Figure 6.

Induction of Wip1 inhibits Cdc2 kinase activity. (A) Wip1 modulates the kinase activity of Cdc2/cyclinB. Immune complexes from the total soluble protein of 12 h un-induced (Lane⁻) and induced (lane+) U2–15 cells using anti-cdc2 monoclonal antibodies were mixed with histone H1 and γ-³²P-ATP at 30°C for 30 min. Autoradiogram (top part) shows much less phosphorylated histone H1 in Lane⁺ than in Lane⁻. [Wb (middle part) for the control of cdc2 protein used in the assay and Coomassie blue-stained blot (bottom part) for H1 loading control]. (B and C) Cdc2 kinase activity is decreased upon Wip1 induction in mouse cells when functional p53 is restored. Similar to the experiment conducted in (A), the corresponding mouse cells used for cell cycle analysis showing a nice G₂/M arrest were used for preparing cell lysates, and subsequently Cdc2 IP and kinase assay were performed. The decreased Cdc2 kinase activity was found when Wip1 was induced in clone 23 cells (B) but not in MF-41 cells (C) at 32°C.