FIG. 8.

Cdc25A is activated in vivo by Cdk2. (A) Analysis of Cdc25A-associated kinase activity. (A, top panel) Control-infected and Ad.p16-infected MCF-7 cells were growth arrested and treated for 12 h with E₂. Lysates were precleared and incubated with GST-Cdc25A-coated beads, and histone kinase activity associating with Cdc25A was measured as given in Materials and Methods. (A, middle panel) The in vitro assay of Cdc25A-associated kinase activity was carried out with the addition of flavopiridol, roscovitine, PD9059, or geldanamycin (all 2.5 μM). DMSO was used as a solvent control. Lysates were from estrogen-treated MCF-7 cells. (A, bottom panel) Immunodepletion analysis of Cdc25A-associated kinase activity is shown. Lysates of estrogen-treated MCF-7 cells were subjected to immunodepletion with the indicated specific antibodies or control goat IgG before incubation with Cdc25A beads and assay. (B) Reversal of Cdc25A inhibition in vivo. MCF-7 cells were infected with Ad.Con and Ad.p16 vectors along with Ad.Con, Ad.cycE, or Ad.Raf-1^caax as indicated. Cdc25A activity was assayed following growth arrest and a 20-h treatment with E₂. (C) Reactivation of Cdc25A in vitro. Cdc25A immunoprecipitates from lysates of estrogen-treated Ad.p16-infected MCF-7 cells prepared as described above were incubated in vitro with soluble, active cyclin A-Cdk2 complexes as described in Materials and Methods and assayed for activation of cyclin B1-Cdc2 complexes. Activity in Cdc25A immunoprecipitates from Ad.Con-infected cells is given for comparison. In panels C and D, relative activity based on densitometry is given above the respective lanes. (D) Activation and inhibition of ectopic Cdc25A activity. MCF-7/tTA cells were transfected with pBI-HACdc25A along with control plasmid (pcDNA3), pBPSTRI-p16 (p16), or dominant-negative Cdk2 vector (DNCdk2). Following growth arrest and E₂ treatment (20 h), Cdc25A activity was assayed in anti-HA immunoprecipitates as given in Materials and Methods. Relative activity is given under each lane. In the lower panel, equal expression of HA-Cdc25A was verified by anti-Cdc25A immunoblot analysis of anti-HA immunoprecipitates prepared from the same lysates.