In this study, we showed that the lack of the two cytoplasmic thioredoxins impairs the nuclear accumulation of two transcriptional regulators, Msn2/4 and Maf1, specifically under H2O2 treatment. The data indicate that during the response to H2O2, the modulation of PKA activity does not have a prevailing role in controlling the nuclear redistribution of both transcriptional effectors. We also show that the presence of the cytoplasmic thioredoxins is required for Maf1 PP2A-dependent dephosphorylation, leading to its nuclear accumulation, whereas a different mechanism seems to apply to Msn2, which still responds to H2O2 in the absence of PP2A activity.
Under oxidative stress conditions, cell viability requires the transcriptional activation of many genes that have protective roles. This includes both specific oxidative stress genes and a common set of genes involved in the general response to stress. The latter group is part of the ESR cluster and is mainly regulated by the Msn2/4 transcription factors. Our observations provide several lines of evidence highlighting the role of the yeast cytoplasmic thioredoxins in the activation of the general response to H2O2. We found that in a yeast strain lacking the two genes encoding the cytoplasmic thioredoxins (trx1Δ and trx2Δ), the transcriptional induction of Msn2/4 target genes and the increased neosynthesis of the corresponding proteins were nearly abolished (Fig. ). The specific requirement of cytoplasmic thioredoxins for signaling the presence of H2O2 was confirmed by monitoring the subcellular localization of Msn2/4 (Fig. ), expressed as GFP fusion proteins under the control of the strong ADH1 promoter, and Maf1 (Fig. ), expressed at its native chromosomal locus.
It is noticeable that in contrast to other stress responses, the nuclear localization of Msn2 and Maf1 occurred in only a fraction of the population. The limited proportion of apparently responsive cells could reflect either that the H2
signaling pathway is less efficient than others in the general response to stress or that the cells display different sensitivities to this environmental condition. It is also possible that this heterogeneity reflects the dynamic behavior of the transcription factors, as recently reported for Msn2 (14
). Indeed, upon particular stress conditions, Msn2 oscillates between the cytoplasm and the nucleus. Thus, when the population is fixed by formaldehyde for observation, it is expected that Msn2 will accumulate in the nucleus in only a portion of the cells.
One of the main questions raised by the thioredoxin-dependent H2
signaling is how the H2
signal goes to and from thioredoxins. Our Msn2-GFP experiments with different thioredoxin pathway mutants provide some interesting clues about the initial mechanisms. The amount of cells displaying a nuclear accumulation of Msn2-GFP in response to H2
treatment is higher for a trr1
Δ strain than for a WT strain (Fig. ). On the contrary, we observed a very low proportion of cells exhibiting a nuclear GFP staining in a tsa1
Δ strain. In accordance with the role of peroxidase reduction through the thioredoxin pathway (Fig. ), a trr1
Δ strain accumulates oxidized thioredoxins in the presence of H2
), whereas thioredoxins are likely to be less oxidized in a tsa1
Δ strain under the same conditions. Based on these results, we suggest a model in which the thioredoxins, in their oxidized form, are essential for signaling the presence of H2
to the effectors of the general stress response (Fig. ). In this working model, cytoplasmic thioredoxins, which are involved in H2
detoxification, are also required for driving the general response to stress in the presence of H2
. Thus, the global yeast response to stress induced by H2
is essentially activated by two highly sensitive peroxidases, Orp1/Gpx3 and Tsa1, directly oxidized by H2
. Orp1 activates the Yap1 transcription factor through a direct redox interaction, leading to the transcription of genes specifically dedicated to the H2
stress response (43
). Similarly, through a direct redox interaction, Tsa1 activates the cytoplasmic thioredoxins (Fig. ), which are required to set up the general response to stress. This response is characterized by the repression of growth-related processes and the induction of cellular protection mechanisms, as illustrated respectively by the nuclear accumulation of Maf1 and Msn2/4. How the H2
signal is transmitted from the oxidized thioredoxins to the transcriptional effectors remains to be determined.
FIG. 6. A working model of the thioredoxin signaling pathway. Accordingly to a model proposed by Ross and colleagues (38), in the course of H2O2 detoxification by the thioredoxin peroxidase Tsa1, the cytoplasmic thioredoxin Trx2 is oxidized. Our results demonstrate (more ...)
Under optimal growth conditions, Msn2 and Maf1 are phosphorylated in vivo, and it is now well established that their nuclear accumulation correlates tightly with modifications of their phosphorylation states. Our observations strongly suggest that at least in the case of Maf1, the H2
redox signal leads to the modification of the phosphorylation/dephosphorylation ratio. It could be speculated that thioredoxins would impact the cAMP/PKA pathway, inducing Msn2/4 and Maf1 nuclear accumulation through a decrease in PKA activity. Indeed, independent studies clearly established a link between the inhibition of PKA activity and the nuclear localization of Msn2/4 (2
) and Maf1 (for a review, see reference 48
). Furthermore, Maf1 and Msn2 are substrates of PKA (4
). In this framework, our results on PKA activity during the H2
response are striking. We observed that addition of cAMP in the culture medium of pde2
Δ cells had no impact on the nuclear accumulation of Msn2-GFP and Maf1 during H2
treatment (Fig. ). In addition, the phosphorylation level of the PKA-consensus sites increased significantly when monitored on immunoprecipitated Maf1 after 30 min of H2
treatment (Fig. ). From these results, we conclude that the ESR response to H2
is not mediated by an inhibition of PKA activity, highlighting the particular regulatory features of the response to H2
compared to other stress conditions.
Our observations showing the rapid accumulation of nuclear Maf1 (Fig. ) and a hypophosphorylated form of Maf1 (Fig. ) while PKA activity remained elevated suggest the involvement of another kinase. We thus propose that through thioredoxins, the presence of H2
leads to the inhibition of at least one other, still-unknown protein kinase activity. Interestingly, upon the transition from glucose to a nonfermentable carbon source, Ciesla and coworkers have also implicated another uncharacterized protein kinase activity, since an altered level of PKA activity affects neither the pattern of Maf1 phosphorylation nor its nuclear accumulation (6
An alternative, and nonexclusive, hypothesis would be that H2
leads to the activation of a phosphatase activity. Indeed, the dephosphorylation step is the crucial event for the nuclear accumulation of these factors. In the case of Msn2, it has been shown that different phosphatase activities control its nuclear accumulation (24
). In particular, upon glucose depletion, the protein phosphatase 1 (PP1) is the direct antagonist of PKA-dependent phosphorylation (9
), whereas the PP2A activity was also implicated in the light-induced oscillation of Msn2 (14
In this report we focus on the Maf1 negative regulator, for which nuclear accumulation depends upon PP2A phosphatase activity under rapamycin treatment (33
). We monitored a clear correlation between Maf1 nuclear accumulation and its dephosphorylation: in the absence of cytoplasmic thioredoxins, Maf1 remains cytoplasmic and its dephosphorylation is impaired. Furthermore, we identify PP2A as the phosphatase responsible for Maf1 dephosphorylation in the presence of H2
. Thus, the PP2A phosphatase appears as a key player in the control of Maf1 nuclear import, since its activity is required in response to rapamycin (33
), NaCl, and H2
(Fig. ). In this respect, our study describes a major difference in the regulation of Msn2 and Maf1 nuclear accumulation, since the H2
response of Msn2 is not affected by the lack of the PP2A phosphatase. Since Msn2 nuclear accumulation very likely requires a dephosphorylation step, this means that an additional phosphatase activity remains to be identified. Thus, the scheme in Fig. indicates that parts of the H2
signaling pathway share a common route through the thioredoxin system and then diverge to activate Msn2 and Maf1, which are transcription factors of the general response to stress and the final integrators of these pathways.