FIG. 9.

Dependence of Mpk1 serine phosphorylation on Mec1/Tel1 and Rad53 and its role in FKS2 transcription. (A) Caffeine-induced phosphorylation of Mpk1 Ser423 is blocked in a rad53Δ mutant, and phosphorylation of both Ser423 and Ser428 are blocked in a Mec1/Tel1 mutant. Yeast strains with the sml1Δ gene (wild type [WT] [strain DL3950]), mec1Δ tel1Δ sml1Δ gene (strains DL3955 to DL3957), and rad53Δ sml1Δ gene (strain DL3953) that had been transformed with plasmid expressing Mpk1-FLAG (p2704), Mpk1-S423A-FLAG (p2824), or Mpk1-S428A-FLAG (p2825) were subjected to caffeine stress. The cell extracts were processed for detection of serine-phosphorylated Mpk1 (phospho-Ser) and total Mpk1 (Mpk1-Flag) as described in the legend to Fig. 7. (B) Blocking Ser423 phosphorylation in a rad53Δ mutant allows caffeine-induced cell wall stress to drive FKS2 transcription. An FKS2-lacZ reporter plasmid (p2052) was transformed into yeast strains with the sml1Δ gene (WT; DL3950) and rad53Δ sml1Δ gene (DL3953). Transformants were grown as described in the legend to Fig. 2A and subjected to thermal or caffeine stress or maintained at 23°C. β-Galactosidase activity was measured in crude extracts. β-Galactosidase specific activity (in units) [β-Gal Sp. Act. (U)] is shown on the y axis. Each value represents the mean and standard deviation (error bar) from three independent transformants.