Figure 3. Chemical rescue of Cdk2^as by 6-BAP.

(A) Wild-type and Cdk2^as/as RPE-hTERT cells were synchronized by contact inhibition and released for 27 hr into medium containing DMSO, 1 μM 3-MB-PP1, or 10 μM of indicated compound. Cyclin A and Cdk2 were detected by immunoblotting, and rescue of Cdk2^as was assessed by calculating ratio of phosphorylated-to-unphosphorylated Cdk2 in each extract.

(B) Cells synchronized as in (A) were released into indicated concentration of 3-MB-PP1 or 6-BAP, collected after 25 hr, and phosphorylated-to-unphosphorylated Cdk2 ratio determined.

(C) The ability of 6-BAP to restore normal cyclin A pairing was determined as in Figure 2A.

(D) Extracts from (C) were labeled with [γ-³²P]N6-(benzyl)-ATP to determine effect of treating cells with 6-BAP on amount of active Cdk2^as in extracts.

(E) Cdk2^as/cyclin A and Cdk2^WT/cyclin A complexes were tested for sensitivity to 3-MB-PP1 by incubation with increasing concentrations of drug prior to histone H1 kinase assay. IC₅₀ for Cdk2^as/cyclin A is ~3 nM.

(F) As in (E) for 3-MB-PP1, IC₅₀ of 6-BAP for Cdk2^as/cyclin A was determined to be ~1 μM.