Volume 15, Issue 1, Pages 129-139 (July 2004) A 2-Cys Peroxiredoxin Regulates Peroxide-Induced Oxidation and Activation of a Stress-Activated MAP Kinase  Elizabeth A. Veal, Victoria J. Findlay, Alison M. Day, Stephanie M. Bozonet, Jennifer M. Evans, Janet Quinn, Brian A. Morgan  Molecular Cell  Volume 15, Issue 1, Pages 129-139 (July 2004) DOI: 10.1016/j.molcel.2004.06.021

Figure 1 Tpx1 Is a Member of the 2-Cys Prx Family of Thioredoxin Peroxidases (A) Thioredoxin peroxidases, such as 2-Cys Prx, are oxidized during the reduction and detoxification of peroxides. The oxidized enzyme is recycled by the sequential oxidation and reduction of thioredoxin and thioredoxin reductase using NADPH. Oxidized and reduced forms of the enzymes are indicated by “ox” and “red.” (B) Alignment of the primary amino acid sequence of Tpx1 from S. pombe (SP) with 2-Cys Prxs from S. cerevisiae (SC), mouse (M), and human (H). Amino acids conserved in all of the presented proteins are indicated (bold). The positions of the conserved cysteine residues are indicated by asterisks. In Tpx1 (SP) these conserved cysteine residues are at positions 48 and 169, respectively. The full-length sequence of each protein is shown except for AOE372 where the homology with Tpx1 begins at the leucine residue at position 74. (C) Δtpx1 cells are sensitive to H2O2. The zone of growth inhibition around a filter soaked with 5 μl of 3% (v/v) H2O2 placed in the center of a Ye5S plate on which 2 × 106 exponentially growing tpx1+ (NT5) and Δtpx1 (VXOO) cells were pipetted along separate lines proceeding radially from the edge of the plate. (D) Overexpression of tpx1+protects cells against peroxides. Equal numbers of exponentially growing tpx1+ cells (KS2096) containing Rep1 vector or Rep1tpx1+ were serially diluted and spotted onto selective plates, one of which contained 1.0 mM tBOOH. Molecular Cell 2004 15, 129-139DOI: (10.1016/j.molcel.2004.06.021)

Figure 2 Tpx1 Is Required for Peroxide-Induced Activation of the Sty1 SAPK Pathway (A) In eukaryotes, conserved SAPK signaling pathways, such as the Sty1 SAPK pathway in S. pombe, regulate gene expression in response to a variety of stimuli including oxidative stress. Sty1 activation is regulated by phosphorylation of conserved threonine and tyrosine residues by the mitogen-activated protein kinase kinase (MAPKK) Wis1 (Warbrick and Fantes, 1991; Shiozaki and Russell, 1995), which in turn is regulated by the MAPKKKs Wak1 (or Wis4/Wik1) (Samejima et al., 1997; Shieh et al., 1997; Shiozaki et al., 1997) and Win1 (Samejima et al., 1998; Shieh et al., 1998). Activated Sty1 phosphorylates the Atf1 transcription factor and is required for the induction of expression of Atf1-regulated genes such as gpx1+, encoding glutathione peroxidase and Atf1- and Pap1-regulated genes such as ctt1+, encoding catalase. Sty1 is indirectly involved in regulation of Pap1. (B, C, and D) The levels of phosphorylation of HA-tagged Sty1 (Sty1-P) were determined by Western blot analyses using lysates prepared from (B and C) tpx1+ (JM1522) cells, containing either (B) Rep1 (vector) or Rep1tpx1+, or (C) Rep41 (vector) or Rep41trr1+ treated with 0.2 mM H2O2 for 0, 5, 10, or 20 min; and (D) tpx1+ (JM1522) and Δtpx1 (VX04) cells treated with 0, 0.1, 0.2, 0.5, or 1.0 mM H2O2 (upper panel) and 0, 1.0, 2.0, 6.0, or 10.0 mM H2O2 (lower panel) for 5 min. Total Sty1 levels are indicated. Molecular Cell 2004 15, 129-139DOI: (10.1016/j.molcel.2004.06.021)

Figure 3 Tpx1 Is Required for Sty1-Dependent Cellular Responses to Peroxide (A) The reduced mobility of phosphorylated 6HisHA-tagged Atf1 was detected by Western blot analysis using lysates prepared from tpx1+ (KS1479) and Δtpx1 (VX05) cells treated with 1.0 mM H2O2 for 0 or 10 min. (B) Northern blot analysis of total RNA extracted from tpx1+ (NT5) and Δtpx1 (VX00) cells treated with 1.0 mM H2O2 for 0, 20, 40, or 60 min. Probes used were specific for gpx1+, ctt1+, and leu1+ (invariant loading control) mRNA. Molecular Cell 2004 15, 129-139DOI: (10.1016/j.molcel.2004.06.021)

Figure 4 Tpx1 Stimulates Sty1 Phosphorylation Specifically in Response to Peroxide The levels of phosphorylation of HA-tagged Sty1 (Sty1-P) were determined by Western blot analyses using lysates prepared from (A) tpx1+ (JM1522) and Δtpx1 (VX04) cells treated with 1.0 mM H2O2 for 0, 5, 10, or 20 min; (B) tpx1+ (KS2096) and Δtpx1 (EV35) cells treated with 0.9 M KCl for 0, 5 or 10 min; and (C) tpx1+ (JM1522) cells, containing either Rep1 vector or Rep1tpx1+, treated with 0.2 M KCl for 0 or 5 min. Total Sty1 levels are indicated. Molecular Cell 2004 15, 129-139DOI: (10.1016/j.molcel.2004.06.021)

Figure 5 Tpx1 Regulation of Sty1 Requires Wis1 but Is Independent of the MAPKKKs The levels of Sty1 phosphorylation (Sty1-P) in cell lysates from (A) mcs4DN cells (JM1700), containing Rep1 vector or Rep1tpx1+, treated with 1.0 mM H2O2 for 0, 5, or 10 min; (B) wis1+ (JM1522) and Δwis1 (GD1682) cells, containing Rep1 vector or Rep1tpx1+, treated with 1.0 mM H2O2 for 0, 5, or 10 min; and (C) wis1+ (KS2096) and wis1DD (KS2088) cells, containing Rep1 vector or Rep1tpx1+, treated with 1.0 mM H2O2 or 0.6 M KCl for 0, 5, or 10 min. Total Sty1 levels are shown. Molecular Cell 2004 15, 129-139DOI: (10.1016/j.molcel.2004.06.021)

Figure 6 Tpx1 Associates Directly with Sty1 (A) Cell lysates were prepared under nonreducing conditions from mid-log cultures of wis1+ (JM1698) and Δwis1 (EV41) cells expressing Sty1-Myc and containing Rep41MHN-tpx1+ (expressing (His)6-Myc-Tpx1) or Rep41MHN vector. Proteins affinity purified from these lysates with Ni2+ NTA-agarose were separated under reducing conditions by SDS PAGE and analyzed by Western blotting with anti-Myc (9E10) antibodies. For comparison, cell lysate equivalent to 2.0% of the input lysate used for Ni2+ purification was also analyzed. (B) Proteins affinity purified with Ni2+ NTA-agarose from cell lysates prepared under nonreducing conditions from untreated and oxidatively stressed (1.0 mM H2O2 for 5 min) Δtpx1 (EV35) cells expressing Sty1(His)6HA and containing Rep1-tpx1+ (lanes 1, 3, and 5), Rep1-tpx1C48S (lane 6), Rep1-tpx1C169S (lane 7), or Rep1 vector control (lanes 2 and 4) were separated by nonreducing SDS PAGE and analyzed by Western blotting with anti-HA antibodies. For comparison, 1 sample (lane 3) was treated with β-mercaptoethanol (β-ME). (C and D) Sty1(His)6HA was partially purified and analyzed under nonreducing conditions (see B) from (C) Δtpx1 (EV35) cells that contained Rep1-tpx1+ or Rep1 vector control and had been treated with 1.0 mM H2O2 or 0.9 M KCl for 5 min or from (D) Δtpx1 (EV35) cells that contained Rep1-tpx1, Rep1-Flagtpx1+, or vector control and had been treated with 1.0 mM H2O2 for 5 min. (E and F) Sty1(His)6HA was partially purified using anti-HA agarose or Ni2+ NTA-agarose under nonreducing conditions from Δtpx1 (EV35) cells that contained Rep1-tpx1, Rep1-Flagtpx1+, Rep1-tpx1C169S, Rep1-Flagtpx1C169S, or vector control and had been treated with 1.0 mM H2O2 for 5 min. Precipitated proteins were separated under nonreducing conditions and analyzed by Western blotting with anti-Flag or anti-HA antibodies as indicated. Arrows indicate in (E) Flag-Tpx1-Sty1(His)6HA and in (F) Flag-Tpx1C169S -Sty1(His)6HA complexes. Molecular Cell 2004 15, 129-139DOI: (10.1016/j.molcel.2004.06.021)

Figure 7 Tpx1 Regulates Peroxide-Induced Activation of Sty1 through Tpx1-Sty1 Disulphide Formation (A) The levels of phosphorylation of Sty1(His)6HA (Sty1-P) were determined by Western blot analysis of Ni2+-affinity purified Sty1(His)6HA prepared from Δtpx1 cells (EV35), containing Rep1 vector, Rep1tpx1+, Rep1tpx1C48S, or Rep1tpx1C169S, treated with 0.2 mM H2O2 for 0, 5, or 10 min. Total Sty1 levels are indicated. (B) Cell lysates were prepared under nonreducing conditions from untreated, and oxidatively stressed (1.0 mM H2O2 for 5 min) Δtpx1 (EV45) cells containing, as indicated, Rep41MHNSty1 or Rep41MHNSty1C35S and Rep2-tpx1+ or Rep2 vector control. Proteins affinity purified from these lysates with Ni2+ NTA-agarose were separated by nonreducing SDS PAGE and analyzed by Western blotting with anti-Myc antibodies. The right-hand panel contains an enlarged view of lanes 3, 4, 7, and 8 showing the formation of the peroxide-induced Tpx1-Sty1 complex (approximately 70 kDa) requires cysteine 35 in Sty1. Asterisk indicates additional peroxide-induced Tpx1-dependent oxidized form of Sty1. These data are representative of several independent experiments. (C) The levels of phosphorylation of Myc-tagged Sty1 (Sty1-P) were determined by Western blot analyses of Ni2+-affinity purified (His)6MycSty1 or (His)6MycSty1C35S prepared from tpx1+ (NT4) or Δtpx1 (VXOO) cells, containing Rep41MHNSty1 or Rep41MHNSty1C35S, treated with 1.0 mM H2O2 for 0, 5, or 10 min. (D) Proposed mechanism for Tpx1-dependent peroxide-induced Sty1 activation requires both Tpx1-Sty1 disulphide bond formation and Wis1-dependent phosphorylation. Molecular Cell 2004 15, 129-139DOI: (10.1016/j.molcel.2004.06.021)