Sir2 histone deacetylase prevents programmed cell death caused by sustained activation of the Hog1 stress-activated protein kinase

Abstract

Exposure of yeast to high osmolarity induces a transient activation of the Hog1 stress-activated protein kinase (SAPK), which is required for cell survival under these conditions. However, sustained activation of the SAPK results in a severe growth defect. We found that prolonged SAPK activation leads to cell death, which is not observed in nma111 cells, by causing accumulation of reactive oxygen species (ROS). Mutations of the SCF(CDC4) ubiquitin ligase complex suppress cell death by preventing the degradation of Msn2 and Msn4 transcription factors. Accumulation of Msn2 and Msn4 leads to the induction of PNC1, which is an activator of the Sir2 histone acetylase. Sir2 is involved in protection against Hog1-induced cell death and can suppress Hog1-induced ROS accumulation. Therefore, cell death seems to be dictated by the balance of ROS induced by Hog1 and the protective effect of Sir2.

Figure 1

Sustained activation of Hog1 causes cell death that is partly suppressed by SCF^CDC4 mutations. (A) Mutations on the SCF^CDC4 complex prevent cell death induced by PBS2^DD or SLN1 inactivation. Cells expressing the PBS2^DD allele under the GAL1 promoter (pGAL1–PBS2^DD) were spotted on glucose (control) or galactose. The sln1Δ and sln1Δ cdc4-1 strains carrying a plasmid expressing the protein tyrosine phosphatase 2 (PTP2) gene under the GAL1 promoter (pGAL1–PTP2). (B) Hog1-mediated cell death is improved in a cdc4-1 mutant strain. Cells as in A were grown in galactose for 24 h. Viability was monitored by counting the colony-forming units in glucose plates. Data represent the mean and standard deviation of three independent experiments. (C) DNA single-strand breaks caused by Hog1 activation are reduced in cdc4-1 mutant cells. Cells were processed with the TdT-mediated dUTP nick end labeling (TUNEL) assay (+) and the presence of single-strand DNA breaks was detected by FACS analysis. Data shown are representative of three independent experiments. (D) Induction of the appearance of SubG1 population of cells on Hog1 activation is reduced in cdc4-1 mutant cells. Cells were grown as in A and analysed for SubG1 population by FACS analysis. Data shown are representative of three independent experiments. (E) Cell death caused by sustained PBS2^DD expression is mediated by Nma111. The indicated strains expressing the PBS2^DD allele were grown in glucose (control) or galactose. (F) Cell viability on permanent Hog1 activation is fully restored in the absence of NMA111. Strains as in E were grown in glucose (control) or galactose for 24 h and colony-forming units were assessed in glucose plates. Data represent the mean and standard deviation of three independent experiments. FACS, fluorescence-activated cell sorting.

Figure 2

Hog1-induced reactive oxygen species accumulation is reduced in a cdc4-1 mutant. (A) Reactive oxygen species (ROS) production causes cell death under sustained Hog1 activation. Indicated strains plated on glucose or galactose, and anaerobiosis (left panel) in the presence of antimycin at 2.5 μg/ml (middle panel) or dinitrophenol at 25 μg/ml (right panel). (B) Hog1-induced ROS accumulation is reduced in a cdc4-1 mutant. Strains were grown in galactose for 12 h. Cells were incubated with DCFH-DA for 30 min and ROS was measured. Data represent the mean and standard deviation of three independent experiments. (C) Hog1 inhibits mitochondrial respiration in response to osmostress. Wild-type and hog1as-mutant strains in YPD plates with or without 0.8 M NaCl were grown for 12 h at 25 °C, and tetrazolium chloride was added as an overlay in the presence (+) of the kinase inhibitor 1NM-PP1 (5 μM). (D) Activation of Hog1 causes a reduction in oxygen consumption independently of SIR2, cdc4-1 and GPD1. Oxygen consumption was measured as in B. Data represent the mean and standard deviation of three independent experiments. DCFH-DA, dichloro-dihydro-fluorescein diacetate.

Figure 3

cdc4-1 mutation increases Msn2- and Msn4-dependent gene expression. (A) Hog1 phosphorylation in response to osmostress or PBS2^DD induction is not altered in a cdc4-1 mutant. Cells were subjected to osmostress (0.4 M NaCl; left panel). Cells were grown in raffinose for 4 h and then shifted to galactose. Hog1 phosphorylation was followed by using antibodies against phospho-p38 mitogen-activated protein kinase or Hog1. (B) Hog1 nuclear accumulation in response to osmostress is not affected in a cdc4-1 mutant. Cells carrying pRS416–GFP–Hog1 were subjected to osmostress (0.4 M NaCl). Data represent the mean and standard deviation of three independent experiments. (C) SCF^CDC4 mutants are slightly osmoresistant in a plate assay. Wild-type and the indicated mutant strains were spotted on YPD plates in the presence of NaCl and sorbitol. (D) SCF^CDC4 mutants are slightly osmoresistant in liquid assays. The indicated mutant strains were grown in the presence of NaCl. Data represent the mean and standard deviation of three independent experiments. (E) Mutations in the SCF^CDC4 complex increase Msn2- and Msn4-dependent gene expression in response to osmostress. Cells were grown in YPD and treated with 0.4 M NaCl for the indicated times. Total RNA collected was assayed by northern blot analysis for transcript levels of CTT1, ALD3, GRE2, STL1 and ACT1 (as a loading control). (F) Degradation of Msn2 is delayed in a cdc4-1 mutant strain in response to osmostress. Wild-type and cdc4-1 mutant cells carrying a monocopy plasmid with haemagglutinin (HA)-tagged MSN2 under the ADH1 promoter were grown to mid-log phase and treated with 0.4 M NaCl. (G) Recruitment of Msn2 at osmoresponsive promoters is extended in a SCF^CDC4 mutant on osmostress. Cells as in F were treated with 0.4 M NaCl. Chromatin immunoprecipitation of Msn2 was assessed by immunoprecipitation with HA antibody. Binding to CTT1 promoter was determined by real-time polymerase chain reaction. Data represent the mean and standard deviation of three independent experiments. (H) Hog1 phosphorylates Msn2 in vitro. Msn2-HA was phosphorylated in an in vitro kinase assay using Hog1as with or without the presence of the kinase inhibitor 1NM-PP1 (5 μM). In both cases, radioactively labelled PE-adenosine triphosphate (ATP) was used. GFP, green fluorescent protein; OD, optical density; PE, phycoerythrin.

Figure 4

MSN2/MSN4, PNC1 and SIR2 counteract Hog1-mediated cell death. (A) Deletion of MSN2/MSN4 and PNC1 eliminates the ability of cdc4-1 mutation to prevent cell death. Cells were spotted onto glucose or galactose plates. Viability was monitored by counting colony-forming units in glucose plates (lower panel). Data represent the mean and standard deviation of three independent experiments. (B) cdc4-1 mutant shows an increase in PNC1 gene expression in response to osmostress. Strains were treated with 0.4 M NaCl and total RNA was probed with PNC1 and ACT1. (C) Mutation in the SCF^CDC4 complex increases Pnc1 protein levels in response to osmostress. Pnc1 and Hog1 were assessed in cells treated as in B. (D) SIR2 deletion eliminates the ability of cdc4-1 mutation to prevent cell death. Strains were grown in the presence of resveratrol in the plates (5 μM). Cells that expressed haemagglutinin (HA)-tagged SIR2 under the GAL1 promoter (pGAL1–SIR2–HA) and those that expressed PBS2^DD were spotted on glucose (control) or galactose plates. (E) Hda2 and Hst1 deacetylases do not mediate the effect of cdc4-1. Strains were spotted onto glucose or galactose plates. (F) Sir2 prevents Hog1-induced apoptosis-like cell death. Strains were grown from raffinose to galactose for 24 h. DNA content was assessed by flow cytometry. Data shown are representative of three independent experiments. (G) Tentative model that depicts the effect of Hog1 and Sir2 in dictating cell-fate determination. Hog1 inhibits mitochondrial respiration, which results in an increase in reactive oxygen species (ROS) accumulation that leads to cell death. In parallel, Hog1 induces PNC1 expression through Msn2 and Msn4 transcription factors, which are regulated by SCF^CDC4. Pnc1 activates Sir2, which mediates a decrease of ROS accumulation. SIR2 activation by the stress-activated protein kinase Hog1 relieves the Hog1-induced oxidative stress to prevent apoptotic cell death. ROS, reactive oxygen species.