Activation of the redox sensor Pap1 by hydrogen peroxide requires modulation of the intracellular oxidant concentration

Abstract

SummaryThe transcription factor Pap1 and the MAP kinase Sty1 are key regulators of hydrogen peroxide-induced responses in Schizosaccharomyces pombe . Pap1 can be activated quickly at low, but not high, hydrogen peroxide concentrations. The MAP kinase Sty1 has been reported to participate in Pap1 activation by the oxidant. Here, we provide biochemical and genetic evidence for the in vivo formation of a hydrogen peroxide-induced disulphide bond in Pap1, which precedes the rapid and reversible nuclear accumulation of the transcription factor. We show that activation of the Sty1 cascade before the oxidative insult, or overexpression of the Sty1-regulated genes ctt1 (encoding catalase) or gpx1 (encoding glutathione peroxidase), can accelerate Pap1 entry even at high doses of hydrogen peroxide. In fact, the lack of Sty1 impedes Pap1 nuclear localization, but only at high doses of the oxidant. We propose that, whereas low doses of hydrogen peroxide lead directly to Pap1 oxidation-activation, high concentrations of the oxidant initially activate the Sty1 pathway, with the consequent increase in scavenging enzymes, which in turn helps to decompose the excess of hydrogen peroxide and achieve an appropriate concentration for the subsequent activation of Pap1. Our results also suggest that activation of Sty1 at high doses of hydrogen peroxide may also be required to trigger other antioxidant activities such as those reverting the overoxidation of cysteine residues at the Pap1 pathway.

A cysteine-sulfinic acid in peroxiredoxin regulates H 2 O 2 -sensing by the antioxidant Pap1 pathway

“…Indeed, Pap1 and Sty1 have complementary roles in the oxidative stress response, Sty1 being more important for cell survival after exposure to high levels of H 2 O 2 and Pap1 being essential for an adaptation response to low concentrations of the oxidant (12). Consistent with their biological roles, the Sty1 MAPK pathway preferentially responds to high doses of H 2 O 2 (1 mM) (12), and Pap1 is oxidized and activated by low H 2 O 2 concentrations (0.2 mM) but, at higher concentrations of the oxidant (1 mM), Pap1 activation is delayed by Ϸ30 min (5,7,13) or even longer (70 min at 5 mM; ref. 7).…”

“…Consistent with their biological roles, the Sty1 MAPK pathway preferentially responds to high doses of H 2 O 2 (1 mM) (12), and Pap1 is oxidized and activated by low H 2 O 2 concentrations (0.2 mM) but, at higher concentrations of the oxidant (1 mM), Pap1 activation is delayed by Ϸ30 min (5,7,13) or even longer (70 min at 5 mM; ref. 7). This late activation of Pap1 at high concentrations of H 2 O 2 is not observed in cells lacking Sty1 (7), suggesting that this regulator induces the expression of one or more gene products important to restore the cell competence in Pap1 activation.…”

“…This late activation of Pap1 at high concentrations of H 2 O 2 is not observed in cells lacking Sty1 (7), suggesting that this regulator induces the expression of one or more gene products important to restore the cell competence in Pap1 activation. These gene products are neither catalase nor glutathione peroxidase, because they, respectively, do not or are only able to very partially correct the defective Pap1 activation of ⌬sty1 cells when overexpressed (7).…”

“…Pap1 is a bZIP transcription factor, homologue of mammalian c-Jun, that, upon activation by H 2 O 2 , triggers a specific antioxidant gene response (4,5). In response to H 2 O 2 , an intramolecular disulfide bond between distant cysteine residues is formed in Pap1, which masks the accessibility of the nuclear exporter Crm1 to the C-terminal nuclear export signal, and results in accumulation of Pap1 in the nucleus and in Pap1-dependent gene expression (6,7). The mitogen-activated protein kinase (MAPK) Sty1, on the contrary, can be activated by different types of stresses, such as H 2 O 2 , heat shock, nutritional starvation or osmotic stress (8,9).…”

The Peroxiredoxin Repair Proteins

“…Under stress conditions with low levels of H 2 O 2 , Pap1 is activated by the formation of an intramolecular disulfide bond that prevents association with Crm1 and results in the nuclear accumulation (Toone et al, 1998;Kudo et al, 1999;Castillo et al, 2002;Quinn et al, 2002). In contrast, elevated levels of peroxide prevent nuclear accumulation and the expression of Pap1-dependent genes (Quinn et al, 2002;Vivancos et al, 2004). Sty1 and Aft1 are also activated at elevated levels of peroxide (Quinn et al, 2002).…”