Multiple kinases contribute to hyperphosphorylation of Ace2 in rts1Δ cells. (A) Samples were taken at the indicated times after addition of 1NM-PP1 to log phase cells, and phosphorylation of Ace2 was assayed by Western blotting. (B) Ace2-3×HA was immunoprecipitated from wild-type, rts1Δ, or cbk1Δ cells and probed with anti-Ace2 antibody or with an antibody that recognizes phosphorylated S122. Ace2-3×HA was immunoprecipitated from the cbk1Δ cells as a control to demonstrate that the phosphospecific antibody recognizes a site phosphorylated by Cbk1. (C) Kinase reactions containing affinity purified Ace2-3×HA, Cbk1-3×HA, and Cdk1/Clb2-3×HA in the indicated combinations were initiated by addition of ATP. Ace2-3×HA phosphorylation was assayed by Western blotting with the anti-HA mouse monoclonal antibody. (D) Cells were grown to log phase at room temperature, and Ace2 phosphorylation was assayed by Western blotting. The arrow indicates a hyperphosphorylated form of Ace2.