(2001) 20, 2336 ± 2346 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Redox control of AP-1-like factors in yeast and beyond

W Mark Toone1, Brian A Morgan2 and Nic Jones*,1

1CRC Cell Regulation Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK; 2School of Biochemistry and Genetics, The Medical School, University of Newcastle, Newcastle-upon-Tyne NE2 4HH, UK

Cells have evolved complex and ecient strategies for example, exposure to UV light, redox active chemicals dealing with variable and often-harsh environments. A or by conditions a€ecting oxygen availability, such as key aspect of these stress responses is the transcriptional hypoxia. activation of encoding defense and repair . In higher eukaryotes, has been In yeast members of the AP-1 family of proteins are implicated in the etiology of a variety of human required for the transcriptional response to oxidative conditions including a number of neurodegenerative stress. This sub-family of AP-1 (called yAP-1) proteins disorders, atherosclerosis, cancer, alcohol-induced liver are sensors of the redox-state of the cell and are damage and ischemia/reperfusion injury, as well as activated directly by oxidative stress conditions. yAP-1 being a major contributor to the ageing process proteins are bZIP-containing factors that share homol- (Halliwell et al., 1992; Finkel and Holbrook, 2000). ogy to the mammalian AP-1 factor complex and bind to However, cells also utilize ROS to their advantage. For very similar DNA sequence sites. The generation of example, in response to pathogen attack cells and the resulting potential for utilize a burst of superoxide and H2O2 to induce oxidative stress is common to all aerobically growing defensive measures (the so called hypersensitive . Furthermore, many of the features of this response) that lead to a collapse of challenged plant response appear to be evolutionarily conserved and cells and the formation of a restricted lesion (Levine et consequently the study of model organisms, such as al., 1994). Furthermore, ROS are good candidates for yeast, will have widespread utility. The important second messengers that travel to distant sites in the structural features of these factors, signaling pathways plant resulting in the acquisition of systemic immunity controlling their activity and the nature of the target to pathogen attack (Alvarez et al., 1998). Mammalian 7 genes they control will be discussed. Oncogene (2001) phagocytic cells employ an O2 ´-generating plasma 20, 2336 ± 2346. membrane NADPH-dependent oxidase capable of producing large amounts of ROS required for host Keywords: AP-1; yeast; redox-control; stress defenses against microorganisms (Segal and Shatwell, 1997). Moreover, as in plant cells, there is increasing evidence that ROS can function as second messengers in mammalian cells. Therefore, characterizing the Introduction mechanisms by which cells sense ROS, produced either as a result of environmental stress or in normal In eukaryotic cells reactive oxygen species (ROS) are physiological signaling, and discovering how that generated by the chemical reduction of oxygen by information is translated into speci®c responses, is cellular oxidases, and mono- and dioxy- fundamental to our understanding of the ability of cells genases, by exposure to UV or other environmental to survive and ¯ourish in an aerobic environment. agents and by incomplete reduction of oxygen to water in the mitochondrial respiratory chain. The major 7 ROS, superoxide anion (O2 ´), hydrogen peroxide Yeast as a model system for stress signaling 7 (H2O2), the hydroxyl radical (OH ) and singlet oxygen (O), are unstable, reactive molecules that can damage The suggestion that ROS can promote physiological lipids, proteins and DNA (Freeman and Crapo, 1982). responses by activating proteins has come from studies Since ROS are constantly being generated, all aero- initially performed on bacteria and more recently in bically growing cells maintain an assemblage of yeast and higher eukaryotes. In eukaryotic cells biochemical and enzymes that facilitate signaling proteins and transcription factors have been the breakdown of ROS and keep the cell in a state of found to respond to ROS. In particular the yeast AP-1 redox balance. Oxidative stress occurs when this (yAP-1) family of proteins function in a redox balance is upset by increases in ROS caused by, for regulated manner (reviewed in Toone and Jones, 1999). These proteins have been identi®ed in a number of fungal species and include the factors Yap1 in *Correspondence: N Jones Saccharomyces cerevisiae, Pap1 in Schizosaccharomyces Redox control of AP-1-like factors WM Toone et al 2337 pombe, Cap1 in Candida albicans and Kap1 in yAP-1 transcription factors are redox-sensitive Kluvermyces lactis (reviewed in Toone and Jones, 1999). All of these factors contain a bZIP DNA Evidence that Yap1 is regulated in a redox sensitive binding dimerization domain, similar to mammalian manner ®rst came from Kuge and colleagues (1997) cJun, which is a component of the AP-1 transcription who demonstrated that the subcellular localization of factor complex (Figure 1). Furthermore, Yap1 and Yap1 changes in response to oxidative stress. Yap1 is Pap1 can bind and activate transcription from an cytoplasmic in unstressed cells but quickly accumulates SV40-derived AP-1 site. In addition to homology in the nucleus following treatment with the oxidants within the DNA binding domain, a subset of yeast diethyl maleate or diamide. This stress-dependent AP-1 proteins share two rich domains called change in subcellular localization depends on the c- the amino terminal- and carboxyl terminal-cysteine rich CRD; when the c-CRD is removed, Yap1 becomes domains respectively (n-CRD and c-CRD; see Figure constitutively nuclear with a concomitant increase in 1); we will refer to these hereafter as yAP-1. Yap1 transcriptional activity. Furthermore, fusion of Genetic analyses indicate that these proteins, like the c-CRD to a normally nuclear Gal4 DNA binding AP-1 factors in higher eukaryotes, have roles in domain, imparts oxidative stress-dependent regulated controlling stress response genes. For example, over- nuclear localization upon the hybrid (Kuge et expression of Yap1 or Pap1 results in increased al., 1997). Similar results have been seen when the c- resistance to a variety of drugs, oxidants and CRD from Pap1 is fused to GFP and expressed in xenobiotic compounds such as cadmium while inacti- mammalian cells (Kudo et al., 1999). Thus, the c-CRD, vation of yAP-1 proteins (Yap1, Pap1, Cap1 and independent of its context in the protein, or even the Kap1) results in an oxidative stress sensitive phenotype within which it is expressed, is capable of (Toone and Jones, 1999). In agreement with these sensing oxidative stress and directing protein localiza- observed phenotypes, and as discussed below, Yap1 tion. and Pap1 are known to regulate genes encoding Clues to the possible function of the c-CRD domain energy-dependent e‚ux pumps as well as a range of came from studies in S. pombe where conditional oxidative stress response genes. in an essential protein called Crm1 suggested

Figure 1 The yAP-1 family of transcription factors. (a) The yAP-1 family of transcription factors contain three recognizable domains, the bZIP domain, important for DNA binding, and cysteine rich domains, located towards the n-terminus (n-CRD) and c- terminus (c-CRD), which are responsible for redox regulation. (b) Comparison of the sequences encompassing the likely n-CRDs (b) and the likely c-CRDs (c) in yAP-1 proteins from S. cerevisiae (Sc), S. pombe (Sp), C. albicans (Ca), K. lactis (K1), N. crassa (Nc) and A. niger (An). The cysteine residues are highlighted in bold. The positions of the n-CRDs and c-CRDs, where known, are indicated. The asterisk (*) indicates the position of stop codons. The sequences for the N. crassa Yap1 homologue came from ®le AL356172 while the A. niger sequence came from the EST ®le BE759168. The position of the NES domain is shown

Oncogene Redox control of AP-1-like factors WM Toone et al 2338 that Crm1 was a negative regulator of Pap1. In Coleman et al., 1999). These studies suggested that particular, crm1 mutants display a drug resistance H2O2 and diamide regulated Yap1 activity by di€erent phenotype similar to that seen when Pap1 is over- mechanisms. Delaunay et al. (2000), Coleman et al. expressed and, moreover, this drug resistance pheno- (1999), Kuge and colleagues, 2001 (personal commu- type depends on the presence of a functional allele of nication) and Hildalgo and colleagues, 2001 (personal pap1+ (Toda et al., 1992). Crm1 homologs have communication) have con®rmed and extended these recently been identi®ed in evolutionarily divergent ®ndings by identifying particular residues in the n- organisms including S. cerevisiae, Xenopus laevis and CRD and c-CRD that are essential for the normal mammalian cells. In each case Crm1 functions as a response of Yap1 and Pap1 to H2O2 stress (see below). nuclear export factor responsible for shuttling proteins H2O2 is a general oxidizing agent that can damage from the nucleus to the cytoplasm (reviewed in Weis, all of the major classes of macromolecules in the cell 1998). These observations suggest that the localization whilst diamide is a sulfhydryl-oxidant thought to act of Yap1 in S. cerevisiae, and Pap1 in S. pombe, are by oxidizing the intracellular pool of . An controlled through regulated nuclear export. In agree- informative way of comparing the e€ects of di€erent ment with this hypothesis GFP-yAP-1 fusion proteins, types of oxidative stress on the cell is to employ whole in S. cerevisiae and S. pombe, are constitutively nuclear genome expressing pro®ling. Gasch et al. (2000), used in crm1 mutant strains (Kuge et al., 1998; Yan et al., expression pro®ling in S. cerevisiae, to show that 1998; Toone et al., 1998). Yan et al. (1998) have also diamide treatment induces a range of e€ects including shown that the Yap1 c-CRD contains a region rich in protein unfolding, due to oxidation of protein hydrophobic amino acid residues that resembles a sulfhydryl groups, oxidative stress, resulting from the nuclear export sequence (NES). Point mutations within sulfhydryl modi®cation and defects in secretion, and this region, designed to disrupt the putative NES, cell wall formation, due to improper disul®de bond block interaction with Crm1 and result in a constitu- formation in the ER. Thus, the mechanisms by which tively nuclear protein. The homologous region of Pap1 diamide treatment regulates yAP-1 activity are likely to has also been extensively mutated with similar results be complex. (Kudo et al., 1999). Interestingly, the interaction The sulfhydryl group of a cysteine residue (-SH) can between the Yap1 c-CRD and Crm1 appears to be be oxidized to form sulfenic acid (-SOH), sul®nic acid redox sensitive: co-precipitation of Crm1 with GST- (SSG), sulfonic acid (-SO3H) or S-glutathionylated Yap1 is signi®cantly enhanced by performing the assay (-SSG) derivatives. Two or more cysteine residues under reducing conditions and a two-hybrid interaction within the same protein can form intramolecular between Yap1 and Crm1 is inhibited by adding disul®de bonds or intermolecular disul®de linkages diamide to the growth media (Yan et al., 1998). Thus, can be formed between proteins. Furthermore, ROS the c-CRD of Yap1 behaves like a redox sensitive can also a€ect other amino acid residues such as nuclear export sequence. dityrosine formation by H2O2/-dependent How is the activity of the c-CRD regulated by reactions (reviewed in Thannickal and Fanburg, alterations in the redox state of the cell? One likely 2000). Recent studies have revealed that intramolecular possibility is that oxidation of conserved cysteine disul®de bond formation is, in fact, a critical facet of residues within the c-CRD masks the NES thereby Yap1 regulation in response to H2O2. Delaunay et al. blocking its interaction with Crm1 and subsequent (2000) found that H2O2, and organic peroxide export from the nucleus (Figure 2). In agreement with treatment results in the formation of a faster migrating, this hypothesis the Yap1-Crm1 interaction becomes oxidized form of the Yap1 protein, on a non-reducing insensitive to diamide treatment when all three cysteine poly-acrylamide gel. Treatment of these oxidized cell residues in the c-CRD are mutated (Yan et al., 1998; extracts with the reducing agent DTT caused Yap1 to Kuge et al., 1997). Since cysteine residues within the migrate at the same rate as the normally non-oxidized n-CRD might also be involved in forming intramole- form of the protein. In addition, the appearance of this cular disul®de bonds with the c-CRD, Yan et al. (1998) faster migrating species mirrors the kinetics of Yap1 mutated all three n-CRD in combination with activation; as measured by the nuclear localization of all but one of the c-CRD cysteines and still observed a Yap1 and the induction of Yap1-regulated redox-sensitive interaction with Crm1. Thus, it was expression. Interestingly, this redox-dependent migra- concluded that it was unlikely that an intramolecular tional shift is not observed following treatment of cells disul®de bond was blocking the interaction between the with varying concentrations of diamide. Yap1 c-CRD and Crm1. Mutational analysis of Yap1 indicates that one It was possible that the ability of yAP-1 proteins to cysteine from the n-CRD (C303) and one cysteine respond to di€erent oxidants relied on a single from the c-CRD (C598) are absolutely required for the mechanism of redox sensing. However, recent data formation of this faster migrating form of Yap1. show that speci®c mutations in Yap1 behave di€erently Moreover, Delaunay et al. (2000) found that other depending on the nature of the oxidant used. In cysteines residues, in particular C310 and C629, are particular, deletions and point mutations of Yap1 in, required for the formation of a fully oxidized form of and around, the n-CRD result in a protein that confers Yap1. The inability of these mutant proteins to form a greater than normal resistance to diamide but fails to faster migrating species correlates with their inability to confer resistance to H2O2 (Wemmie et al., 1997; accumulate in the nucleus following H2O2 stress or to

Oncogene Redox control of AP-1-like factors WM Toone et al 2339

Figure 2 yAP-1 transcription factors are controlled by oxidative stress-dependent changes in subcellular localization. Under non- stressed conditions yAP-1 factors are continually imported into the nucleus and are immediately exported in a Crm1-dependent manner. Under conditions of oxidative stress the interaction between yAP-1 and Crm1 is blocked and the accumulates in the nucleus. This redox inhibition of export derives from two cysteine rich domains that are conserved amongst yAP- 1 family members. Disruption of these domains can lead to constitutive nuclear localization

activate the expression of an H2O2-responsive gene. result in nuclear localization of Yap1 under non- This data strongly suggests that an intramolecular stressed conditions (Izawa et al., 1999; Carmel-Harel disul®de bond forms in response to H2O2 although it and Storz, 2000; Delaunay et al., 2000). Furthermore, remains to be directly demonstrated. this increased nuclear accumulation of Yap1 correlates S Kuge and colleagues, 2001 (personal communica- with an apparent increase in oxidation of cysteines in tion) have used mass spectrometric analysis to provide the n-CRD and c-CRD (Delaunay et al., 2000). The physical evidence for the involvement of disul®de glutathione system does not appear to have a role in linkages in the regulation of Yap1. They show that a H2O2-dependent Yap1 regulation since mutations in disul®de bond between Cys598-Cys620 forms within the glutathione synthase gene (GSH1), or in genes 1 min of H2O2 treatment. In addition, they observe a encoding (GRX1, GRX2), do not a€ect second disul®de bridge, between Cys303 and Cys598/ Yap1 localization or Yap1-regulated Cys629, which may be required for prolonged Yap1 (Izawa et al., 1999). However, inactivation of Yap1 nuclear localization following H2O2 exposure. In following release from diamide-induced stress appears response to diamide treatment all three potential normal in thioredoxin de®cient cells suggesting that a disul®de bonds can form in the Yap1 c-CRD. (S second reduction system may be active in reversing the Kuge, 2001, personal communication). Thus, di€erent e€ects of diamide oxidation (S Kuge, 2001, personal Yap1 modi®cations may be needed for the temporal communication). Oxidative stress causes the Yap1- and oxidant speci®c regulation of Yap1. dependent induction of genes encoding thioredoxin (TRX2), (TRR1) and thioredoxin peroxidase (TSA1); suggesting that the nuclear locali- Thioredoxin dependent deactivation of yAP-1 zation of Yap1 is regulated by a negative feedback loop (Kuge and Jones, 1994; Morgan et al., 1997; Lee Release from oxidative stress results in rapid-re- et al., 1999) (Figure 3). Thus, following H2O2 stress, localization of Yap1 from the nucleus back to the Yap1 is oxidized resulting in the nuclear localization of cytoplasm implying that yAP-1 proteins are transiently the protein and the subsequent induction of the activated by oxidative stress and can revert to an thioredoxin system genes. As the levels of H2O2 inactive state once the stress has dissipated. (S Kuge, subside, Yap1 is no longer subject to oxidation and 2001, personal communication). Recent evidence in- any remaining disul®des within the existing Yap1 dicates that deactivation (reduction) of Yap1 following protein are reduced by thioredoxin. This results in oxidative stress is mediated by a thioredoxin-based cytoplasmic localization of Yap1 and loss of Yap1- system. Thioredoxin is a small protein containing dependent gene expression. In support of this model, it redox active cysteines within its active site. Mutations has been demonstrated that the oxidation of Yap1 can that a€ect thioredoxin or thioredoxin reductase activity be reversed in crude lysates by the addition of

Oncogene Redox control of AP-1-like factors WM Toone et al 2340 thioredoxin, thioredoxin reductase and NADPH (De- ability of yAP-1 to activate gene expression from launay et al., 2000). certain promoters. The role of thioredoxin peroxidase (Tsa1) in the regulation of Yap1 is less clear. Unlike deletion of the thioredoxin/thioredoxin reductase encoding genes, ROS responsive signaling pathways in yeast studies have shown that deletion of the TSA1 gene has no obvious a€ect on the nuclear localization of Although the evidence suggests that yAP-1 proteins Yap1 (Inoue et al., 1999; Delaunay et al., 2000; Ross can directly sense changes in the cellular redox state, a et al., 2000) or on the basal level expression of Yap1 number of observations suggest that stress-induced target genes, TRX2 and TRR1 (Delaunay et al., 2000; signaling pathways also participate in controlling the Ross et al., 2000); although there is one con¯icting oxidative stress response. Yap1 is phosphorylated when report that suggests an increase in basal level it enters the nucleus, although the nature of the expression of Yap1-dependent genes in a tsa1 strain and the signi®cance of this is (Inoue et al., 1999). In contrast, in response to very unknown (Delaunay et al., 2000). In S. pombe, the low levels of H2O2 (0.1 mM), induction of TRX2 and Sty1 stress-activated protein kinase pathway is required TRR1 is much reduced in a tsa1 strain while over- for the H2O2-dependent accumulation of Pap1 in the expression of TSA1 results in faster than normal nucleus. However, there is no evidence, as yet, to show induction of TRX2 and TRR1 (Ross et al., 2000). that Pap1 is a direct target of Sty1 (Toone et al., 1998). Furthermore, in S. pombe, Tsa1 appears to be Sty1 is activated by a range of adverse stimuli in required for the induction of Pap1-regulated gene addition to H2O2 (reviewed in Millar, 1999). Moreover, + expression in response to H2O2 (Findlay et al., 2001, inactivation of sty1 results in hyper-sensitivity to a unpublished observation). Thus, thioredoxin perox- variety of stresses such as heavy metals, UV and other idase may also have a role in controlling yAP-1 DNA damaging agents that might act via ROS activity (Figure 3). intermediates (Millar et al., 1995; Shiozaki and Russell, It is important to mention that certain mutations 1995; Degols et al., 1996; Degols and Russell, 1997; within Yap1 can lead to constitutive nuclear localiza- Shieh et al., 1997). The components of the Sty1 tion of Yap1 without activation of Yap1-target genes, pathway are homologous to in the S. cerevisiae like TRX2 (Coleman et al., 1999). Similarily, over- Hog1 osmosensing MAPK pathway and to the expression of Pap1 in S. pombe results in the induction mammalian JNK and p38 stress-activated protein of only a subset of known Pap1 target genes (WM kinase cascades (reviewed in Toone and Jones, 1998; Toone, 2001, unpublished observation). In contrast, Millar, 1999). Sty1 (also known as Phh1 and Spc1) is inactivation of the thioredoxin system in S. cerevisiae activated by phosphorylation on conserved (T-171 and results in nuclear localization of Yap1 and the Y-173) residues by the MAPKK, Wis1. In turn Wis1 is induction of Yap1-dependent target genes, including activated by stress-induced phosphorylation of the TRX2. Thus, oxidation of Yap1 may be required not kinase by the MAPKKKs Wak1 (also known as only for yAP-1 nuclear localization but also for the Wis4 and Wik1) (Samejima et al., 1997; Shieh et al.,

Figure 3 A model for redox regulation of Yap1 activity. In response to H2O2 Yap1 becomes oxidized. This results in Yap1 nuclear localization and the induction of the expression of speci®c genes which includes the TRX2 gene, encoding thioredoxin. Following the elimination of H2O2 reduced Yap1 is no longer oxidized and free thioredoxin reduces the remaining oxidized Yap1 deactivating the protein

Oncogene Redox control of AP-1-like factors WM Toone et al 2341 1997; Shiozaki et al., 1997) and Win1 (Samejima et al., Recent studies have revealed that the H2O2-depen- 1998). dent activation of the Sty1 pathway also involves a In addition to Pap1, Sty1 regulates a second bZIP `two-component'-related response regulator protein protein called Atf1 in response to stress. Atf1 is a called Mcs4. Mcs4 binds to the MAPKKK, Wak1, direct target of Sty1 phosphorylation and is also and controls activation of Sty1 in response to H2O2 required to anchor Sty1 in the nucleus following stress (Cottarel, 1997; Shieh et al., 1997; Nguyen et al., 2000; (Wilkinson et al., 1996; Shiozaki and Russell, 1996; Buck et al., 2001). Mcs4 is controlled by two histidine Gaits et al., 1998). Both Sty1 and Atf1 are necessary (sensor) kinases, Mak2 and Mak3, that sense peroxide for the induction of gene expression in response to stress and initiate a multi-step phosphorelay (Buck et oxidative stress, nutrient deprivation, osmotic stress al., 2001). This phosphorelay connects kinase and and heat stress (Wilkinson et al., 1996; Shiozaki and receiver domains within Mak2 and Mak3 with the Russell, 1996). The di€ering roles played by Pap1 and intermediary protein Mpr1, and the receiver domain on Atf1 in controlling the S. pombe oxidative stress Mcs4 itself. The phosphorylation status of Mcs4 then response are discussed below. The amino acid sequence regulates the activity of Wak1 (Figure 4). This pathway of Atf1 shows good similarity to the DNA binding is most similar to the `two-component'-like pathway dimerization domain of mammalian ATF2 (Takeda et that lies upstream of the osmosensing Hog1 MAPK al., 1995). This homology, and the homology between cascade in S. cerevisiae (Posas et al., 1996; Posas and Pap1 and mammalian cJun, are intriguing given that Saito, 1998; Buck et al., 2001). Mak2 and Mak3 are both cJun and ATF2 are also targets for the stress- likely to be direct sensors of H2O2. Although the activated protein kinases p38 and JNK in mammalian mechanism by which they sense H2O2 is not known, cells. Mak2 and Mak3 contain one repeat of a PAS/PAC domain and a GAF domain adjacent to their histidine kinase domain similar to the redox sensing protein Two-component signaling pathways control the oxidative FixL from Rhizobium meliloti (Buck et al., 2001). stress response in yeast Interestingly, Mak2 and Mak3 are only important for activation of Sty1 in response to low levels of H2O2 Two-component systems are the exposure since acute H2O2 exposure activates Sty1 in a major signaling pathways by which bacteria respond to Wak1- and Win1-dependent manner but independent environmental stimuli. In recent years a number of of the histidine kinases (Quinn et al., 2001, unpublished two-component-like systems have also been identi®ed observations). In addition to the peroxide sensors, in eukaryotes, although not to date in animal cells. The Mak2 and Mak3, there is a third histidine kinase in S. archetypal two-component pathway consists of a pombe called Mak1, whose role in the oxidative stress membrane-located histidine kinase, which detects a response remains unclear. However, there is evidence speci®c stimulus, homodimerizes and phosphorylates that Mak1 plays an antagonistic role to that of Mak2 the partner protein on a histidine residue (Stock et al., and Mak3 in modulating the response of Sty1 to H2O2 1989). This phosphate is then transferred to an aspartic (Quinn et al., 2001, unpublished observations). It is acid residue within the receiver domain of the response also possible that one or more of these histidine kinases regulator that a€ects the appropriate response. control the activity of the Prr1 response regulator. Two-component response regulator proteins, Skn7 and Prr1, have been found to function in collaboration with Yap1 in S. cerevisiae, and Pap1 in S. pombe, Transcriptional response to oxidative stress in yeast respectively (Morgan et al., 1997; Ohmiya et al., 1999; Quinn, 2001, unpublished data). Skn7 and Prr1 contain The function of these stress response pathways is the homologous receiver domains as well as putative DNA transcriptional induction of stress response genes that binding domains similar to eukaryotic heat shock encode proteins that protect the cell from the factor (HSF)- suggesting that they are, in fact, deleterious consequences of exposure to ROS. Down- transcription factors. Inactivation of Skn7 or Prr1 stream targets of yAP-1 transcription factors have been results in a stress-sensitive phenotype similar to that identi®ed in a number of organisms. In S. cerevisiae,a seen by inactivating the corresponding yAP-1 factor. combination of whole genome expression pro®ling and Furthermore, epistasis analysis indicates that in S. proteomics is providing us with a global picture of the cerevisiae and S. pombe, these factors function in the gene expression changes associated with an increasing same pathways as Yap1 and Pap1, respectively number of stress responses. Godon et al. (1998) used a (Morgan et al., 1997; Quinn et al., 2001, unpublished comparative two-dimensional gel electrophoresis ap- data). In agreement with these observations both Skn7 proach to identify proteins whose expression pattern and Prr1 are required for the H2O2-dependent changed in response to H2O2 treatment. They found expression of Yap1- and Pap1-dependent genes and, that the synthesis of 115 proteins was stimulated by in the case of S. cerevisiae, Skn7 has been found to H2O2 treatment, while the synthesis of 52 other bind adjacent to Yap1 on the TRX2 promoter proteins was repressed. Stress-induced proteins were (Morgan et al., 1997). However, the mechanism by found to be synthesized at di€erent rates following which these proteins modulate yAP-1-dependent gene H2O2 exposure, resulting in the identi®cation of at least expression is unknown. three kinetic classes with synthesis peaking at 15, 20

Oncogene Redox control of AP-1-like factors WM Toone et al 2342

Figure 4 Pathways controlling the S. pombe response to H2O2 stress. These include the Mak1 and Mak2/3 histidine (sensor) kinases, the Mcs4 response regulator and the Sty1 stress-activated protein kinase pathway as well as the transcription factors Pap1, Prr1 and Atf1, which are regulated by this signaling pathway(s)

and 45 min. Moreover, the synthesis of individual methodology, have identi®ed the complete set of genes proteins responded di€erently to increasing doses of that are induced by a variety of environmental stimuli, H2O2: for example, synthesis of heat shock proteins including several di€erent kinds of oxidative stress. increased with increasing doses of H2O2: whereas the They demonstrated that the transcriptional response to synthesis of thioredoxin reductase was independent of di€erent stresses involved the induction or repression the level of H2O2 exposure. Thus, it appears that of a common set of genes (*900 in total), which they distinct regulatory mechanisms are contributing to the called the environmental stress response (ESR), as well H2O2 response in S. cerevisiae. The role of Yap1 in as changes in the expression of a set of genes speci®c to controlling the synthesis of these proteins has not been the stress imposed. Interestingly, the environmental assessed. However, a similar approach has recently stress response, although common to all stresses, was been used to evaluate the role of Yap1 and Skn7 in initiated by di€erent factors depending on the nature of controlling the response to cadmium. Vido et al. (2001) the stress stimuli. Thus, genes such as TRX2 encoding found that 57 proteins were induced in response to thioredoxin, were induced following H2O2 stress in a cadmium while 43 were repressed. The induction of Yap1-dependent manner but following heat shock their glutathione synthase (Gsh1) and a glutathione-cad- induction required another transcription factor com- mium transporter (Ycf1), as well as nine other proteins, plex called Msn2/4. By comparing the transcript including thioredoxin and thioredoxin reductase, were pro®les of wt and yap17 strains, Yap1 was shown to found to require Yap1. Interestingly, in response to control at least 70 genes in response to H2O2 stress. cadmium, Skn7 appears to be acting as a repressor Activation of the H2O2 stress response in S. pombe since inactivation of SKN7 results in a cadmium relies on a co-ordinated response involving Pap1, Prr1 resistant phenotype and the induction of some of the and Atf1, in conjunction with the Sty1 pathway. same genes which occurs when over-expressing Yap1. Interestingly, these factors apparently control di€erent Two-dimensional gel based techniques, although target genes depending on the level of oxidative stress powerful, only analyse approximately 20% of the imposed. Thus, Pap1 and Prr1 primarily control the expressed proteins. Recent studies utilizing DNA response to low levels of H2O2 stress (adaptive microarrays have found that over-expression of Yap1, response) while Atf1 and Sty1 induce expression of in the absence of stress, results in the induction of 17 target genes in response to high levels of H2O2 stress genes, encoding a high proportion of oxidoreductases (acute response) (Quinn et al., 2001, unpublished (DeRisi et al., 1997). This approach, however, is likely observations). In agreement with these observations to underestimate Yap1-dependent target genes that Sty1, and its target Atf1, are only fully activated require stress-dependent activation of Yap1 ancillary (phosphorylated) by treatment with high concentra- factors, such as Skn7. Gasch et al. (2000), using similar tions of H2O2. In contrast, Pap1 localizes to the

Oncogene Redox control of AP-1-like factors WM Toone et al 2343 nucleus in response to low, but not high levels of H2O2. Hinnebusch, 1988). Recent evidence indicates that Consequently, Pap1 only activates target genes in Gcn4 may have a role in controlling other forms of response to low levels of H2O2. The mechanism by stress. Pascual-Ahuir et al. (2001) have shown that in which this occurs is not known but may indicate that response to osmotic stress the HAL1 gene is controlled the Pap1 c-CRD is sensitive to speci®c levels of H2O2 through the combined actions of Gcn4, the Hog1 MAP (Quinn et al., 2001, unpublished observations). Pap1 is kinase and an Atf1-like transcription factor called required for the expression of genes involved in the Sko1- a situation reminiscent of the interactions synthesis and maintenance of glutathione (gsh1+, gcs1+ between the Pap1, Sty1-Atf1 pathways in S. pombe. and pgr1+), the thioredoxin system (trx2+, trr1+ and Gcn4 is also activated in response to UV irradiation tpx1+), catalase (ctt1+), ABC transporters (pmd1+ and via a pathway that includes Rpn11, a regulatory hba2+) and genes involved in iron transport (®o1+) subunit of the 26S proteosome (Stitzel et al., 2001). (reviewed in Toone and Jones, 1999). With the Rpn11 is related to Pad1 in S. pombe, which has been imminent completion of the S. pombe genome sequen- identi®ed as a regulator of Pap1 activity (Shimanuki et cing project it will soon be possible to identify al., 1995; Spataro et al., 1997). Although it is not clear complete sets of stress response genes, allowing how proteosome subunits control yAP-1 activity this comparisons of these with response genes in S. connection may be conserved among other yAP-1 cerevisiae, and to determine the role of Pap1, Prr1 family members. and Atf1 in their expression. A further level of conservation exists between yAP-1 and two-component response regulators. Analysis of the available databases has revealed potential Skn7/ Evolutionary conservation of fungal AP-1-like proteins Prr1 homologs in C. albicans and Aspergillus nidulans (B Morgan, 2001, unpublished observations). Thus, it The S. cerevisiae genome contains eight potential AP-1- is possible that co-operation between yAP-1 and Skn7/ like proteins based on homology within the putative Prr1 homologs is a general property of the fungal bZIP domains. However, oxidative stress-dependent oxidative stress response. Moreover, given the role of changes in subcellular protein localization has only these proteins in mediating the oxidative stress been described for Yap1 (Fernandes et al., 1997). response and in maintaining drug resistance, Yap1 Yap2, which like Yap1, when over-expressed results in and/or Skn7/Prr1 proteins might represent a possible increased resistance to certain cytotoxic agents, also target for developing new antifungal therapies. contains a c-CRD but has no obvious n-CRD (Figure 1). Upon further sequence analysis we have found that Acr1, or Yap8, which was originally identi®ed as a ROS responsive factors in other systems factor that can confer resistance to arsenic compounds, contains an n-CRD as well as conserved residues in a Although a comprehensive summary of oxidant putative c-CRD (Bobrowicz et al., 1997) (Figure 1). sensitive factors and pathways is beyond the scope of Hence it will be interesting to study Yap2 and Yap8 this review, a precis of ROS responsive signaling for stress-dependent changes in localization and to proteins and transcription factors in other organisms investigate the role of the conserved cysteine residues in is given below. In Escherichia coli a transcription factor their regulation. called OxyR is required for the induction of H2O2 yAP1-like proteins containing conserved c-CRDs response genes (Storz et al., 1990). Moreover, like and n-CRDs have also been described in S. pombe, Yap1, H2O2 activates OxyR by formation of intramo- C. albicans and K. lactis (reviewed in Toone and Jones, lecular disul®de linkages between Cys-199 and Cys-208. 1999) Furthermore, C. albicans Cap1 has recently been Disul®de bond formation presumably causes a con- shown to localize to the nucleus in an oxidant and c- formational change in OxyR that results in enhanced CRD-dependent manner (Zhang et al., 2000). Database DNA binding and the activation of H2O2 response searches show that yAP-1 homologs also exist in genes (Zheng et al., 1998; Zheng and Storz, 2000). Aspergillus niger and Neurospora crassa (Figure 1). Interestingly, OxyR, like Yap1, induces the expression Homologous domains have not been described in of target genes whose products facilitate reduction and animal or plant cell proteins and therefore it is possible deactivation of the transcription factor (reviewed in that this branch of the AP-1 family of proteins, along Carmel-Harel and Storz, 2000). A second factor in E. with their unique mode of regulation, is fungal speci®c. coli called SoxR, controls the response to superoxide However, it is worth reiterating that the c-CRD generating redox-cycling agents such as paraquat and domain can regulate protein localization in mammalian menadione. SoxR forms a homodimer containing two cells (Kudo et al., 1999). Hence, it is not unreasonable redox-active iron- [2Fe-2S] centers that are 7 to propose that similar mechanisms of redox regulation sensitive to oxidation by O2 ´. Oxidation of the iron- of protein activity and nuclear localization operate in sulfur centers activates SoxR resulting in the induction higher eukaryotes. of genes that combat the toxic e€ects of superoxide Gcn4 is another AP-1-like protein in S. cerevisiae, (Ding et al., 1996; Gaudu and Weiss, 1996; Hidalgo et which was originally identi®ed as a transcription factor al., 1997). involved in controlling amino acid biosynthetic genes Other redox-sensitive factors have been described in in response to amino acid starvation (reviewed in eubacteria. FNR, a transcription factor in E. coli

Oncogene Redox control of AP-1-like factors WM Toone et al 2344 responsible for the regulation of a large number of ASK1 activating the JNK and p38 pathways (Saitoh et target genes following the aerobic to anaerobic shift, is al., 1998; Liu et al., 2000). Thus, there are at least two also regulated by reversible oxidation of iron-sulfur mechanisms by which mammalian AP-1 can respond to centers. FixL, a histidine kinase that controls nitrogen redox controls. ®xation genes in Rhizobium meliloti, is negatively regulated by oxygen through a heme-sensing PAS domain. NifL, a ¯avoprotein in proteobacteria, utilizes Summary FAD as a redox sensitive co-factor and in its oxidized state inhibits NifA, a transcriptional activator of Oxidative stress, resulting from excessive production of nitrogen ®xation genes. The function and proposed ROS, can result in damage to all the major mechanism of action of these and other bacterial intracellular macromolecules and, in humans, may redox-sensitive regulatory proteins was reviewed re- cause or provoke speci®c pathological conditions. cently by Bauer et al. (1999). Consequently, aerobically growing cells have devised In animal cells a variety of cytokines and growth mechanisms for sensing oxidative stress and inducing factors have been reported to generate intracellular the expression of genes that encode proteins involved ROS in nonphagocytic cells (reviewed in Thannickal in increasing the reducing potential of the cell and in and Fanburg, 2000). Furthermore, a number of repairing ROS-induced damage. The genetic tractabil- mammalian transcription factors bind DNA in a redox ity of yeast provides an ideal model for characterizing dependent manner. Regulation of these factors, which the oxidative stress response in eukaryotic cells. include NF1 (Bandyopadhyay and Gronostajski, 1994), In yeast a conserved family of AP-1-like factors are Sp-1 (Wu et al., 1996), c-Mby (Myrset et al., 1993), p53 critical for the response to a variety of oxidants. (Rainwater et al., 1995), erg-1 (Huang and Adamson, Recent evidence indicates that these factors are 1993) and NF-kB (Schreck et al., 1992), in most cases regulated by oxidation of cysteine residues located in is thought to depend on the presence of critical cysteine conserved n- and c-terminal domains. Furthermore, in residues. In an interesting parallel with yeast AP-1 the Yap1 transcription factor of the S. cerevisiae, redox dependent processes also regulate the mamma- di€erent cysteine residues are required for Yap1 to lian AP-1 complex. Mammalian AP-1 consists of respond to di€erent oxidants. In response to H2O2 homo- and heterodimers of the Jun, Fos or ATF disul®de bonds form between the n- and c-terminal family members. AP-1 DNA binding activity is a€ected domains, which mask a nuclear export sequence, by reversible oxidation of a single cysteine residue allowing the transcription factor to accumulate in the within the DNA binding domain of fos and jun nucleus. Based on , similar controls proteins. Reduction of this residue stimulates DNA are likely to operate to control yAP-1 factors in other binding in vitro and treatment of cells with various yeasts. Inactivation of Yap1 utilizes the reducing antioxidants stimulates the DNA-binding and transac- potential of thioredoxin operating in an apparent tivation abilitities of AP-1 (Abate et al., 1990). In vivo feedback loop. Thus, yAP-1 factors join transcription the redox state of the transcription factor may be factors from E. coli, such as SoxR and OxyR, which regulated by thioredoxin and an AP endonuclease are activated by oxidation of critical cysteine residues. called Ref1. In response to certain stimuli, including Analysis of these proteins will provide a paradigm for oxidative stress, thioredoxin translocates to the nucleus mechanisms by which proteins in higher eukaryotes facilitating its interaction with Ref-1 and the Ref-1 sense and respond to changes in the redox state of the mediated reduction of the critical AP-1 cysteine residue cell. (Hirota et al., 1997). In higher eukaryotes and in the ®ssion yeast, S. Mammalian AP-1, like Pap1 in S. pombe, is also pombe, ROS induce a stress-activated protein kinase regulated by a redox sensitive MAP kinase pathway. pathway required for AP-1 activity. The S. pombe Sty1 Transcriptional activation of the c-Jun protein is pathway senses H2O2 by a redox sensitive two- strongly enhanced by phosphorylation of two component multistep phosphorelay. Additional two- residues within its activation domain by the c-Jun-N- component response regulators, exempli®ed by Skn7 terminal kinase (JNK) (reviewed in Tibbles and and Prr1, are conserved in other fungal species, and Woodgett, 1999). JNK and p38 kinases constitute the function as transcription factors operating in conjunc- two major families of stress-activated MAP kinases in tion with yAP-1. Future studies, characterizing the mammalian cells. These pathways are homologous to mechanisms by which these pathways respond to the Sty1 pathway in S. pombe and, like Sty1, are di€erent kinds of oxidative stress and how this activated by range of environmental stresses including information is conveyed to the transcription machin- osmotic stress, heat stress, DNA damage and changes ery, will contribute to our understanding of ROS in the cellular redox potential (reviewed in Tibbles and responsive signaling pathways in other systems. Woodgett, 1999). It is not precisely known how JNK is activated by changes in the redox status of the cell. However, the ASK1 MAPKKK, which lies upstream Acknowledgments of both JNK and p38, is controlled via an inhibitory We would like to thank Rachel J Morgan, Janet Quinn and interaction with thioredoxin. Treatment with either Ann Marie Flenniken for help in making the ®gures and TNF-a or ROS causes thioredoxin to dissociate from critically reading the manuscript.

Oncogene Redox control of AP-1-like factors WM Toone et al 2345 References

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