FIGURE 3.

Plk1 phosphorylates ASAP on S289 in vitro. A, purified, recombinant GST-ASAP, α-casein, or GST was incubated with purified His₆-tagged wild-type Plk1 (His₆-Plk1-WT) or the kinase-dead mutant (N281A) (His₆-Plk1-KD) and [γ-³²P]ATP. The kinase reaction mixtures were then analyzed by SDS-PAGE and autoradiography (upper panel); the gel was also stained with Coomassie Blue (lower panel). B, purified GST, GST-ASAP fragments G1–G6 or GST-ASAP were used in a kinase assay with His₆-Plk1-WT as described in A and analyzed by SDS-PAGE and autoradiography (upper panel); the Coomassie Blue-stained gel is shown in the lower panel. C, in vitro kinase assays were performed using purified GST-ASAP, GST-ASAP mutants (S289A or S369A/S370A), or GST as described in A and analyzed by SDS-PAGE and autoradiography (upper panel); the Coomassie Blue-stained gel is shown in the lower panel.