then stimulated with PDGF (25 ng/ml) for the indicated times. Cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated PDGF receptor (p-PDGFR), phosphorylated PLCγ (p-PLCγ), or to total forms of PLCγ or PrxI. (upper panel). The relative extent of phosphorylation of PDGFR or PLC-γ was estimated from the immunoblot band intensities (lower panels); data are means ± SEM from three independent measurements. ∗∗p < 0.01, ∗p < 0.05 versus corresponding wild-type PrxI value.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effects of PTK Inhibitors on PrxI PhosphorylationShow full caption(A) Serum-deprived HER cells were incubated for 30 min in the absence (Con.) or presence of 5 μM STI571 and then for the indicated times in the additional presence of PDGF (25 ng/ml) or EGF (50 ng/ml). Cell lysates (25 μg protein) were then subjected to immunoblot analysis with antibodies to phosphorylated or total PrxI or to phosphotyrosine (p-Tyr).(B and C) Serum-deprived A431 cells were incubated for 30 min in the absence (Con.) or presence of 1 μM AG1478 (B) or 10 μM PP1 (C) and then for the indicated times in the additional presence of EGF (50 ng/ml). Cell lysates (25 μg protein) were then subjected to immunoblot analysis with antibodies to phosphorylated or total forms of PrxI, EGFR, or Src.(D) Serum-deprived Ramos cells were incubated for 30 min in the absence (Con.) or presence of 10 μM PP1 and then for 5 min in the additional absence or presence of antibodies to IgM (15 μg/ml), after which cell lysates (25 μg protein) were subjected to immunoblot analysis with antibodies to phosphorylated or total forms of Src or PrxI.See also Figure S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Phosphorylation of PrxI Associated with Confined Membrane MicrodomainsShow full caption(A–D) Serum-deprived A431 cells (A), NIH 3T3 cells (B), Ramos B cells (C), and Jurkat T cells (D) were stimulated for 5 min with EGF (50 ng/ml), PDGF (25 ng/ml), antibodies to IgM (15 μg/ml), or antibodies to CD3 (10 μg /ml), respectively, after which detergent-soluble (S) and -resistant (R) fractions were prepared from cell lysates and subjected to immunoblot analysis with antibodies to the indicated proteins. In (C) and (D), antibodies to phosphotyrosine were used to detect phosphorylated nonreceptor PTKs (p-Syk, p-Lyn, p-ZAP70, p-Lck, and p-Fyn), with the size of molecular markers (in kilodaltons) being indicated on the right.(E) Serum-deprived A431 cells were incubated for 5 min in the absence or presence of EGF (50 ng/ml), fixed, and subjected to immunofluorescence staining with antibodies to phosphorylated PrxI (green) and to Flot2 (red). Merged images with nuclei stained with 4′,6-diamidino-2-phenylindole (blue) are also shown. The regions indicated by the arrows are shown at higher magnification in the insets. The scale bar represents 20 μm.See also Figure S4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Serum-deprived NIH 3T3 cells were stimulated for the indicated times with PDGF (25 ng/ml). Cell lysates (25 μg) were then subjected to immunoblot analysis with antibodies to phosphorylated or total PrxI. (B) Serum-deprived HER cells were stimulated with PDGF (25 ng/ml) or EGF (50 ng/ml) for the indicated times, after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated or total PrxI. (C) Lysates (250 μg of protein) of NIH 3T3 cells that had been stimulated with PDGF (25 ng/ml) in the presence of 100 μM sodium pervanadate for 10 min were subjected to 2D-PAGE followed by immunoblot analysis with antibodies to phosphorylated or total forms of PrxI or PrxII. The positions of phosphorylated and unphosphorylated PrxI are indicated. Tyr194-phosphorylated PrxI is detected at two spots, probably because PrxI undergoes an additional unknown modification when stimulated with PDGF in the presence of pervanadate. The amount of Tyr194-phosphorylated was estimated" @default.
• W2009476974 created "2016-06-24" @default.
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• W2009476974 date "2010-02-01" @default.
• W2009476974 modified "2023-10-17" @default.
• W2009476974 title "Inactivation of Peroxiredoxin I by Phosphorylation Allows Localized H2O2 Accumulation for Cell Signaling" @default.
• W2009476974 cites W128647466 @default.
• W2009476974 cites W1522219769 @default.
• W2009476974 cites W1921965616 @default.
• W2009476974 cites W1965078724 @default.
• W2009476974 cites W1968463439 @default.
• W2009476974 cites W1973986741 @default.
• W2009476974 cites W1974966731 @default.
• W2009476974 cites W1985776151 @default.
• W2009476974 cites W1986191025 @default.
• W2009476974 cites W1989021197 @default.
• W2009476974 cites W1994863097 @default.
• W2009476974 cites W2001923529 @default.
• W2009476974 cites W2003761660 @default.
• W2009476974 cites W2005478135 @default.
• W2009476974 cites W2017265125 @default.
• W2009476974 cites W2017642315 @default.
• W2009476974 cites W2021938810 @default.
• W2009476974 cites W2025894702 @default.
• W2009476974 cites W2031302525 @default.
• W2009476974 cites W2031729422 @default.
• W2009476974 cites W2044949501 @default.
• W2009476974 cites W2046764130 @default.
• W2009476974 cites W2048569323 @default.
• W2009476974 cites W2049713754 @default.
• W2009476974 cites W2056955418 @default.
• W2009476974 cites W2066589243 @default.
• W2009476974 cites W2067593902 @default.
• W2009476974 cites W2079557671 @default.
• W2009476974 cites W2084893631 @default.
• W2009476974 cites W2085512015 @default.
• W2009476974 cites W2088411222 @default.
• W2009476974 cites W2091787667 @default.
• W2009476974 cites W2099981464 @default.
• W2009476974 cites W2102472842 @default.
• W2009476974 cites W2102619095 @default.
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