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. 2010 Mar 5;285(10):7505-16.
doi: 10.1074/jbc.M109.040840. Epub 2010 Jan 8.

Hydrogen peroxide-sensitive cysteines in the Sty1 MAPK regulate the transcriptional response to oxidative stress

Alison M Day  1 , Elizabeth A Veal
Affiliations

Hydrogen peroxide-sensitive cysteines in the Sty1 MAPK regulate the transcriptional response to oxidative stress

Alison M Day et al. J Biol Chem. .

Abstract

MAPK are activated by and orchestrate responses to multiple, diverse stimuli. Although these responses involve the increased phosphorylation of substrate effector proteins, e.g. transcription factors, the mechanisms by which responses are tailored to particular stimuli are unclear. In the fission yeast Schizosaccharomyces pombe, the Sty1 MAPK is crucial for changes in gene expression that allow adaptation to many forms of environmental stress. Here, we have identified two cysteine residues in Sty1, Cys-153 and Cys-158, that are important for hydrogen peroxide-induced gene expression and oxidative stress resistance but not for other functions of Sty1. Many Sty1-dependent changes in gene expression are mediated by the Atf1 transcription factor. In response to stress, Sty1 increases Atf1 levels by (i) promoting increases in atf1 mRNA and by (ii) directly phosphorylating and stabilizing Atf1 protein. Although dispensable for phosphorylation and stabilization of Atf1 protein, we find that both Cys-153 and Cys-158 are required for increases in atf1 mRNA levels and Atf1-dependent gene expression in response to hydrogen peroxide but not osmotic stress. Indeed, our data indicate that oxidation of Sty1, by formation of a disulfide bond between Cys-153 and Cys-158, is important for maintaining atf1 mRNA stability at high concentrations of hydrogen peroxide. Together, these data reveal that redox regulation of cysteine thiols in Sty1 is involved in a stress-specific mechanism regulating transcriptional responses to oxidative stress. Intriguingly, the conservation of these cysteine residues in other MAPK raises the possibility that similar mechanisms may ensure appropriate responses to hydrogen peroxide in other eukaryotes.

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Figures

FIGURE 1.
FIGURE 1.
Sty1 is susceptible to reversible hydrogen peroxide-induced cysteine thiol oxidation. A and B, proteins extracted from wild-type cells (CHP429) treated for 10 min with the indicated concentrations of hydrogen peroxide were reacted (as indicated by +) with the thiol-active agent AMS and then analyzed by Western blotting with anti-Sty1 antibodies. A, following AMS treatment, the mobility of Sty1 prepared from untreated cells was decreased (Sty1+AMS), consistent with cysteines in Sty1 being in the reduced thiol state in untreated cells (Sty1red). Following exposure to increasing concentrations of hydrogen peroxide, a more mobile form of AMS-modified Sty1 was detected (Sty1ox), consistent with an increasing proportion of Sty1 becoming oxidized and hence less reactive with AMS. B, electrophoretic separation of Sty1 from hydrogen peroxide-treated cells into two forms is dependent on treatment of protein samples with AMS (as indicated by +), suggesting that the more mobile hydrogen peroxide-induced form of Sty1 is an oxidized, AMS-resistant form of Sty1 (Sty1ox). C, the absence of oxidized Sty1 (Sty1ox) when proteins extracted from wild-type cells (CHP429) exposed to 25 mm hydrogen peroxide for 10 min were treated with DTT (as indicated by +) prior to AMS treatment suggests that hydrogen peroxide-induced thiol oxidation of Sty1 is reversible. D and E, proteins extracted from wild-type cells (CHP429) treated, as indicated, for 10 min with 25 mm (D) or 6.0 mm (E) hydrogen peroxide were treated sequentially (as depicted in supplemental Fig. S2A) with (i) the thiol-modifying agent IAA, (ii) DTT (as indicated by +), and then (iii) AMS (as indicated by +). Analysis of these proteins by Western blotting with anti-Sty1 antibodies revealed that following DTT treatment, a proportion of IAA-modified Sty1 (Sty1+IAA) from hydrogen peroxide-treated cells was AMS-modified (Sty1+IAA+AMS), decreasing its mobility. Experiments were repeated at least twice, and representative experiments are shown.
FIGURE 2.
FIGURE 2.
Cysteines 153 and 158 are required for hydrogen peroxide-induced oxidation of Sty1 and resistance to oxidative stress. A, Sty1 contains six cysteine residues. The positions of these cysteines (black) in the primary sequence of Sty1 are indicated relative to the protein kinase domain (pale gray) and its key features, the ATP binding site (dark gray) and MAPKK phosphorylation sites (Thr-171 and Tyr-173). N, N terminus; C, C terminus. B–D, Western blot analysis of proteins extracted from WT (AD38) and sty1C153S (AD41) cells (B), Δsty1 cells (AD22) containing pRep41sty1 (WT) or pRep41sty1C158S (C) wild-type (AD38) and sty1C153SC158SS (AD44) cells (D) treated for 10 min with 25 mm hydrogen peroxide. Proteins were sequentially (i) IAA-modified and (ii) DTT-treated (as indicated by +) followed by (iii) AMS modification of DTT-reduced cysteine thiols before SDS-PAGE and Western blotting with anti-Sty1 antibodies. E, equal numbers of exponential phase WT (AD38), Δsty1 (AD22), and sty1C153SC158S (AD44) cells were serially diluted and spotted onto plates containing, as indicated, 1 m KCl, 250 mm CaCl2, 1.0 mm H2O2, or Cd2+ (0.1 mm CdSO4). F, the growth and survival of exponential phase WT (AD38), Δsty1 (AD22), and sty1C153SC158S (AD44) cells (grown in rich media) following treatment with 1.0 or 25 mm hydrogen peroxide for the indicated length of time. Error bars indicate the S.D. Experiments were repeated three times, and representative experiments are shown.
FIGURE 3.
FIGURE 3.
Cysteines 153 and 158 of Sty1 are dispensable for hydrogen peroxide-induced phosphorylation of Sty1 (Sty1-P) (A) or Atf1 (B), but as shown in C–E, required for hydrogen peroxide-induced increases in levels of Atf1 protein (C) and atf1 mRNA (D) due to an essential role in preventing hydrogen peroxide-induced destabilization of atf1 mRNA (E). A, Western blot analysis of the level of phosphorylated Sty1 in WT (AD38) and sty1C153SC158S (AD44) cells following treatment for 0, 10, or 20 min with 1.0 mm H2O2. Total Sty1 levels (anti-Sty1) are also shown. B, phosphorylation of Atf1 was detected as a decrease in Atf1-HA mobility following exposure of WT (AD69) and sty1C153SC158S (AD59) cells to 1.0 mm H2O2 by Western blot analysis of cell lysate using anti-HA antibodies. Tubulin levels are shown as a loading control. C, Western blot analysis using anti-HA antibodies of phosphatase-treated Atf1-HA purified from WT (AD69) and sty1C153SC158S (AD59) cells treated for 0, 40, or 60 min with 1.0 mm H2O2. A nonspecific band (*) indicates that the reduced H2O2-induced increase in the levels of Atf1 in sty1C153SC158S cells does not reflect differences in loading. The experiments in A–C were repeated three times, and representative experiments are shown. D and E, Northern blot analysis of RNA-extracted from WT (AD38) and sty1C153SC158S (AD44) cells treated for 0, 20, 40, or 60 min with 1.0 mm hydrogen peroxide (D) or 0.6 m KCl and with 300 μg/ml 1,10-phenanthroline and then with 1.0 mm H2O2 or 0.6 m KCl for 0, 20, 40, or 60 min (E). In D, a representative Northern blot analysis of atf1 and leu1 (loading control) mRNA levels is shown together with graphs showing the mean -fold induction of atf1 mRNA at 20 and 40 min relative to time 0 for each strain (calculated from PhosphorImager analysis of atf1 mRNA relative to leu1 mRNA data obtained in multiple (n) independent experiments; 1.0 mm H2O2 n = 10, 0.6 m KCl n = 3). Error bars indicate the S.E. Statistical analysis of the data indicates that the induction of atf1 mRNA by H2O2 in sty1C153SC158S when compared with wild-type cells was significantly reduced (Student's t test; at 20 min, p = 0.030, at 40 min, p = 0.0068, whereas at 0 min, p = 0.197) but that the osmotic stress (0.6 m KCl)-induced increase in atf1 mRNA was no lower in sty1C153SC158S than wild-type cells. In E, the linear decay curve for atf1 mRNA was obtained by plotting log 2 (atf1m RNA levels/25 S rRNA levels) against time. This experiment was repeated three times, and a representative experiment is shown.
FIGURE 4.
FIGURE 4.
Cysteines 153 and 158 in Sty1 are required to limit the hydrogen peroxide-induced destabilization of atf1 mRNA at increasing concentrations of hydrogen peroxide (A and B), allowing Atf1-dependent gene expression (C). A, Northern blot analysis of atf1 mRNA levels in WT (AD38) and sty1C153SC158S (AD44) cells before and after exposure to 1.0, 6.0, and 25 mm H2O2 reveals that the induction of atf1 mRNA decreases as the concentration of hydrogen peroxide increases in both wild-type cells and sty1C153SC158S cells. The graph shows the mean -fold induction of atf1 mRNA levels relative to leu1 mRNA (loading control) determined from PhosphorImager analysis of data obtained in three separate experiments. Error bars indicate the S.E. B, Northern blot analysis of atf1 mRNA levels in WT (AD38) and sty1C153SC158S (AD44) cells treated with 300 μg/ml 1,10-phenanthroline and for 0, 10, 20, or 40 min with 1.0, 6.0, or 25 mm H2O2 reveals that the half-life of atf1 mRNA is lower in sty1C153SC158S (AD44) than in WT (AD38) cells following treatment with 1.0, 6.0, or 25 mm H2O2. The half-life of atf1 mRNA in each strain and under each condition was calculated from the decay curves (log 2 atf1 mRNA/25 S rRNA against time), and mean values determined from at least four experiments are shown. Error bars indicate the S.E. C, Northern blot analysis of RNA from WT (AD38) and sty1C153SC158S (AD44) cells treated for 0, 20, 40, 60, or 80 min with 1.0 mm H2O2 revealed a significantly smaller H2O2-induced increase in gpx1 and pyp2 mRNA levels (relative to leu1 mRNA loading control) in sty1C153SC158S cells when compared with WT. Graphs show mean -fold induction of each mRNA at 0, 20, and 40 min calculated from data obtained in at least six independent experiments. (Two-way analysis of variance analysis: pyp2, WT versus sty1C153SC158S, p = 0.0222, n = 9; gpx1, WT versus sty1C153SC158S, p = 0.0237, n = 6).
FIGURE 5.
FIGURE 5.
The role of cysteines 153 and 158 of Sty1 in stabilization of atf1 mRNA is sufficient to account for their role in resistance to hydrogen peroxide. A, equal numbers of WT (AD38), sty1C153SC158S (AD44), Δatf1 (AD65), and Δatf1sty1C153SC158S (AD66) cells were serially diluted and spotted onto plates containing 1.0 mm hydrogen peroxide as indicated. B, survival of WT (AD38), sty1C153SC158S (AD44), Δatf1 (AD65), and Δatf1sty1C153SC158S (AD66) cells treated with 25 mm hydrogen peroxide for the indicated length of time. C, survival of WT (AD38) cells containing pRep41, sty1C153SC158S (AD44) cells containing pRep41, and sty1C153SC158S (AD44) cells containing pRep41atf1 following treatment with 25 mm hydrogen peroxide for the indicated lengths of time. Error bars indicate the S.D. Experiments were repeated three times, and representative experiments are shown.
FIGURE 6.
FIGURE 6.
Mutation of cysteines 153 and 158 of Sty1 prevents any H2O2-induced increase in Atf1 protein levels and greatly increases the oxidative stress sensitivity of cells containing an ATF1 mutant (ATF1-11M) that is not stabilized by H2O2-induced phosphorylation by Sty1 (21). A, Western blot analysis using anti-HA antibodies of Atf1-HA nickel-purified from atf1-HA sty1+(AD69), atf1-HA sty1C153SC158S (AD59), atf1-11M-HA sty1+ (NJ543), or atf1-11M-HA sty1C153SC158S cells (AD70) treated with 1.0 mm hydrogen peroxide for 0, 40, or 60 min. Western blot analysis with anti-tubulin antibodies was used to confirm similar protein contents of cell lysate from which Atf1-HA was purified. B, equal numbers of exponential phase atf1-HA sty1+ (AD69), atf1-HA sty1C153SC158S (AD59), atf1-11M-HA sty1+ (NJ543), and atf1-11M-HA sty1C153SC158S cells (AD70) were serially diluted and spotted onto plates containing 0.75 mm hydrogen peroxide as indicated (+H2O2). Experiments were repeated twice, and representative experiments are shown.
FIGURE 7.
FIGURE 7.
Cysteines 153 and 158 of Sty1 and phosphorylation of Atf1 act independently to maintain Atf1 protein levels and increase oxidative stress resistance. A model demonstrating the two Sty1-dependent mechanisms that have evolved in S. pombe to increase levels of the transcription factor Atf1 in response to hydrogen peroxide is shown. Following exposure to increasing concentrations of hydrogen peroxide, Sty1 becomes increasingly phosphorylated (P) and reversibly oxidized by formation of a disulfide bond between cysteines 153 and 158. This enables Sty1 to [1] inhibit the hydrogen peroxide-induced destabilization of atf1 mRNA and [2] phosphorylate Atf1 inhibiting Atf1 protein degradation (21). Together, these mechanisms lead to increased levels of Atf1 and thus increased Atf1-dependent gene expression and oxidative stress resistance.
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