FIG. 2.

Pcr1 is a phosphoprotein. (A) Pcr1 is phosphorylated under unstressed conditions. Native extracts from MS7 cells (Δpcr1) transformed with p138.41x, expressing HA-Pcr1, were prepared and incubated with (+) or without (−) lambda phosphatase in the presence or absence of phosphatase inhibitors, as indicated. Pcr1 was detected after SDS-PAGE followed by Western blotting using polyclonal anti-Pcr1 antibodies. (B) Pcr1 is dephosphorylated upon oxidative stress. Strains MS7 with p138.41x (Δpcr1 + pHA-pcr1) or 972 (WT) were treated with 1 mM H₂O₂ stress for the times indicated. Native (to detect HA-Pcr1) or boiled (to detect endogenous Pcr1 in strain 972) protein extracts, obtained as described in Materials and Methods, were analyzed by Western blot analysis with anti-Pcr1 antibodies. Phosphorylated (Pcr1-P) and unphosphorylated (Prc1) protein forms, either HA tagged or untagged, are indicated with arrows. (C) Dephosphorylation of Pcr1 does not occur upon KCl stress. Strain MS7 with p138.41x (Δpcr1 + pHA-pcr1) was treated with 0.4 M KCl for 15 or 30 min or left untreated. The phosphorylation status of HA-Pcr1 was analyzed as panel B. (D) H₂O₂-dependent dephosphorylation of Pcr1 is Sty1 dependent. Strain NT224 transformed with p138.41x (Δsty1 + pHA-pcr1) was treated with 1 mM H₂O₂ stress for the times indicated. The phosphorylation status of HA-Pcr1 was analyzed as for panel B.