Pkc1-dependent phosphorylation of Ste5-Ser185 inhibits signaling and prevents lysis during mechanostress. (A and B) Expression of the FIG1-qV reporter was measured by FACS in ste5Δ cells expressing GFP-tagged WT Ste5 (diamond), Ste5^S185A (square), or Ste5^S185D (triangle) and plotted in arbitrary units at the times after addition of α-factor. The cells were transformed with a plasmid expressing either WT (Pkc1, A) or dominant-active (Pkc1^R398A, B) Pkc1 from the inducible GAL1-promoter for 2 h. Expression of Pkc1^R398A inhibits pheromone signaling in a Ste5^Ser185-dependent manner. (C) Cell lysis upon mechanostress in cells expressing WT Ste5 or Ste5^S185A mutant was analyze in cells treated as in Fig. 1 A; ≥150 shmooing cells from three independent experiments were analyzed for each condition and are shown as percentage of lysed cells. The error bars indicate SEM, and significance was validated with a t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) Dispersal of either tV-tagged WT Ste5 or Ste5^S185A from shmoo tips upon mechanostress in cells treated as in Fig. 1 A. Loss of Ste5-tV at shmoo tips was quantified in ≥150 cells from three independent experiments for each condition and is shown as percentage of cells with dispersed Ste5 localization. The error bars indicate SEM, and significance was validated with a t test (**, P ≤ 0.01; ***, P ≤ 0.001).