FIGURE 7.

Pah1p phosphorylation by PKA or Pho85p-Pho80p reduces subsequent phosphorylation by PKC. Wild type and the indicated phosphorylation site mutants were expressed and purified from E. coli. The Pah1p enzymes were phosphorylated with PKC (A), PKA (B), or Pho85p-Pho80p (C) using [γ-³²P]ATP without or with prephosphorylation by the indicated protein kinases using unlabeled ATP. Following the reactions, Pah1p was separated from labeled ATP by SDS-PAGE. The polyacrylamide gel was dried and subjected to phosphorimaging and ImageQuant analysis. To control for loading, the dried gel was swollen with water, stained with Coomassie Blue, and subjected to image analysis. The relative amount of the phosphorylated wild type enzyme was arbitrarily set at 100%. The data reported are the average of four independent experiments ±S.D. (error bars).