Hydrogen Peroxide-sensitive Cysteines in the Sty1 MAPK Regulate the Transcriptional Response to Oxidative Stress*

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.

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.
In eukaryotes, mitogen-activated protein kinases (MAPK) 2 play vital roles in orchestrating appropriate responses to a vast array of stimuli, including growth factors, cytokines, and environmental stress. (For a recent review, see Ref. 1.) MAPK acti-vation by these stimuli is important for changes in gene expression that promote cell growth, apoptosis, or survival/ adaptation. Consequently, MAPK play important roles in regulating many fundamental biological processes (1). For instance, in metazoans, p38 and JNK MAPK are important for innate immunity and inflammation (2,3). Conversely, MAPK in pathogenic fungi trigger adaptive changes that aid survival/ evasion of the host immune system and are important for virulence (4,5). The association of MAPK activity with various diseases, including cancer, has generated great medical interest in targeting drugs to regulate MAPK activity (6). However, as MAPK are activated by multiple stimuli and important for a large number of processes, drugs that target the kinase activity have inevitable side effects (7). The identification/understanding of mechanisms by which MAPK tailor responses to particular stimuli will be an important step toward developing drug treatments that target specific aspects of MAPK function.
The amenability to genetic manipulation and presence of fewer functionally redundant MAPK genes have allowed studies in the model eukaryote, the fission yeast Schizosaccharomyces pombe, to yield important insight into MAPK function/regulation (8). For instance, the S. pombe ortholog of the p38 and JNK families of MAPK, Sty1 (Spc1, Phh1), is important for cell cycle progression and also essential for cell survival and adaptation in response to environmental change (9 -12). Significantly, like its mammalian counterparts, p38 and JNK, Sty1 is activated in response to a variety of stresses including heat, oxidative stress, osmotic stress, nutrient limitation, and UV (9 -12). In response to these stimuli, signaling mechanisms are initiated that lead to phosphorylation of these MAPK and consequent activation of their kinase activity (9 -15). Among the key substrates that are phosphorylated by p38, JNK and Sty1 MAPK are ATF transcription factors (16 -19). For instance, Sty1 phosphorylates Atf1 (18,19). As a heterodimer with a second bZip transcription factor, Pcr1, Atf1 regulates the expression of many genes. Indeed, global analysis of mRNA has identified a Sty1/Atf1-dependent core environmental stress response that is activated by a variety of stress stimuli and important for protection against different stress conditions, including osmotic and oxidative stress (20). Stress-induced activation of the Sty1 kinase increases the phosphorylation of Atf1, inhibiting its degradation and thus leading to increased Atf1 levels and increased transcription of Atf1 target genes (21). Under oxidative stress conditions, in addition to increasing Atf1 protein stability, Sty1 is also required for atf1 mRNA stability (22).
In addition to the core environmental stress response, subsets of Sty1-regulated genes are only induced in response to certain specific stresses (20). Indeed, different sets of genes have been shown to be induced in response to different levels of the same stress. For instance, the transcriptional response to low levels of hydrogen peroxide (0.07 mM H 2 O 2 ) is significantly different from the response to higher levels (0.5 and 6 mM H 2 O 2 ) (23,24). Although the mechanisms by which Sty1 tailors responses to specific stimuli are poorly understood, this differential response to different levels of hydrogen peroxide is partly orchestrated through the use of different transcription factors. The hydrogen peroxide-specific transcriptional response to low levels of hydrogen peroxide is mediated by the AP-1 (activator protein 1)-like transcription factor, Pap1, with Atf1 more important for the response to higher levels of hydrogen peroxide (23,24).
The hydrogen peroxide-induced activation of Pap1 involves reversible oxidation of cysteine thiols in Pap1 (25). Indeed, the susceptibility of particular cysteine residues to hydrogen peroxide-induced oxidation has been shown to regulate the activity of an increasing number of proteins (26,27). Notably, cysteine thiol oxidation has also been implicated in stress-sensing mechanisms leading to activation of MAPK. For instance, oxidation of cysteine thiols in the MAPKKK (mitogen-activated protein kinase kinase kinase), Ask1, and MEKK1 is involved in the regulation of downstream MAPK, JNK, and p38 (28,29). Moreover, previous work in our laboratory revealed that hydrogen peroxide-induced activation of Sty1 is associated with the formation of mixed disulfide bonds between Sty1 and the typical 2-Cys peroxiredoxin Tpx1 (30).
Here, we have explored the possibility that redox regulation of cysteines in Sty1 might also be involved in tailoring downstream signaling events to generate hydrogen peroxide-specific responses in S. pombe. Indeed, we have identified two redoxsensitive cysteine residues in Sty1 that are required specifically for the response to hydrogen peroxide. We show that these two cysteines are required for the hydrogen peroxide-induced increase in Atf1 levels and hence for the hydrogen peroxideinduced expression of Atf1-dependent genes and oxidative stress resistance. Our data support a role for reversible oxidation of these cysteine residues in preventing hydrogen peroxide-induced destabilization of atf1 mRNA.

EXPERIMENTAL PROCEDURES
Yeast Growth Conditions-The strains used in this study are shown in Table 1. Strains were grown either in rich medium (YE5S) or in synthetic minimal medium (Edinburgh minimal medium 2) with appropriate supplements (31). Strains were grown at 30°C, and experiments were performed using exponential phase liquid cultures grown with constant aeration.
Yeast Strains-Strain AD22 (⌬sty1) was generated by homologous recombination using a disruption cassette generated by PCR amplification of wild-type his7 gene (35) flanked by DNA homologous to the regions immediately upstream and downstream of the sty1 open reading frame. This cassette was introduced into CHP429 and HIS ϩ transformants selected for on media containing the appropriate supplements. Integration at the correct genomic location was verified by PCR.
Analysis of Proteins by Western Blotting-Protein concentrations were estimated using the bicinchoninic acid protein assay (Perbio) for denatured samples or the Coomassie Blue protein assay (Perbio) for non-denatured samples. Equal amounts of protein were separated by SDS-PAGE on 10% gels and electroblotted to nitrocellulose membrane (Schleicher & Schuell). Nonspecific interactions were blocked with 10% bovine serum albumin in TBST (20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.1% Tween 20) before incubation with primary antibodies diluted 1 to 1000 with 5% bovine serum albumin in TBST. Following TBST washes, horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies were utilized in conjunction with ECL (Amersham Biosciences) and x-ray film (Fujifilm). For determination of Sty1 phosphorylation, 50 mM NaF and 2 mM sodium vanadate were included as phosphatase inhibitors in all blotting and antibody incubation steps. TAT1 anti-tubulin antibodies (Cancer Research UK) were used as a loading control.
Sensitivity Tests-For spot tests, equal numbers of exponentially growing cells (ϳ1 ϫ 10 7 ) were subjected to 10-fold serial dilutions and then spotted onto YE5S agar containing the indicated concentrations of stress-inducing agents using a 96-well replicating tool. Plates were incubated at 30°C for up to 4 days. To assay recovery/survival following exposure to H 2 O 2 , similar numbers of exponentially growing cells in liquid culture were treated with 1.0 or 25 mM H 2 O 2 . Duplicate samples containing the appropriate numbers of cells were plated onto YE5S agar at the indicated time points to give between 30 and 300 colonies/ plate. Plates were incubated at 30°C for up to 4 days, after which time colonies were counted. Experiments were repeated, and two-factor analysis of variance confirmed that differences between curves were statistically significant (p Ͻ 0.0001).

High Concentrations of Hydrogen Peroxide Induce Reversible
Oxidation of Cysteines in Sty1-We have previously shown that during hydrogen peroxide-induced activation of the Sty1 MAPK, a small proportion of Sty1 becomes oxidized through formation of a mixed disulfide with the peroxiredoxin, Tpx1 (30). In Saccharomyces cerevisiae, hydrogen peroxide-induced activation of the Yap1 transcription factor involves formation of a mixed disulfide with the peroxiredoxin, Gpx3, as an essential but transient step preceding the formation of multiple intramolecular disulfide bonds in Yap1 (39). Hence, we examined the possibility that other oxidation events might occur at cysteines in Sty1 in response to hydrogen peroxide. First, we examined whether the oxidation state of cysteines in Sty1 changed when cells were exposed to hydrogen peroxide. Proteins were extracted from wild-type cells under acidic conditions to preserve the in vivo redox state of cysteine thiols, reacted with the thiol-modifying agent AMS, and examined by Western blotting using anti-Sty1 antibodies. AMS modification of reduced cysteine thiols increases the molecular mass by ϳ0.6 kDa per covalently modified cysteine, allowing electrophoretic separation of forms containing different numbers of modified cysteines. In unstressed cells, AMS treatment produces an ϳ3.6-kDa shift in the electrophoretic mobility of Sty1, consistent with all six cysteine residues being reduced (Fig. 1A). Similarly, when cells were exposed to low levels of hydrogen peroxide, there was no decrease in the ability of Sty1 to bind AMS, indicating that all the cysteines in Sty1 remain reduced. However, following exposure of cells to concentrations of hydrogen peroxide above 1.0 mM, a form of Sty1 with an increased electrophoretic mobility was detected (Fig. 1A). This lower molecular weight form of Sty1 was only detected when samples were treated with AMS ( Fig. 1B), strongly suggesting that it repre-sents a form of Sty1 in which one or more cysteines is oxidized, reducing its capacity to bind AMS. As the levels of hydrogen peroxide increased, the proportion of oxidized to reduced protein also increased such that the greatest proportion of oxidized to reduced protein was observed at the highest concentration of hydrogen peroxide tested (25 mM) (Fig. 1A). At this highest concentration of hydrogen peroxide (25 mM), Sty1 protein levels were also apparently reduced. However, when proteins were extracted from cells treated with 25 mM hydrogen peroxide under non-denaturing conditions at pH 7.5, comparable levels of Sty1 were detected by Western analysis (supplemental Fig. S1B). This suggests that the reduced levels of Sty1 detected following acid lysis of 25 mM hydrogen peroxidetreated cells reflects a hydrogen peroxide-induced increase in the formation of acid-precipitated, SDS-resistant insoluble forms of Sty1 rather than a decrease in total Sty1 protein levels.
It is possible that this oxidized form of Sty1 (Fig. 1A) is produced by irreversible oxidation of one or more cysteine thiols in Sty1. However, when proteins from hydrogen peroxide-treated cells were pretreated with the reducing agent DTT prior to reaction with AMS, only reduced forms of Sty1 were detected (Fig. 1C). The DTT reversibility of this oxidation suggests that the oxidized form of Sty1 is produced by formation of one or more disulfide bonds between cysteines in Sty1 rather than by the irreversible oxidation of cysteine thiols to sulfinic or sulfonic acid derivatives. To examine the possibility that the oxidized form of Sty1 contains one or more disulfide bonds, protein extracts prepared from wild-type cells, before or following treatment with 25 mM hydrogen peroxide, were treated stepwise with (i) the thiol-modifying agent iodoacetamide to alkylate any reduced cysteine residues, (ii) DTT to reduce any disulfide bond(s), and (iii) AMS to bind to any cysteine residues reduced by DTT (supplemental Fig. S2A). Again, following exposure to 25 mM hydrogen peroxide, lower levels of Sty1 were detected, reflecting the increase in SDSinsoluble Sty1 in hydrogen peroxide-treated cells following acid lysis (Fig. 1D). Consistent with a stress-induced increase in Sty1 phosphorylation, we noted that Sty1 mobility was slightly decreased in cells exposed to hydrogen peroxide (Fig. 1, D and  E). Prior reduction with DTT of samples from hydrogen peroxide-treated cells containing oxidized Sty1 increased the amount of Sty1 detected and, importantly, also allowed AMS modification of Sty1 (Fig. 1D). A similar AMS-dependent band was also detected following DTT treatment of Sty1 extracted from cells treated with 6.0 mM hydrogen peroxide (Fig. 1E). These data are 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 ϩ) 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 (Sty1ϩAMS), consistent with cysteines in Sty1 being in the reduced thiol state in untreated cells (Sty1 red ). Following exposure to increasing concentrations of hydrogen peroxide, a more mobile form of AMS-modified Sty1 was detected (Sty1 ox ), 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 ϩ), suggesting that the more mobile hydrogen peroxide-induced form of Sty1 is an oxidized, AMS-resistant form of Sty1 (Sty1 ox ). C, the absence of oxidized Sty1 (Sty1 ox ) when proteins extracted from wild-type cells (CHP429) exposed to 25 mM hydrogen peroxide for 10 min were treated with DTT (as indicated by ϩ) 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 ϩ), and then (iii) AMS (as indicated by ϩ). Analysis of these proteins by Western blotting with anti-Sty1 antibodies revealed that following DTT treatment, a proportion of IAA-modified Sty1 (Sty1ϩIAA) from hydrogen peroxide-treated cells was AMS-modified (Sty1ϩIAAϩAMS), decreasing its mobility. Experiments were repeated at least twice, and representative experiments are shown.
consistent with the reversible hydrogen peroxide-induced oxidation of Sty1 involving formation of one or more intramolecular disulfide bonds.
The genetic amenability of S. pombe allowed us to investigate which of the six cysteine residues in Sty1 are required for the formation of hydrogen peroxide-induced disulfide bond(s) in Sty1 ( Fig. 2A). First, a series of S. pombe mutant strains was generated, each expressing, in place of wild-type Sty1, a Sty1 mutant protein in which a single cysteine residue was substituted with serine. We then examined the ability of each of these Sty1 mutant proteins to form a disulfide bond in response to hydrogen peroxide. Previous work in our laboratory has shown that cysteine 35 of Sty1 is able to form a hydrogen peroxideinduced disulfide bond with Tpx1 (30). However, serine substitution of cysteine 35 did not prevent the AMS modification of DTT-treated Sty1 C35S extracted from hydrogen peroxidetreated cells (supplemental Fig. S2B). This indicates that cysteine 35 is not involved in the hydrogen peroxide-induced for-mation of intramolecular disulfide bond(s) in Sty1. Similarly, Sty1 mutant proteins in which cysteine 13, cysteine 202, or cysteine 242 was replaced with serine were also able to form Sty1 intramolecular disulfide bonds (supplemental Fig. S2, B and C). In contrast, the reduced effect of AMS on the mobility of DTT-treated oxidized Sty1 C153S suggests that cysteine 153 in Sty1 is required for normal Sty1 oxidation ( Fig. 2B and supplemental Fig. S2D). However, pretreatment with DTT still increased AMS binding to IAAmodified Sty1 C153S , suggesting that an additional cysteine is reversibly oxidized even in the absence of cysteine 153 ( Fig. 2B and supplemental Fig. S2D). We examined the possibility that this cysteine might be cysteine 158. Interestingly, although serine substitution of the other cysteines in Sty1 had a negligible effect upon the levels of Sty1 protein ( Fig.  2B and supplemental Fig. S2, B, C, and D), the levels of Sty1 C158S protein produced were far higher than wild type (supplemental Fig. S2D) as a result of increased sty1 C158S mRNA levels. 3 Furthermore, when protein levels were adjusted for this (by loading 10-fold less protein onto the gel), we found that DTT treatment did not allow Sty1 C158S extracted from hydrogen peroxidetreated cells and modified with IAA to bind AMS (supplemental Fig.  S2D, lower panel). This indicates that as well as its role in inhibiting sty1 gene expression, this cysteine was also required for hydrogen peroxide-induced oxidation of Sty1. It was possible that the insensitivity of Sty1 C158S to hydrogen peroxide-induced oxidation might be due to the increased amount of Sty1 C158S protein present. To eliminate this possibility we examined whether Sty1 C158S protein was DTT reversibly oxidized in cells expressing Sty1 and Sty1 C158S protein from another promoter (nmt) and thus containing equivalent amounts of Sty1 protein (32) (Fig. 2C). Consistent with cells expressing Sty1 from its normal promoter ( Fig. 1 and supplemental Fig. S2, B-D), DTT treatment of IAA-modified Sty1 and Sty1 C158S extracted from hydrogen peroxide-treated cells expressing Sty1 and Sty1 C158S from the Rep41 nmt promoter allowed wild-type Sty1, but not Sty1 C158S protein, to be modified by AMS (Fig. 2C). Thus, our data suggest that the hydrogen peroxide-induced oxidation of Sty1 may involve reversible oxidation of cysteine 158.  As substitution of cysteine 153 with serine also reduced the AMS reactivity of Sty1 (Fig. 2B), this suggested that cysteine 153 might participate in formation of a disulfide with cysteine 158. However, the DTT-induced AMS reactivity of Sty1 C153S suggests that in the absence of cysteine 153, cysteine 158 still becomes oxidized, possibly through formation of a disulfide with an alternative cysteine. The absence of any AMS-reactive form following DTT treatment of IAA-modified Sty1 C153SC158S , in which both cysteine 153 and cysteine 158 are replaced with serine, is consistent with this possibility (Fig. 2D). Taken together, these data suggest that in response to hydrogen peroxide, cysteine 158 in Sty1 becomes reversibly oxidized. Moreover, our data suggest that in wild-type Sty1, this reversible oxidation is likely to involve formation of a disulfide bond between cysteines 153 and 158. Intriguingly, not only did Sty1 C153SC158S not undergo oxidation, but it was also expressed at normal levels, suggesting that cysteine 153 is required for the increased Sty1 protein levels in the sty1 C158S mutant.
Cysteines 153 and 158 of Sty1 Are Required for Resistance to Oxidative Stress-Sty1 is important for cell cycle progression and for resistance to a variety of stress conditions, including osmotic and oxidative stress. Having determined that cysteines 153 and 158 play important roles in regulating Sty1 levels and redox sensitivity, we examined whether substitution of cysteines 153 and 158 with serine affected the ability of cells to grow under a variety of different stress conditions that inhibit the growth of ⌬sty1 mutant cells (10,11). Although Sty1 C158S is insensitive to thiol oxidation, to eliminate the possibility that the increased levels of Sty1 C158S might be responsible for any phenotypes associated with the single mutation, these investigations were undertaken with Sty1 C153SC158S . Importantly, sty1 C153SC158S cells were of a similar size and grew at a similar rate to wild-type (WT) cells, suggesting that neither cysteine is important for Sty1 function in controlling cell cycle progression (9) (supplemental Fig. S3 and Fig. 2E). Indeed, the similar growth of wild-type and sty1 C153SC158S mutant cells on plates containing 1 M potassium chloride or 250 mM calcium chloride (Fig. 2E) suggests that these cysteines are also dispensable for Sty1 function in resistance to osmotic stress and calcium. However, although much less sensitive than the ⌬sty1 mutant, the reduced growth of sty1 C153SC158S cells when compared with the WT control strain, on plates containing hydrogen peroxide or the heavy metal cadmium (Fig. 2E) suggested that these cysteines might be important for resistance to oxidative stress. Indeed, this was also evident in the substantially reduced capacity of the sty1 C153SC158S cells to recover and grow following transient exposure to either a level of hydrogen peroxide (1.0 mM) that is sublethal to wild-type cells or an acutely toxic level (25 mM) (Fig. 2F). Together, these data suggest that cysteines 153 and 158 are specifically important for Sty1 function under oxidative stress conditions.
Cysteines 153 and 158 of Sty1 Are Not Required for Hydrogen Peroxide-induced Phosphorylation of Sty1 or Its Substrate, Atf1-Activation of the kinase activity of Sty1 by dual phosphorylation of threonine 171 and tyrosine 173 by the MAPKK, Wis1, is essential for Sty1-dependent phosphorylation of downstream substrates in response to a variety of stress conditions, including hydrogen peroxide (11). Hence, it was possible that the increased oxidative stress sensitivity of cells expressing Sty1 C153SC158S was due to reduced hydrogen peroxide-induced phosphorylation of Sty1 C153SC158S . However, we found that Sty1 C153SC158S was inducibly phosphorylated to an even greater extent than wild-type Sty1 in response to 1.0 mM H 2 O 2 (Fig.  3A). Thus, Cys-153 and Cys-158 are not required for hydrogen peroxide-induced phosphorylation of Sty1.
Although dispensable for Sty1 phosphorylation, it was possible that serine substitution of Cys-153 and Cys-158 reduced the kinase activity of Sty1, thus reducing the phosphorylation of substrate proteins such as the bZip transcription factor Atf1 (18,19). We therefore examined whether cysteines 153 and 158 of Sty1 were required for hydrogen peroxide-induced phosphorylation of Atf1. As expected, the mobility of HA epitopetagged Atf1 (Atf1-HA) extracted from WT cells treated with hydrogen peroxide was significantly decreased, consistent with hydrogen peroxide-induced Atf1-HA phosphorylation (Fig.  3B) (21). The similar decrease in Atf1-HA mobility observed when cells expressing Sty1 C153SC158S were treated with hydrogen peroxide suggests that hydrogen peroxide-induced phosphorylation of Atf1 is unimpaired by serine substitution of these cysteines (Fig. 3B). From this we conclude that cysteines 153 and 158 are not required for Sty1-dependent, hydrogen peroxide-induced phosphorylation of Atf1.
Cysteines 153 and 158 Are Required for Hydrogen Peroxideinduced Increases in Atf1 Levels-The stress-induced phosphorylation of Atf1 by Sty1 inhibits degradation of Atf1, thus leading to an increase in Atf1 protein levels (21). However, despite apparently normal hydrogen peroxide-induced phosphorylation, we noticed that hydrogen peroxide appeared to induce a smaller increase in Atf1-HA levels in sty1 C153SC158S mutant than in WT cells (Fig. 3B). To examine possible effects on Atf1 protein levels more closely, we examined the levels of Atf1-HA in phosphatase-treated samples prepared from wild-type and Sty1 C153SC158S mutant cells before and after exposure to 1.0 mM H 2 O 2 . As expected, in wild-type cells, there was a significant increase in the levels of Atf1 following exposure to hydrogen peroxide. However, a much smaller increase in Atf1 levels was detected in cells expressing Sty1 C153SC158S (Fig. 3C). This suggests that although not necessary for H 2 O 2 -induced phosphorylation of Atf1, cysteines 153 and 158 of Sty1 are required to ensure increased levels of Atf1 protein in response to peroxide stress.
Cysteines 153 and 158 of Sty1 Are Required for Hydrogen Peroxide-induced Increases in atf1 mRNA-In addition to stabilizing Atf1 by phosphorylation, Sty1 also promotes oxidative stress-induced increases in the levels of atf1 mRNA (21,22). To examine the possibility that the failure to effectively increase Atf1 protein levels in sty1 C153SC158S mutant cells (Fig. 4, B and C) was due to a failure to increase atf1 mRNA levels in response to hydrogen peroxide, we compared the levels of atf1 mRNA in wild-type and sty1 C153SC158S cells before and after treatment with hydrogen peroxide. As expected, atf1 mRNA levels were increased following exposure of wild-type cells to hydrogen peroxide or osmotic stress (0.6 M KCl) (Fig. 3D). However, the hydrogen peroxide-induced increase in atf1 mRNA levels was significantly (p Յ 0.03) lower in sty1 C153SC158S cells (Fig. 3D). This implies that the reduced levels of Atf1 protein following hydrogen peroxide treatment of sty1 C153SC158S cells may be due, at least in part, to a failure to effectively increase atf1 mRNA levels. There was no significant difference between the levels of atf1 mRNA in untreated sty1 C153SC158S and wild-type cells (Fig. 3D). Moreover, osmotic stress induced similar increases in atf1 mRNA levels in both wild-type cells and sty1 C153SC158S mutant cells (Fig. 3D). Together, these data are consistent with cysteines 153 and 158 being specifically required for hydrogen peroxide-induced increases in atf1 mRNA levels.
Previous studies have suggested that Sty1 influences atf1 mRNA levels by affecting mRNA stability (22). However, it was possible that the reduced levels of atf1 mRNA in sty1 C153SC158S cells could reflect either reduced transcription or reduced atf1 mRNA stability. To distinguish between these two possibilities, we examined the effect of hydrogen peroxide on atf1 mRNA levels in wild-type and sty1 C153SC158S mutant cells treated with the transcriptional inhibitor 1,10-phenanthroline (40). As expected, treatment with 1,10-phenantholine prevented any increase in atf1 mRNA levels in either wild-type or sty1 C153SC158S cells following H 2 O 2 treatment, indicating that transcription had been effectively blocked (Fig. 3E). However, although in wild-type cells atf1 mRNA levels remained relatively constant up to 60 min after exposure to hydrogen peroxide, in sty1 C153SC158S cells treated with 1,10-phenanthroline, atf1 mRNA levels decreased (Fig. 3E). This suggests that atf1 mRNA was less stable in these cells than in wild-type cells. Indeed, although the stability of atf1 mRNA was similar in untreated and osmotically stressed sty1 C153SC158S and wildtype cells (Fig. 3E and data not shown), following treatment Error bars indicate the S.E. Statistical analysis of the data indicates that the induction of atf1 mRNA by H 2 O 2 in sty1 C153SC158S when compared with wild-type cells was significantly reduced (Student's t test; at 20 min, p ϭ 0.030, at 40 min, p ϭ 0.0068, whereas at 0 min, p ϭ 0.197) but that the osmotic stress (0.6 M KCl)-induced increase in atf1 mRNA was no lower in sty1 C153SC158S 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. with 1.0 mM hydrogen peroxide, the half-life of atf1 mRNA was substantially decreased in sty1 C153SC158S cells when compared with wild-type cells. These data suggest that cysteines 153 and 158 in Sty1 are required for atf1 mRNA stability following exposure to 1.0 mM H 2 O 2 .
Hydrogen Peroxide-induced Oxidation of Sty1 Is Associated with Maintenance of atf1 mRNA Stability-Having found that cysteines 153 and 158 in Sty1 (i) are required for atf1 mRNA stability following exposure to hydrogen peroxide (Fig. 3E) and (ii) become oxidized in response to hydrogen peroxide (Figs. 1 and 2), we examined whether oxidation of these cysteines was important for hydrogen peroxide-induced increases in atf1 mRNA levels. When we compared the levels of atf1 mRNA in wild-type cells treated with 1.0, 6.0, and 25 mM H 2 O 2 , we found that treatment with 1.0 mM H 2 O 2 , at which oxidation of Sty1 is minimal (Fig. 1A), produced a much bigger increase in atf1 mRNA levels than with 6.0 or 25 mM (Fig. 4A), at which an increasing proportion of Sty1 is oxidized (Fig. 1A). This smaller increase in atf1 mRNA as H 2 O 2 concentrations increase suggested that oxidation of cysteines 153 and 158 might prevent Sty1 from maintaining atf1 mRNA stability. However, if either serine substitution or oxidation of cysteines 153 and 158 in Sty1 prevents Sty1-dependent stabilization of atf1 mRNA, then it would be expected that the induction of atf1 mRNA in sty1 C153SC158S cells would be reduced to a similar extent at both low and high concentrations of H 2 O 2 . In contrast, we found that as H 2 O 2 concentrations increase, there was an even smaller increase in atf1 mRNA levels in sty1 C153SC158S cells than in WT cells such that atf1 mRNA levels actually decreased following exposure of sty1 C153SC158S cells to 25 mM H 2 O 2 (Fig. 4A). This suggested that oxidation of Sty1 at these concentrations of H 2 O 2 might be important for maintaining atf1 mRNA stability. Consistent with this, although exposure to increasing levels of hydrogen peroxide reduced the half-life of atf1 mRNA in wildtype cells, it produced a much greater decrease in atf1 mRNA stability in sty1 C153SC158S cells (Fig. 4B). Indeed, we found that the half-life of atf1 mRNA was still significantly reduced in sty1 C153SC158S cells exposed to 25 mM hydrogen peroxide when compared with wild-type cells similarly treated (at 25 mM hydrogen peroxide, half-lives of atf1 mRNA are: wild-type, 8.00 Ϯ 0.95 min; sty1 C153SC158S , 4.60 Ϯ 0.41 min; Student's t test p ϭ 0.00938) (Fig. 4B). Thus, our data indicate that the oxidation of cysteines 153 and 158 in Sty1 that occurs at these concentrations of hydrogen peroxide (Fig. 1A) is important for maintenance of atf1 mRNA stability following oxidative stress.
Cysteines 153 and 158 of Sty1 Are Required for Hydrogen Peroxide-induced Atf1-dependent Gene Expression-Our data suggest that the smaller hydrogen peroxide-induced increase in Atf1 levels in sty1 C153SC158S cells when compared with wildtype (Fig. 3C) is due to the reduced capacity of Sty1 C153SC158S to maintain atf1 mRNA stability under these conditions (Figs. 3E and 4B). We next examined whether the reduction in Atf1 levels in hydrogen peroxide-treated sty1 C153SC158S mutant cells was sufficient to reduce the hydrogen peroxide-induced expression of Atf1-dependent genes, encoding the glutathione peroxidase, gpx1, and the protein tyrosine phosphatase pyp2. As expected, levels of both gpx1 and pyp2 mRNA increased in wild-type cells following exposure to 1.0 mM H 2 O 2 (Fig. 4C). gpx1 and pyp2 mRNA levels also increased following exposure of sty1 C153SC158S mutant cells to 1.0 mM H 2 O 2 . However, the increases in each mRNA were markedly reduced when compared with wild-type cells (p Ͻ 0.025) (Fig. 4C). This suggests that the role of cysteines 153 and 158 of Sty1 in atf1 mRNA stability is important for the Sty1-dependent transcriptional response to hydrogen peroxide and may be responsible for the reduced oxidative stress resistance of sty1 C153SC158S mutant cells (Fig. 2, E and F). Indeed, as negative feedback regulation of Sty1 involves dephosphorylation by Pyp2 (10,11), it is possible that the reduced levels of pyp2 expression may also be responsible for the slight increase in hydrogen peroxideinduced Sty1 phosphorylation detected in sty1 C153SC158S mutant cells (Fig. 3A).
The Role of Cysteines 153 and 158 in Oxidative Stress Resistance Is Atf1-dependent-It was possible that the reduced H 2 O 2 -induced expression of Atf1-dependent genes might account for the increased hydrogen peroxide sensitivity of sty1 C153SC158S mutant cells, or alternatively, that these cysteines have additional roles that contribute to their essential role in oxidative stress resistance. To examine these possibilities, we compared the sensitivity of sty1 C153SC158S , ⌬atf1, and ⌬atf1sty1 C153SC158S cells to hydrogen peroxide. The sensitivity of ⌬atf1sty1 C153SC158S cells was similar to ⌬atf1 cells, both on plates containing low levels of hydrogen peroxide (Fig. 5A) and following exposure to high levels of hydrogen peroxide in liquid culture (Fig. 5B). This indicates that loss of cysteines 153 and 158 of Sty1 does not further reduce the oxidative stress resistance of cells lacking Atf1. The Atf1 dependence of the oxidative stress-protective role of cysteines 153 and 158 is consistent with their function in oxidative stress resistance being predominantly through their role in maintaining atf1 mRNA stability. Indeed, the reduced ability of sty1 C153SC158S cells to survive transient exposure to 25 mM hydrogen peroxide was completely rescued by ectopic expression of Atf1 (pRep41 atf1), suggesting that increased expression of Atf1 is sufficient to restore wild-type levels of hydrogen peroxide resistance to sty1 C153SC158S cells (Fig. 5C). Together, these data also indicate that the Sty1-dependent maintenance of atf1 mRNA stability, for which Cys-153 and Cys-158 are required, makes an important contribution to the role of Sty1 in oxidative stress resistance.
Sty1 Regulates atf1 mRNA Stability and Atf1 Phosphorylation by Independent Mechanisms to Increase Atf1 Protein Levels and Oxidative Stress Resistance-Stress-induced phosphorylation of Atf1 by Sty1 increases the stability of Atf1 protein such that a mutant in which 11 consensus MAPK phosphorylation sites have been substituted (Atf1-11M) is no longer phosphorylated or stabilized by Sty1 (21). Our data suggest that cysteine 153 and cysteine 158 in Sty1 act to increase Atf1 protein levels independently from Sty1-dependent phosphorylation of Atf1 (Fig. 3). A prediction of this model is that serine substitution of these cysteines in Sty1 in cells containing the Atf1-11M mutant form of Atf1 will prevent stressinduced stabilization of atf1 mRNA and thus further reduce the levels of Atf1 protein. To test this hypothesis, we constructed a strain containing both the atf1-11M-HA and the sty1 C153SC158S alleles and compared the levels of Atf1-HA protein in these cells with those in the atf1-11M-HA and sty1 C153SC158S parent strains before and after treatment with hydrogen peroxide. This Western blot analysis revealed the hydrogen peroxide-induced increase in Atf1-HA levels observed in wild-type cells is reduced to a similar extent by Ser substitution of Cys-153 and Cys-158 in Sty1 (sty1 C153SC158S ) as by mutation of all the consensus MAPK phosphorylation sites on Atf1 (Atf1-11M) (Fig. 6A). Furthermore, in atf1-11M-HA sty1 C153SC158S cells, the hydrogen peroxide-induced increase in Atf1 levels was completely abolished, indicating that Sty1-dependent phosphorylation of Atf1 and stabilization of atf1 mRNA act independently and are together vital for the hydrogen peroxide-induced increase in Atf1 levels (Fig. 6A). This was also reflected in the heightened oxidative stress sensitivity of atf1-11M-HA sty1 C153SC158S mutant cells, in which both Sty1-dependent phosphorylation of Atf1 and Sty1-dependent stabilization of atf1 mRNA are defective when compared with atf1-11M-HA and sty1 C153SC158S cells (Fig. 6B).
Thus, we propose that S. pombe have evolved to use the Sty1 MAPK in two independent mechanisms to increase Atf1 levels in response to hydrogen peroxide. Firstly, formation of a disulfide between cysteines 153 and 158 in Sty1 is required to maintain atf1 mRNA stability. Secondly, Sty1mediated direct phosphorylation of Atf1 increases Atf1 protein stability (21) (Fig. 7). The evolution of multiple mechanisms to increase Atf1 levels in response to oxidative stress indicates the critical role that Atf1 plays in survival and adaptation to oxidative stress conditions.

DISCUSSION
Activation of MAPK by phosphorylation is a key step in the initiation of biological responses to a multitude of stimuli (1).
ATF transcription factors are important mediators of MAPKdependent transcriptional responses in mammals and yeast (16,23,24). Phosphorylation by MAPK increases ATF activity by multiple mechanisms, including the inhibition of ATF protein degradation (21,41). In S. pombe, the Sty1 MAPK regulates Atf1 to elicit both core stress responses common to different stimuli and also responses specific to particular stress conditions (20). However, it is unclear how different stimuli, which produce similar increases in phosphorylation of Sty1 and Atf1, produce different responses appropriate for adaptation to a particular stimulus. Here, we have identified two cysteines in Sty1 that are specifically required to increase Atf1 levels and Atf1-dependent transcription in response to hydrogen peroxide. Under oxidative stress conditions, in addition to phosphorylating and stabilizing Atf1 protein, Sty1 is also required to maintain atf1 mRNA stability (22). Indeed, we find that cysteines 153 and 158 in Sty1 are dispensable for phosphorylation of Atf1 but instead required to inhibit the hydrogen peroxideinduced destabilization of atf1 mRNA (Figs. 3E, 4B, and 7).
Previous studies have suggested that phosphorylation by Sty1 and heterodimerization with Pcr1 together act to increase the protein stability of Atf1 (21). In addition, at least two Sty1dependent mechanisms are important for maintaining atf1 mRNA stability following oxidative stress. One of these mechanisms involves the RNA-binding proteins Csx1 and Upf1, which act in the same pathway to increase atf1 mRNA stability (22,42). However, Sty1 is also required for atf1 mRNA stability, even in the absence of Csx1 (22). Indeed, we find that cysteines 153 and 158 in Sty1 regulate atf1 mRNA stability independently from Csx1 (data not shown). Thus, Sty1 is involved in multiple   (21). Together, these mechanisms lead to increased levels of Atf1 and thus increased Atf1-dependent gene expression and oxidative stress resistance. mechanisms for increasing Atf1 levels in response to stress. The evolution of several mechanisms for increasing Atf1 levels following stress reflects the important role of this transcription factor in stress survival/adaptation (18,19). In addition, it suggests that constitutively high levels of Atf1 do not benefit, or indeed may adversely affect, cells under non-stress conditions. Strikingly, the two cysteines identified here are important for hydrogen peroxide-induced increases in atf1 mRNA and oxidative stress resistance but not for osmotic stress-induced increases in atf1 mRNA or osmotic stress resistance (Figs. 2 and  3). This raises the possibility that the regulation of Atf1 by different mechanisms in response to different stresses allows S. pombe to tailor the levels of Atf1 appropriately to initiate distinct transcriptional responses to a particular stress. Indeed, cells containing an Atf1 mutant protein that is not phosphorylated by Sty1 in response to stress are also sensitive to oxidative stress but have normal resistance to osmotic stress (21). Together, these data suggest that Sty1-dependent increases in Atf1 levels are particularly important for oxidative stress resistance.
Intriguingly, we have also found that Sty1 is sensitive to hydrogen peroxide-induced oxidation. The DTT reversibility of this oxidation and its rapid reversal following removal from hydrogen peroxide (data not shown) suggest that it involves the formation of disulfide bond(s) between cysteine thiols in Sty1. Indeed, our analysis of this oxidation in Sty1 mutants, in which each cysteine is substituted with serine, suggest that cysteines 153 and 158 form an intramolecular disulfide in response to high levels of hydrogen peroxide. Comparisons with the crystal structures of other MAPK suggest that both Cys-153 and Cys-158 are likely to be on the surface of Sty1, where they are accessible to hydrogen peroxide, and moreover that it is structurally feasible for a disulfide bond to form between them. 4 Given the important role of these cysteines in the response of cells to hydrogen peroxide, it was possible that this oxidation might be important for the function of Sty1 in increasing atf1 mRNA stability. Indeed, as hydrogen peroxide concentrations increase, the greater decline in atf1 mRNA stability in sty1 C153SC158S than in wild-type cells (Fig. 4) suggests that the increased oxidation of these cysteines in wild-type Sty1 is important for maintenance of atf1 mRNA stability under these conditions (Fig. 4). Although the mechanism by which formation of a disulfide bond between cysteines 153 and 158 in Sty1 could prevent hydrogen peroxide-induced destabilization of atf1 mRNA is not clear, this suggests that redox regulation of these cysteines allows S. pombe to modulate the response to high levels of hydrogen peroxide.
Previous transcriptional profiling studies have suggested that Atf1-dependent gene expression is reduced at higher concentrations of hydrogen peroxide (24). Here, we show that this could reflect smaller increases in atf1 mRNA levels as hydrogen peroxide concentrations increase (Fig. 4A). It is possible that the reduced "protective" transcriptional response at these higher levels may allow commitment to an alternative cellular response, e.g. apoptosis rather than initiation of mechanisms to ensure survival. Thus, under these conditions, regulation of atf1 mRNA stability may be critical in determining whether or not cells survive. In support of this, the survival of sty1 C153SC158S cells is greatly reduced following exposure to hydrogen peroxide levels that substantially reduce atf1 mRNA stability in these cells (Figs. 2, 3E, 4B, and 5). Furthermore, the ability of ectopic expression of atf1 to restore normal oxidative stress resistance to sty1 C153SC158S cells suggests that reduced Atf1 levels are responsible for the sensitivity of these cells to hydrogen peroxide (Fig. 5C).
The critical role that SAPK (stress-activated protein kinase) and bZip transcription factors also play in coordinating responses to stress in other eukaryotes suggests that this regulatory mechanism might be conserved. Indeed, Cys-153 is conserved in all MAPK, and Cys-158 is also conserved in fungal orthologs of Sty1, such as Hog1, which is important for the virulence of opportunistic human pathogens, Candida albicans and Cryptococcus neoformans (supplemental Fig. S4) (4,43). As hydrogen peroxide is generated by mammalian immune cells as a potent weapon against pathogens, the ability to activate responses to hydrogen peroxide is likely to be important for the virulence of these fungi. Hence, these cysteines, particularly Cys-158, which is not conserved in mammalian p38/JNK MAPK, may represent a potential target for the treatment of fungal infections.
Although cysteine 158 is not conserved in mammalian p38 and JNK MAPK, it is conserved in the ERK family of MAPK (supplemental Fig. S4). This raises the intriguing possibility that ERK may have a role in regulating mRNA stability. ERK is activated in response to growth factors, many of which have been shown to lead to the generation of hydrogen peroxide as a second messenger. Hydrogen peroxide promotes signal transduction by causing the reversible inactivation by thiol oxidation of a catalytic cysteine in protein tyrosine phosphatases (44). If cysteines 153 and 158 are important for ERK1/2 function, then it is an intriguing possibility that hydrogen peroxide may also regulate signal transduction by reversible oxidation of these cysteine thiols.
In summary, together with previous studies (21,22), our data suggest that Sty1 is involved in multiple mechanisms to ensure that adequate levels of Atf1 protein are produced and maintained to initiate appropriate levels of antioxidant gene expression in response to hydrogen peroxide. Moreover, our studies reveal that the function of the MAPK activity of Sty1 in increasing Atf1 protein stability (21) can be genetically separated from the role of Sty1 in prevention of hydrogen peroxide-induced destabilization of atf1 mRNA (22). Significantly, both activities are important for adaptation and survival of S. pombe cells following exposure to oxidative stress. These data imply that distinct dose-and stimuli-specific responses can be initiated by MAPK by regulating the levels of a single transcription factor by multiple mechanisms. It is not clear whether the stability of atf mRNA is stress-regulated in other eukaryotes. However, the conserved role of bZip transcription factors as effectors of MAPK signaling pathways raises the intriguing possibility that similar mechanisms may be employed to ensure appropriate patterns of gene expression in response to different stimuli in mammals.