Figure 1: Phosphorylation of fimbrin (Sac6) by Cdk1.

(a) Purification of cyclin–Cdk1 complexes from yeast. Coomassie blue staining shows Clb5–ΔN–Cdk1, Clb3–Cdk1 and Clb2–ΔN–Cdk1 purifed from yeast. (b) Normalization of kinase activity using histone H1. An amount of 2 μg of histone H1 was incubated with 12 ng of Clb5–ΔN–Cdk1, 11 ng of Clb3–Cdk1 and 6.5 ng of Clb2–ΔN–Cdk1 in the presence of [γ³²P]-ATP at room temperature (25 °C) for 20 min. (c) Sac6 phosphoryaltion using same H1 kinase activities of cyclin–Cdk1 complexes. An amount of 1 μg of Sac6 protein was incubated with 6 ng Clb5–ΔN–Cdk1, 5.5 ng of Clb3–Cdk1 and 3.25 ng of Clb2–ΔN–Cdk1 in the presence of [γ³²P]-ATP at room temperature for 20 min. (d) Identification of Sac6 residue T103 as a Clb2–Cdk1 target using mass spectrometry. (e) Clb2–ΔN–Cdk1 phosphoryaltes WT Sac6 but not Sac6-T103A. An amount of 1 μg Sac6 or Sac6-T103A was incubated with different cyclin–Cdk1 complexes. Phosphorylation was analysed by autoradiography following SDS–PAGE in b,c and e. (f,g) Asynchronous and nocodazole arrested SAC6 and sac6(T103A) cells were collected, respectively, and subjected to immunoprecipitation (IP). Proteins were resolved on either regular or phosphate-affinity (Phos-tag), and blotted using Sac6, metaphase cyclin Clb2 and loading control Pgk1 antibodies. Numbers in g correspond to lane numbers in f. (h,i) SAC6 and sac6(T103A) cells, carrying ADH1pr-GAL4BD-hER-VP16 (GEV), were induced with 100 nM of β-estradiol, collected and subjected to IP. (j,k) SAC6 cells were collected from asynchronous and nocodazole arrested cells. Nocodazole arrested cells were treated with or without additional 1-NM-PP1 before being subjected to IP. The intensity ratio of phospholyated Sac6 species to total Sac6 in f,h and j (upppest panel, Phos-tag gel) was analysed and is shown in the graphs g,i and k, respectively. Symbol (p) indicates the phosphorlyated species of Sac6 on phos-tag gel in f,g,i and k.