FIG. 4.

Phosphorylation and activity of yeast CDKs in cak1 mutants. The indicated mutant strains were grown to mid-log phase at 25°C and shifted to 37°C for 3 h. Epitope-tagged Cdc28 (A), Kin28 (B and C), Pho85 (D), and Srb10 (E) were immunoprecipitated from cell lysates and analyzed by immunoblotting with anti-HA antibodies (top panels) or kinase activity toward the indicated substrate (bottom panels). Cdc28 phosphorylation does not correlate with kinase activity in panel A, presumably because only a small fraction of Cdc28 is associated with cyclin and is active in asynchronous cells, and kinase activity should be normal even if only this fraction is phosphorylated.