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. 2013 Mar;33(5):1057-72.
doi: 10.1128/MCB.00834-12. Epub 2012 Dec 28.

Yeast Protein Phosphatase 2A-Cdc55 Regulates the Transcriptional Response to Hyperosmolarity Stress by Regulating Msn2 and Msn4 Chromatin Recruitment

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Yeast Protein Phosphatase 2A-Cdc55 Regulates the Transcriptional Response to Hyperosmolarity Stress by Regulating Msn2 and Msn4 Chromatin Recruitment

Wolfgang Reiter et al. Mol Cell Biol. .
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Abstract

We have identified Cdc55, a regulatory B subunit of protein phosphatase 2A (PP2A), as an essential activating factor for stress gene transcription in Saccharomyces cerevisiae. The presence of PP2A-Cdc55 is required for full activation of the environmental stress response mediated by the transcription factors Msn2 and Msn4. We show that PP2A-Cdc55 contributes to sustained nuclear accumulation of Msn2 and Msn4 during hyperosmolarity stress. PP2A-Cdc55 also enhances Msn2-dependent transactivation, required for extended chromatin recruitment of the transcription factor. We analyzed a possible direct regulatory role for PP2A-Cdc55 on the phosphorylation status of Msn2. Detailed mass spectrometric and genetic analysis of Msn2 showed that stress exposure causes immediate transient dephosphorylation of Msn2 which is not dependent on PP2A-Cdc55 activity. Furthermore, the Hog1 mitogen-activated protein kinase pathway activity is not influenced by PP2A-Cdc55. We therefore propose that the PP2A-Cdc55 phosphatase is not involved in cytosolic stress signal perception but is involved in a specific intranuclear mechanism to regulate Msn2 and Msn4 nuclear accumulation and chromatin association under stress conditions.

Figures

Fig 1
Fig 1
PP2A-Cdc55 is required for high level expression of hyperosmolarity stress-induced genes. (A) Transcript levels of CTT1, HSP12, and PGM2. Wild-type (W303) and cdc55Δ mutant cells were grown to early exponential phase and treated with 0.4 M NaCl for the indicated times. Total RNA was extracted and analyzed for expression by Northern blot analysis. Hybridization signals from at least three independent experiments were quantified with a PhosphorImager and normalized against values for IPP1. The highest levels in the wild type were set to 1. (B) Hierarchical cluster analysis of the mRNA profile of the selected genes (as described in the text) of wild-type (W303), cdc55Δ, msn2Δ msn4Δ, and msn2Δ msn4Δ cdc55Δ strains (Treeview data; see Table S6 in the supplemental material). (C) Hierarchical cluster analysis of the mRNA profile of the selected genes (as described in the text) of msn2Δ msn4Δ and msn2Δ msn4Δ cdc55Δ strains carrying plasmid pMSN2Msn2ΔNES (Treeview data; see Table S6). (D) Correlation matrix of different genotypes. Pearson′s correlation coefficients were calculated for average fold induction of the selected genes and used to generate a cluster matrix. (E) Average fold induction of wild-type and mutant strains over 10, 20, and 30 min. (F) Venn diagram of Msn2- and Cdc55-dependent genes within the stress-inducible selected set. The probability was calculated as cumulative hypergeometric distribution. The cutoffs for the different groups are indicated.
Fig 2
Fig 2
PP2A-Cdc55 regulates nuclear retention of Msn2 and -4. (A and B) Hyperosmotic stress-induced Msn2 and Msn4 nuclear localization is shortened in the cdc55Δ mutant. Msn2-GFP and Msn4-GFP were both expressed under the control of the ADH1 promoter. Fluorescence was recorded in nonfixed cells treated with 0.4 M NaCl at the indicated time points. Representative images are shown. (C) Msn2-GFP was expressed under the control of its native promoter, and fluorescence was recorded after treatment with 0.4 M NaCl for the indicated time points. (D) Cdc55 does not control Hog1 nuclear localization. Cells expressing Hog1-GFP were treated with 0.4 M NaCl for the times indicated. (E) Cdc55 does not change the activity of the Hog1 regulated transcription factor Hot1. Cells were subjected to hyperosmolarity stress and mRNA levels of the Hot1-dependent gene STL1 was determined as described in the legend to Fig. 1A.
Fig 3
Fig 3
PP2A-Cdc55 regulates Msn2 in the nucleus. (A) Mutant cells msn2Δ msn4Δ and msn2Δ msn4Δ cdc55Δ carrying the plasmid pMSN2Msn2ΔNES-GFP were treated with 0.4 M NaCl and fluorescence signals were monitored at indicated time points. (B) Comparable nuclear abundance of Msn2ΔNES in msn2Δ msn4Δ and msn2Δ msn4Δ cdc55Δ cells. Msn2ΔNES was expressed from its native promoter. Quantification of Msn2ΔNES-GFP fluorescence intensity (as described in Materials and Methods) in nuclei is shown. (C) The msn2Δ msn4Δ and msn2Δ msn4Δ cdc55Δ strains carrying either YCplac 111 (empty vector), pMSN2Msn2, or pMSN2Msn2ΔNES were treated with 0.4 M NaCl for the indicated times. CTT1 and HSP12 mRNA levels were analyzed by quantitative real-time PCR using IPP1 as a reference. (D) msn2Δ msn4Δ and msn2Δ msn4Δ cdc55Δ strains carrying the plasmid pMSN2Msn2ΔNES-TAP were treated with 0.4 M NaCl for the indicated times and cross-linked with formaldehyde. Association of Msn2ΔNES-TAP with the promoter regions of CTT1 and HSP12 was detected using quantitative real-time PCR. Signals were normalized to the signals obtained with primer pairs specific to the TEL region on the right arm of chromosome III (+269425/269624). (E) Same settings as described for panel D, except that association of RNA Pol II with promoters was monitored by ChIP of the largest Pol II subunit, Rpb1. (F) msn2Δ msn4Δ and msn2Δ msn4Δ cdc55Δ strains carrying the plasmid pMSN2Msn2ΔNES-TAP were treated with 0.4 M NaCl for the indicated times and cross-linked with formaldehyde. Micrococcal nuclease digestion-generated DNA fragments corresponding to mononucleosomes. Isolated DNA was quantified by 15 overlapping amplicons of CTT1 and 16 overlapping amplicons of HSP12. A well-positioned nucleosome within the VCX1 coding region, unchanged by hyperosmotic stress, is shown as an inset (MSQ, mean starting quantity). The relative occupancy of nucleosomes was determined using the peak of this VCX1 nucleosome (forward, TGC GTG TGC ATC CCT ACT GA; reverse, AAG TGG TCT TCC TTG CCA TGA).
Fig 4
Fig 4
PP2A-Cdc55 does not dephosphorylate detectable phosphorylation sites of Msn2. (A) Scheme of the 22 phosphorylation sites identified on Msn2 by mass spectrometry. Shown are the integrated MS results obtained with purified Msn2. Two different protein purification methods were applied to generate MS samples: (i) immunoaffinity purification of Msn2-HA (samples used for phosphorylation site mapping only) and (ii) tandem affinity purification using Msn2-HTBeaq (36) (site mapping and quantitative analysis). Both Msn2 variants were expressed at endogenous levels. Position of phosphorylation sites are indicated by bars and bold type. Regulated sites are indicated in red (phosphoserines 582 and 620 could be quantified only via phosphospecific antibodies; see panels C and D and reference 19). Mass spectrometry sequence coverage is underlined. Predicted PKA consensus motifs are indicated with asterisks (identified by at least two different prediction tools) and plus signs (identified by only one prediction tool). A degree sign indicates a Snf1 kinase target site (19). Phosphorylation sites previously listed as ambiguous are labeled with an “a.” Tyrosine 621 has been mapped by automated phosphorylation site allocation; however, a strong neutral loss of phosphoric acid upon collision-induced dissociation indicates that S620 is phosphorylated, which is in accordance with previous observations (19). (B) The phosphorylation state of Msn2 changes shortly and transiently during hyperosmotic stress. Cells expressing endogenous Msn2-HTBeaq (36) were either treated with 0.4 M NaCl or starved for glucose for the indicated times. Msn2 was purified via histidine-biotin tandem affinity purification. Phosphorylation kinetics of Msn2 phosphorylation sites during hyperosmotic stress were analyzed with selective reaction monitoring (SRM) technology. Levels of the unstressed sample (0 min) were set to 1. Only results obtained with nonphosphorylated peptides are shown. (C) Phosphorylation dynamics of PKA consensus motifs of Msn2 during hyperosmotic stress. Wild-type and cdc55Δ cells were grown to mid-exponential phase and treated with 0.4 M NaCl for the indicated times. Protein extracts were analyzed using purified phosphorylation-specific antibodies raised against peptides containing phosphorylated serines of Msn2 (S288 and S620). Msn2 levels were controlled with an anti-Msn2 antiserum. msn2Δ and msn2Δ msn4Δ mutant cells were included as negative controls. (D) Wild-type and cdc55Δ cells were grown to mid-exponential phase and starved for glucose for the indicated times. Readdition of glucose is indicated with a plus sign. Phosphorylation of Msn2 was monitored by Western blotting using phosphorylation-specific antibodies.
Fig 5
Fig 5
Serine 686 of the DNA binding domain is a potentially regulatory site. (A) msn2Δ msn4Δ cdc55Δ cells carrying either pADH1Msn2-GFP, pADH1Msn2S686A-GFP, or pADH1Msn2S686D-GFP were harvested at exponential growth or after 60 min exposure to 0.4 M NaCl. Catalase units were determined as described in reference . (B) Scheme of GST-purified zinc finger (ZF) domains of Msn2 and Msn2-S686A and Msn2-S686D mutants analyzed in band shift assays. The fusion proteins were expressed from plasmids pGGM-ZF (GAL1 promoter driven, GST-tagged zinc finger domain of Msn2), pGGM-ZFS686A, and pGGM-ZFS686D, respectively. (C) Expression of Msn2p zinc finger domains fused with a GST tag was induced using the GAL1 promoter. The different domains were purified from whole-cell extracts by GST pulldowns. Quantities of loaded proteins were controlled with Western blots. (D) GST-purified Msn2 zinc finger domains were incubated with a 32P-labeled DNA-oligomer of the CTT1 promoter. Reactions were loaded on band shift gels. Binding of the probe was visualized using a PhosphorImager. (E) msn2Δ msn4Δ and msn2Δ msn4Δ cdc55Δ cells carrying the plasmid pMSN2Msn2ΔNESS686A were treated with 0.4 M NaCl. mRNA levels of CTT1, HSP12, and PGM2 were quantified on Northern blots. (F) W303 wild-type and cdc55Δ cells carrying pADH1Msn2S686D-GFP were treated with 0.4 M NaCl, and fluorescence signals were monitored at the indicated time points.
Fig 6
Fig 6
Graphical representation of the possible regulatory role of PP2A-Cdc55 for Msn2 and Msn4. The transcriptional response to hyperosmotic stress (as well as the nuclear retention time of Msn2/4) is shortened in the absence of CDC55. Furthermore, hyperosmotic stress, in contrast to glucose starvation, leads to only a very transient dephosphorylation of the transcription factor's regulatory sites. This fluctuation in the phosphorylation pattern is PP2A-Cdc55 independent and possibly participates in a mechanism that promotes initial nuclear import. PP2A-Cdc55 instead contributes to an intranuclear mechanism promoting maintenance of nuclear accumulation and promoter association of Msn2/4 for the duration of the hyperosmotic stress response.

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