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. 2020 Feb 3;39(5):e103444. doi: 10.15252/embj.2019103444

Figure 8. The yeast pheromone‐to‐Hog1 crosstalk and the basal activity of the mammalian p38 MAPK might be suppressed by the same mechanism.

Figure 8

  1. KT299 (MAT a hkr1Δ msb2Δ ssk2/22Δ) was exposed to the indicated concentrations of α‐factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting.
  2. KT299 (MAT a ssk2/22Δ hkr1Δ msb2Δ) was exposed to the indicated concentrations of α‐factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay.
  3. Average values of four independent experiments from (B) were plotted.
  4. HM06‐1 (MAT a ΔS/O/H/M ssk2/22Δ) was exposed to the indicated concentrations (log scale) of α‐factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay. Average values of three or more independent experiments were plotted.
  5. KT306 (MAT a hkr1Δ msb2Δ ssk2/22Δ hog1Δ) was transformed with pRS416‐Hog1 (WT) or pRS416‐Hog1‐N149H D162G (N/H D/G) and was exposed to 10 μM α‐factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos‐tag band‐shift assay. Average of three independent experiments is plotted.
  6. A schematic model showing that the lack of osmotic enhancement of the Pbs2‐Hog1 reaction prevents the pheromone‐to‐Hog1 crosstalk.
  7. Typical FRET (YFP/CFP ratio) images showing p38 activation. HeLa cells carrying the p38 reporter PerKy‐p38 (Tomida et al, 2015) were stably transfected with an expression vector for the indicated p38α mutant proteins. FRET analysis was performed as described in Materials and Methods.
  8. Distribution of p38 activity in individual cells from sets (a)–(d) in (G).
Data information: (B) Representative results from three independent experiments. (C–E) Error bars are SEM: (C) n = 4; (D), n = 3 or more; and (E) n = 3. (G) Scale bars: 20 μm. (H) Statistics, Student's two‐tailed t‐test.Source data are available online for this figure.