FIG. 3.

Mutation of T162 abolishes Kin28 phosphorylation, activity, and function. (A) Alignment of activating loop regions in Cdc28, Kin28, and Kin28A. The activating phosphorylation site (T169) in Cdc28 is indicated by an asterisk. (B) HA epitope-tagged wild-type Kin28 (WT) or Kin28A (A) was expressed under the control of the GAL1-10 promoter in wild-type or kin28-3 cells at 25°C. Anti-HA immunoprecipitates were prepared from cell lysates and subjected to immunoblotting to detect Kin28 (top). Kin28-associated CTD kinase activity was measured in parallel immunoprecipitates (bottom). Lane C contains a control immunoprecipitation from cells lacking epitope-tagged Kin28. The asterisk indicates a background band phosphorylated in CTD kinase assays in the absence of Kin28. (C) Growth of wild-type or kin28-3 strains containing KIN28 (WT) or kin28A (A) under the control of the GAL1-10 promoter at 25°C (top) or 37°C (bottom) on plates containing dextrose (DEX) or galactose (GAL).