Role of mitogen-activated protein kinase Sty1 in regulation of eukaryotic initiation factor 2alpha kinases in response to environmental stress in Schizosaccharomyces pombe

Abstract

The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2alpha, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2alpha kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2alpha kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2alpha phosphorylation. Finally, the MAPK Sty1, but not the eIF2alpha kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2alpha kinase pathways for a particular range of stress responses.

FIG. 1.

In vitro and in vivo activity of S. pombe eIF2α kinases Hri1 and Hri2. (A) His-tagged purified Hri1 (lane 2) and purified rabbit HRI (as a control, lane 1) were assayed for their ability to phosphorylate eIF2α, as described in Materials and Methods. Proteins were resolved in SDS-PAGE, and phosphoproteins were visualized by autoradiography of the dried gel. The position of Hri1, HRI, and eIF2α are indicated on the right, and the positions of molecular weight markers are indicated on the left. (B) His-tagged purified Hri2 was assayed for its ability to phosphorylate eIF2α and phosphoproteins were resolved and visualized as described in panel A. The position of Hri2 and eIF2α are indicated on the right, and positions of molecular weight markers are indicated on the left. (C) Cell extracts from S. pombe strains modified to express HA-tagged Hri1 or Hri2 were subjected to immunoprecipitation with anti-HA monoclonal antibody. The immune complexes were assayed for their ability to phosphorylate eIF2α, and proteins were resolved in SDS-PAGE and transferred to a PVDF membrane. Phosphoproteins were visualized by autoradiography of the dried membrane (upper panel), and HA-tagged proteins were detected by Western blotting with anti-HA antibody (lower panel). The position of HA-tagged Hri1 and Hri2 and eIF2α are indicated on the right, and positions of molecular weight markers are indicated on the left. (D) J80 and J82 strains of S. cerevisiae transformed with empty pEMBLyex4 and pYX212 vectors or with plasmids encoding S. pombe Hri1 and Hri2 or S. cerevisiae GCN2 were grown on SD+aa (lower panel) or SD+3-AT (upper panel) plates at 30°C for 3 days. Shown are the results of representative experiments of at least three independent experiments with similar results.

FIG. 2.

Effect of oxidative stress on eIF2α phosphorylation by distinct eIF2α kinases. Role of the MAPK Sty1. (A and B) Gcn2 is the primary eIF2α kinase activated in response to oxidative stress; the MAPK Sty1 negatively regulates Gcn2 activity during oxidative stress. (C) Hyperactivation of Sty1 reduces the eIF2α phosphorylation induced in response to oxidative stress. (D) Quantification of the levels of phosphorylated eIF2α (ratio eIF2α-P/eIF2α referring to untreated cells, whose value was set as 1) in panel C. Wild-type (WT) S. pombe cells or cells of strains lacking the different eIF2α kinases and/or the MAPK Sty1 (Δsty1) and its negative regulator Pyp1 (Δpyp1) were subjected to oxidative stress (1.5 mM H₂O₂), as indicated. Phosphorylation of eIF2α and Sty1 were analyzed in the cell extracts by Western blotting with phospho-specific antibodies, as described in Materials and Methods. Shown are the durations of the stress, the names of the strains, and the antibody used for the Western blot (right). In panel B, the length of treatment with H₂O₂ was 15 min. In panel C, WT and Δsty1 strains transformed to overexpress a recombinant Sty1-HA-His₆ protein were used; experiments were performed in EMM alone or EMM supplemented with 0.5 μM thiamine to repress recombinant protein expression. Shown are the results of representative experiments of at least three independent experiments with similar results.

FIG. 3.

Effect of the deletion of the different eIF2α kinases and Sty1 on eIF2α phosphorylation and cell viability during oxidative stress. (A) Differential activation of eIF2α kinases in response to oxidative stress. Role of the MAPK Sty1. Wild-type (WT) S. pombe cells or cells of strains lacking the different eIF2α kinases and/or the MAPK Sty1 and its negative regulator Pyp1, were subjected to oxidative stress (1.5 mM H₂O₂), as indicated. Phosphorylation of eIF2α and Sty1 were analyzed in the cell extracts by Western blotting with phospho-specific antibodies, as described in Materials and Methods. Shown are the length of the stress and the name of the strains (top) and the antibody used for the Western blot (right). (B) Cell viability during oxidative stress in the absence of the eIF2α kinases and/or Sty1. The same cell strains as in panel A were grown on YES-agar plates either containing or not containing H₂O₂ 0.2, 0.75 or 1.5 mM, as indicated, and incubated for 48 h at 32°C. Shown are the results of representative experiments of at least three independent experiments with similar results.

FIG. 4.

Effect of the deletion of the different eIF2α kinases and Sty1 on eIF2α phosphorylation and cell viability during genotoxic stress by MMS. (A) Differential activation of eIF2α kinases in response to genotoxic stress by MMS. (B) Gcn2 is the primary eIF2α kinase activated in response to DNA damage-induced stress caused by MMS, without any effect due to the absence or the overactivation of the MAPK Sty1. (C and D) Sty1 is not activated in response to DNA damage-induced stress caused by MMS. Wild-type (WT) S. pombe cells or cells of strains lacking the different eIF2α kinases and/or the MAPK Sty1 and its negative regulator Pyp1, were subjected to oxidative stress (1.5 mM H₂O₂) or DNA damage-induced stress (0.02% MMS), as indicated. Phosphorylation of eIF2α and Sty1 were analyzed in the cell extracts by Western blotting with phospho-specific antibodies, as described in Materials and Methods. Shown are the durations of the stress and the names of the strains and the antibody used for the Western blot (right). In panel C, the Δsty1 strain transformed to overexpress a recombinant Sty1-HA-His₆ protein was used; experiments were performed in EMM alone or EMM supplemented with 0.5 μM thiamine to repress recombinant protein expression. In panel D, cells expressing endogenous HA-tagged Sty1 protein were used. (E) Cell viability during genotoxic stress in the absence of the eIF2α kinases and/or Sty1. Cell strains were grown on YES-agar plates either containing or not containing 0.02% MMS, as indicated, and incubated for 48 h at 32°C. Shown are the results of representative experiments of at least three independent experiments with similar results.

FIG. 5.

Effect of heat shock on eIF2α phosphorylation by distinct eIF2α kinases. Role of the activation of the type 2A protein phosphatases in the dephosphorylation of eIF2α. (A and B) Hri2 is the primary eIF2α kinase activated in response to heat shock at 40 and 48°C. (C and D) The type 2A protein phosphatases are activated during heat shock at 48°C and dephosphorylate eIF2α. Wild-type (WT) S. pombe cells or cells of strains lacking the different eIF2α kinases and/or the protein phosphatases 1-1 (dis2) and 2A (ppa2) were subjected to heat shock (48°C), as indicated. Phosphorylation of eIF2α was analyzed in the cell extracts by Western blotting with a phospho-specific antibody, as described in Materials and Methods. Shown are the durations of the stress (in minutes), the names of the strains, and the antibody used for the Western blot (right). In panel D, the length of stress was 5 min; also indicated is the name of the strain and the pretreatment with okadaic acid (top) and the antibody used for the Western blot (right). Shown are the results of representative experiments of at least three independent experiments with similar results.

FIG. 6.

Effect of the deletion of the different eIF2α kinases and Sty1 on eIF2α phosphorylation and cell viability during heat shock. (A) Differential activation of eIF2α kinases in response to heat shock. Role of the MAPK Sty1. Wild-type (WT) S. pombe cells or cells of strains lacking the different eIF2α kinases and/or the MAPK Sty1 and its negative regulator Pyp1 were subjected to heat shock (48°C), as indicated. Phosphorylation of eIF2α and Sty1 were analyzed in the cell extracts by Western blotting with phospho-specific antibodies, as described in Materials and Methods. Shown are the durations of the stress, the names of the strains (top), and the antibody used for the Western blot (right). (B and C) Cell viability during heat shock in the absence of the eIF2α kinases and/or Sty1. The same cell strains as in panel A plus a strain lacking the hri2 gene (Δhri2) were grown on YES-agar plates; for the heat shock, plates were incubated at 40°C for 24 h and then incubated for 48 additional hours at 32°C. Shown are the results of representative experiments of at least three independent experiments with similar results.

FIG. 7.

Changes in mRNA and protein levels of the distinct eIF2α kinases in response to oxidative stress and heat shock. (A, B, and C) Levels of hr1, hri2, and gcn2 mRNAs increase after oxidative stress and 40°C heat shock, but decrease after 48°C heat shock. (D, E, and F) Levels of Hri1, Hri2, and Gcn2 proteins increase after oxidative stress and 40°C heat shock but decrease after 48°C heat shock. Wild-type (WT) S. pombe cells or cells of strains expressing the different HA-tagged eIF2α kinases were subjected to oxidative stress (1.5 mM H₂O₂) or heat shock (40 and 48°C). The levels of the distinct mRNAs were quantified by quantitative real-time RT-PCR, as described in Materials and Methods. Values show the fold induction compared to untreated control cells, whose level of mRNA was set as 1. The results show the means of three to five independent experiments plus the standard deviations. HA-tagged proteins levels and phosphorylation of eIF2α were analyzed in the cell extracts by Western blotting with anti-HA or phospho-specific antibodies, as described in Materials and Methods. Shown are the durations of the stress (minutes) and the names of the antibody used for the Western blot (right). Shown are the results of representative experiments of at least three independent experiments with similar results.