Research ArticleCell Biology

MAPK feedback encodes a switch and timer for tunable stress adaptation in yeast

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Science Signaling  13 Jan 2015:
Vol. 8, Issue 359, pp. ra5
DOI: 10.1126/scisignal.2005774
  • Fig. 1 The Hog1 signaling profile is a linear transducer that converts dose to duration.

    (A) Validation of the Phos-tag method for resolving dually phosphorylated and activated (top band) or nonphosphorylated and unactivated (bottom band) Hog1. Cells untreated (−) or treated for 5 min (+) with 550 mM KCl were lysed, resolved by Phos-tag SDS-PAGE, and immunoblotted with Hog1 antibodies. Hog1T174A and Hog1Y176F, mutants lacking one of two phosphorylation sites; Hog1T100A, analog-sensitive mutant; ptc1Δ and ptc2Δ, serine/threonine phosphatase mutants; ptp2Δ and ptp3Δ, tyrosine phosphatase mutants; pbs2Δ and hog1Δ, MAPKK and MAPK mutants, respectively. Data are representative of two experiments. (B) Hog1 activation over time. Wild-type (WT) cells were treated with 550 mM KCl, lysed, and probed by immunoblotting with Hog1 antibodies. Top, Phos-tag SDS PAGE. Bottom, identical samples processed by SDS-PAGE in the absence of Phos-tag. pbs2Δ cells served as a negative control. Data are representative of two experiments. (C) Hog1 signaling profile. WT cells were exposed to the indicated concentrations of KCl. Percentage of dually phosphorylated Hog1 (ppHog1) was calculated by dividing intensity of the upper band by the total intensity of all Hog1 bands in each lane. Data are means ± SEM (n > 3 experiments). (D) Hog1T100A activation by the indicated concentrations of KCl in the presence of the inhibitor 1-NA-PP1. Lysates were resolved by Phos-tag SDS-PAGE and immunoblotted with Hog1 antibodies. Data are representative of two experiments. (E) Hog1T100A signaling profile in the presence of the kinase inhibitor 1-NA-PP1 (n = 2). (F) Data from (C) and (E) are presented as area under the curve. (G) Diagram of potential positive and negative feedback represented by green and red lines, respectively.

  • Fig. 2 Feedback phosphorylation of Ste50 is dose-dependent.

    (A). Ste50 phosphorylation over time. WT and the indicated mutant strains were treated with 550 mM KCl, harvested, and immunoblotted for Ste50. Top, Phos-tag SDS PAGE. Bottom, identical samples in the absence of Phos-tag. (B) Ste50 phosphorylation profile. WT cells were treated as in (A) with the indicated concentrations of KCl. Red, mean Ste50 distribution measured from unstimulated cells; black, mean Ste50 distribution measured for each dose and time in the variable matrix. Shading, ±SEM (red and gray) (n = 3). (C) Integration of the Ste50 phosphorylation profile. Data are presented as mean area under the curve ± SEM.

  • Fig. 3 Modeling positive and negative feedback by Hog1.

    Mathematical model of activated Hog1 (ppHog1, y axis) over time (x axis) as a function of six input signal strengths. (A) Hog1 signaling profile. Model includes Hog1 activation that induces feedback. (B) Hog1T100A signaling profile. Model allows Hog1 activation but has no feedback. (C) Integration of Hog1 signaling profile. The total amount of activated Hog1 produced for each input signal (x axis) is computed by integrating each curve in (A) (y axis).

  • Fig. 4 Hog1 encodes dose-to-duration signaling through graded phosphorylation.

    (A) Integration of the Hog1 signaling profile for the Ste505A strain. WT is shown for reference (see Fig. 1). Data from fig. S3 are presented as area under the curve for Ste505A. (B) Representative data from the signaling in (A). Lysates were resolved by Phos-tag SDS-PAGE and immunoblotted for Hog1. (C) Transcription reporter data for WT and Ste505A. Data are mean relative fluorescence ± SEM (n > 4). 0, untreated control. (D) Comparison of transcriptional output to total Hog1 activity. Computational transformation of data in (C), where x-axis values are replaced using Hog1 duration as determined in (A) for WT and Ste505A. See also fig. S3 for the Hog1 signaling profile for the Ste505A strain used to compute (A).

  • Fig. 5 The Hog1 signaling profile can be reengineered through component gene deletions.

    (A) Diagram of the Hog1 signaling pathway. Gray circles, pathway component deletions without effect. Black circles, essential pathway components that were not evaluated. The other colored circles represent genes that altered either the Hill slope, EC50, or both for the transcriptional reporter response. (B) Summary of transcription reporter data. 8XCRE-LacZ Hill slope and EC50 for each mutant strain plotted relative to WT (black dot) and color-coded as in (A). Only significant (P < 0.05) changes are displayed. (C) Rank order of change in transcription reporter EC50 and Hill slope for each mutant strain relative to WT. Data are mean relative fluorescence ± SEM (n > 4). Statistical significance was calculated by two-tailed t test (*P < 0.05).

  • Fig. 6 The Hog1 signaling profile is insufficient to predict downstream output.

    (A) Transcription reporter dose-response curves for WT, ssk1Δ, rga1Δ, and hog1Δ strains. Data are mean relative fluorescence ± SEM (n > 4). (B) Integration of Hog1 signaling profiles for ssk1Δ (top) and rga1Δ (bottom) strains. WT is shown for reference (see Fig. 1). (C) Top, representative data from (B). Bottom, representative time course for WT and rga1Δ strains in the nonlinear range for the mutant. Lysates were resolved by Phos-tag SDS-PAGE and immunoblotted for Hog1. (D) Comparison of transcriptional output to total Hog1 activity. Computational transformation of data in (A), where x-axis values correspond to the Hog1 duration values determined in (B) for WT, ssk1Δ, and rga1Δ.

  • Fig. 7 Hog1 executes a tiered adaptive protein induction program over time.

    (A) mRNA content quantified from WT cells every 15 min after the initial stimulation with 700 mM NaCl (54) and sorted with respect to time until >2-fold log2 change was achieved. Scale is log2 fold change with respect to unstimulated cells. (B) GO analysis of clusters 1, 2, 3, and 4 from (A). Numerals represent total number of unique genes in each category. (C) GFP-tagged protein abundance measured by flow cytometry. Candidates were selected at random from those identified in (A) and treated with 0, 350, or 650 mM KCl for 30 min. Each data point represents change in mean GFP intensity relative to the unstimulated control for each strain; all data represent >10,000 individual cell counts. (D) Mean change in protein abundance (left) and CV (right) for cells treated with 350 or 650 mM KCl for 30 min. The parent cluster for each protein is represented by the colors gray (cluster 1), yellow (cluster 2), cyan (cluster 3), and red (cluster 4). Mean values for each cluster are indicated.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/359/ra5/DC1

    Fig. S1. The antibodies generated against phosphorylated p38 recognize monophosphorylated and dually phosphorylated Hog1.

    Fig. S2. Negative feedback only model fails to capture the Hog1 signaling profile.

    Fig. S3. Hog1 signaling profile in the Ste505A strain.

    Fig. S4. Hog1 signaling profile in ssk1Δ and rga1Δ yeast.

    Fig. S5. Gating parameters for flow cytometry data.

    Table S1. Transcription factor binding and interaction analysis.

    Table S2. List of yeast strains used in this study.

    Table S3. List of parameter values used in modeling.

    Database S1. Annotated microarray data with GO analysis and organization.

    Database S2. Induction of 95 proteins from clusters 1 to 4 in response to 0, 350, or 650 mM KCl.

  • Supplementary Materials for:

    MAPK feedback encodes a switch and timer for tunable stress adaptation in yeast

    Justin G. English, James P. Shellhammer, Michael Malahe, Patrick C. McCarter, Timothy C. Elston, Henrik G. Dohlman*

    *Corresponding author. E-mail: hdohlman{at}med.unc.edu

    This PDF file includes:

    • Fig. S1. The antibodies generated against phosphorylated p38 recognize monophosphorylated and dually phosphorylated Hog1.
    • Fig. S2. Negative feedback only model fails to capture the Hog1 signaling profile.
    • Fig. S3. Hog1 signaling profile in the Ste505A strain.
    • Fig. S4. Hog1 signaling profile in ssk1Δ and rga1Δ yeast.
    • Fig. S5. Gating parameters for flow cytometry data.
    • Table S1. Transcription factor binding and interaction analysis.
    • Table S2. List of yeast strains used in this study.
    • Table S3. List of parameter values used in modeling.
    • Legends for database S1 and S2

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 537 KB

    Other Supplementary Material for this manuscript includes the following:

    • Database S1 (Microsoft Excel format). Annotated microarray data with GO analysis and organization.
    • Database S2 (Microsoft Excel format). Induction of 95 proteins from clusters 1 to 4 in response to 0, 350, or 650 mM KCl.

    [Download databases S1 and S2]


    Citation: J. G. English, J. P. Shellhammer, M. Malahe, P. C. McCarter, T. C. Elston, H. G. Dohlman, MAPK feedback encodes a switch and timer for tunable stress adaptation in yeast. Sci. Signal. 8, ra5 (2015).

    © 2014 American Association for the Advancement of Science

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