FIG. 7.

The double mutants' intrinsic activity is independent of either Thr174 or Tyr176 phosphorylation. (A) Cells of the hog1Δ strain harboring MET3-based plasmids encoding Hog1 double mutants (activating mutation) with a mutated phosphoacceptor (phosphoacceptor mutation) were used. Cultures were grown in the presence of methionine to logarithmic phase, counted, washed with water, resuspended in inducing (methionine-free) media, serially diluted to the specified numbers, and plated on the indicated plates (+Met or −Met). Plates were scanned 48 h after plating. (B) Western blot analysis of whole-cell lysates using anti-phospho-Tyr (left panel) or of immunoprecipitated Hog1 proteins using anti-phospho-Thr (right panel). The positive control (pos. con.) is a lysate extracted from hog1Δ cells carrying Hog1 single mutant D170A 10 min after 1 M salt induction.