Multistep phosphorelay proteins transmit oxidative stress signals to the fission yeast stress-activated protein kinase

Abstract

In response to oxidative stress, eukaryotic cells induce transcription of genes required for detoxification of oxidants. Here we present evidence that oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase in the fission yeast Schizosaccharomyces pombe. The fission yeast mpr1(+) gene encodes a novel protein with a histidine-containing phosphotransfer domain homologous to the budding yeast Ypd1. Spc1 activation upon oxidative stress is severely impaired in the Deltampr1 mutant as well as in the mpr1HQ strain, in which the putative phosphorylation site Mpr1-His221 is substituted with glutamine. In response to oxidative stress, Mpr1 binds to the Mcs4 response regulator that functions upstream of the Spc1 cascade, suggesting that Mcs4 is a cognate response regulator for Mpr1. Unexpectedly, when exposed to hydrogen peroxide, Deltampr1 cells can induce the catalase gene ctt1(+), one of the transcriptional targets of the Spc1 pathway, and survive oxidative stress in the absence of significant Spc1 activation. We have found that Pap1, a bZIP transcription factor homologous to human c-Jun, can mediate induction of ctt1(+) expression upon oxidative stress independently of the Spc1 stress-activated protein kinase. These studies show that oxidative stress stimuli are transmitted by multiple pathways to induce specific gene expression.

Figure 1

Mpr1 is a fission yeast homologue of the budding yeast Ypd1 protein, a response regulator phosphotransferase. (A) Alignment of the amino acid sequence of Mpr1 with that of Ypd1. Identical residues are shown by shaded boxes. An asterisk marks the phosphorylation site His-64 of Ypd1, which is also conserved in Mpr1. The nucleotide sequence of mpr1⁺ is available in the GenBank database under accession number AL034352 (ORF, SPBC725.02). (B) Restriction map of the mpr1⁺ locus and the mpr1::his7⁺ construct for gene disruption. The HindIII–XhoI fragment encoding residues 136–281 was replaced with the his7⁺ marker gene (Apolinario et al., 1993) to disrupt the mpr1⁺ gene. Restriction enzyme sites: Hd, HindIII; RV, EcoRV; Sl, SalI; Xh, XhoI.

Figure 2

Mpr1 is important for activation of Spc1 in response to oxidative stress. Wild-type (KS1376) and Δmpr1 (CA279) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with either high-osmolarity stress induced by 0.6 M KCl (A) or oxidative stress induced by 0.3 mM H₂O₂ (B). Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. Δmpr1 mutant cells showed a significant defect in Spc1 activation upon oxidative stress, whereas osmostress induced strong activation of Spc1 in Δmpr1 mutants and wild-type cells.

Figure 3

The putative histidine phosphorylation site, His-221, is required for Mpr1 function in oxidative stress signaling to Spc1. The mpr1HQ mutant gene with the His-221→Gln substitution was created by site-directed mutagenesis and fused to the HA6H tag, which was used to replace the mpr1⁺ locus of an spc1:HA6H strain. The resultant strain (CA403) and the control strain, which has the wild-type mpr1⁺ locus tagged with HA6H (CA385), were grown to midlog phase at 30°C in YES medium and then treated with osmostress induced by 0.6 M KCl (Os) and oxidative stress induced by 0.3 mM H₂O₂ (Ox) for the indicated times. Spc1 as well as wild-type and mutant Mpr1 were purified on Ni-NTA beads and analyzed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The Mpr1 and Mpr1HQ proteins were detected as weak bands below the Spc1 bands in anti-HA immunoblotting. Stress-induced Spc1 activation in the mpr1:HA strain was indistinguishable from that in strains expressing untagged Mpr1, indicating that the HA6H tag does not disturb the Mpr1 function.

Figure 4

Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4⁺ tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H₂O₂ (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to GSH-Sepharose beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H₂O₂. Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.

Figure 5

The Prr1 response regulator is not important for Spc1 activation after oxidative stress. Wild-type (KS1376), Δprr1 (CA425), Δmcs4 (CA220), and Δmcs4 Δprr1 (CA423) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H₂O₂. Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies.

Figure 6

Δmpr1 cells are not sensitive to oxidative stress. (A) Wild-type (PR109), Δspc1 (KS1366), and Δmpr1 (CA288) cells were grown to midlog phase in YES medium at 30°C, and H₂O₂ was added at concentrations of 0, 1, and 2 mM. After 1 h of incubation at 30°C, cells were washed, diluted, and plated on YES agar medium. The survival of each strain was evaluated in terms of its colony-forming ability after 3 d of incubation at 30°C. Results from an average of three independent experiments are shown. (B) Oxidative stress–induced expression of the catalase gene, ctt1⁺, in Δmpr1 cells. The strains used in A were treated with 0.3 mM H₂O₂ in YES medium, and aliquots of cells were harvested at the indicated times for Northern hybridization analysis of the ctt1⁺ mRNA. The leu1⁺ probe served as a control. ctt1⁺ expression was strongly induced in Δmpr1 cells but was not detectable in Δspc1 cells.

Figure 7

Oxidative stress but not osmostress can induce ctt1⁺ expression independently of Spc1 activation. (A) Wild-type (KS2096) and wis1DD (KS2088) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with either high-osmolarity stress induced by 0.6 M KCl (Os) or oxidative stress induced by 0.3 mM H₂O₂ (Ox). Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The level of active Spc1 does not change in wis1DD cells even after osmostress and oxidative stress. (B and C) Wild-type (PR109), wis1DD (KS2081), and Δwis1 (JM544) cells were grown to midlog phase in YES medium and treated with oxidative stress induced by 0.3 mM H₂O₂ (B) and high-osmolarity stress induced by 0.6 M KCl (C). Aliquots of cells were harvested at the indicated times for Northern hybridization analysis with the ctt1⁺ and leu1⁺ probes. In wis1DD cells, ctt1⁺ expression was increased significantly in response to oxidative stress but not osmostress.

Figure 8

Two transcription factors, Atf1 and Pap1, regulate expression of ctt1⁺ in response to oxidative stress. (A) Wild-type (PR109), Δatf1 (KS1497), Δpap1 (TP108-3c), and Δatf1 Δpap1 (CA334) strains were grown to midlog phase in YES medium at 30°C and treated with 0.3 mM H₂O₂. Aliquots of cells were harvested at the indicated times for Northern hybridization analysis with the ctt1⁺ and leu1⁺ probes. Compared with wild-type cells, ctt1⁺ expression after oxidative stress was significantly impaired in Δatf1 and Δpap1 mutants and was not detectable in Δatf1 Δpap1 double mutants. (B) The strains used in A were grown to midlog phase in YES medium at 30°C, and H₂O₂ was added at concentrations of 0, 1, and 2 mM. After 1 h of incubation at 30°C, cells were washed, diluted, and plated on YES agar medium. The survival of each strain was evaluated in terms of its colony-forming ability after 3 d of incubation at 30°C. Results from an average of three independent experiments are shown. The Δatf1 mutant showed higher sensitivity to H₂O₂ than the wild-type cells, and this sensitivity was further accentuated in the Δpap1 background.

Figure 9

Pap1 can regulate ctt1⁺ expression upon oxidative stress independently of the Spc1 pathway. (A) Wild-type (PR109), wis1DD (KS2081), and wis1DD Δpap1 (CA356) strains were grown to midlog phase in YES medium at 30°C and treated with 0.3 mM H₂O₂. Aliquots of cells were harvested at the indicated times for Northern hybridization analysis with the ctt1⁺ and leu1⁺ probes. In wis1DD cells, which have constitutive Spc1 activity, ctt1⁺ induction upon oxidative stress was dependent on Pap1. (B) Northern analysis similar to that described for A was performed with Δatf1 (KS1497), Δatf1 Δpap1 (CA334), and Δatf1 Δspc1 (KS1533). In the Δatf1 background, the induction of ctt1⁺ upon oxidative stress was dependent on Pap1 but not Spc1.

Figure 10

Oxidative stress signaling pathways that lead to transcription of the ctt1⁺ gene. A multistep phosphorelay composed of an unknown sensor kinase, Mpr1, and the Mcs4 response regulator transmits oxidative stress signals to the Spc1 MAPK cascade. Oxidative stress induces partial activation of Spc1 even in the Δmcs4 Δmpr1 strain, suggesting that oxidative stress can be transmitted to the Spc1 cascade by additional pathways. Activated Spc1 induces transcription of ctt1⁺ and other stress-response genes through the Atf1 transcription factor. The Pap1 transcription factor can also mediate ctt1⁺ expression in response to oxidative stress independently of the Spc1 pathway.