FIG. 3.

Binding of Skp2 to cyclin A-Cdk2 and Skp1. (A) Skp2 wild type (WT) and mutants. The Skp2 truncation and site-directed point mutants were constructed as described in Materials and Methods. The position of the F box (∼112 to 152) is indicated. Abilities to bind GST-Skp1 and GST-Cdk2-cyclin A, and inhibition of cyclin A-Cdk2 kinase activity, are indicated (see text). ND, not determined. CΔ96 does not contain an H6 tag and hence cannot be purified from bacteria for cyclin A-Cdk2 inhibition assay. (B) Binding of Skp1 and cyclin A-Cdk2 to Skp2 truncation mutants. Wild-type Skp2 (WT) or truncated mutants of Skp2 were translated in a coupled transcription-translation rabbit reticulocyte lysate system in the presence of [³⁵S]methionine. The amount of ³⁵S label in the translated proteins was quantitated by SDS-PAGE (17.5% gel) and phosphorimagery (lanes 1 to 8). The proteins were adjusted to the same amount of labeling (the strongest band was about 10% of the input for the binding experiments) and incubated with bacterially expressed GST-Skp1 (lanes 9 to 16) or GST-Cdk2 and PA-cyclin A (lanes 17 to 24). The GST fusion proteins and associated proteins were then precipitated with GSH-agarose and analyzed by SDS-PAGE (17.5% gel) and phosphorimagery. No significant binding to these proteins was detected when GST was used (data not shown). The positions of molecular size standards (in kilodaltons) are shown on the left.