(A) PLK1 phosphorylates CDK-phosphorylated RAD9. PLK1 or PLK1-KR (K82R) (0.3 pmol) was incubated with GST-RAD9 (3A) (10 pmol; phosphorylated with the CDK complex (0.5 pmol) for 30 min at 30 ˚C), at 30 ˚C for the indicated time points in the presence of roscovitine (10 µM). Samples were obtained and western blot analyses using α-RAD9 (GST-RAD9) and α-pT292 (pT292) antibodies were performed. (B) Top: Samples for mass spectrometry were prepared by an in vitro CDK-PLK1 kinase assay. GST-RAD9 (3A) (5 µg) was incubated with CDK2-Cyclin A2 (0.5 µg), followed by an incubation with PLK1 (5 µg) in a 100 µl reaction. Phosphorylated GST-RAD9 was pulled down using GSH-agarose beads. A 10% portion of the captured materials was subjected to SDS-PAGE and silver staining. Bottom: Mass spectrometry analysis of the PLK1-phosphorylated GST-RAD9 (3A), excised from the bands corresponding to band 0 to band 3 at the top. The total counts of the reliable MS/MS spectra (confidence ≥95%) corresponding to the peptides originating from GST-RAD9 (3A), in which Thr292 (pT292), Thr312 (pT312), Thr313 (pT313), Ser324 (pS324), or Ser326 (pS326), was phosphorylated. (C), (D) PLK1 phosphorylates Thr313 (pT313) and Ser326 (pS326) of RAD9, when RAD9 was pre-phosphorylated by CDK on Thr 292 (pT292). GST-RAD9 (10 pmol) was incubated with or without CDK2-CyclinA2 (0.5 pmol) prior to incubations with different amounts of PLK1 (0, 0.3, 0.8, 2.6, 8 pmol). The western blot is shown in (C). GST-RAD9 (3A) (10 pmol) or GST-RAD9 (3A)-T292A (10 pmol) was incubated with different amounts of PLK1 (0, 0.3, 0.8, 2.6, 8 pmol). The western blot is shown in (D). α-RAD9 (GST-RAD9), α-pT292 (pT292), α-pT313 (pT313), and α-pS326 (pS326) antibodies were used for the western blot analyses in (C) and (D). When the GST-RAD9 was incubated with PLK1, roscovitine (10 µM) was added to inhibit the remaining CDK. (E) 293A T-REx cells stably expressing RAD9-WT-mH (WT) or RAD9-S291A/T292A-mH (T292A) were collected, two hours after UV-irradiation (1 J/m2, 10 J/m2). To inhibit the cellular PLK1, BI2536 (2 µM) was added for 15 min, prior to the harvest. Cell lysates were subjected to immunoprecipitation using α-myc antibody-coated agarose beads. Western blotting analyses of the input (Input) and immunoprecipitates (IP) were performed, using α-RAD9 (RAD9), α-pT292 (pT292), α-pT313 (pT313), and α-pS326 (pS326) antibodies. See also Figure 3—figure supplement 1. PLK1 phosphorylates RAD9 on Thr 313 and Ser 326, Figure 3—figure supplement 2. PLK1 phosphorylates RAD9 on Thr 313 and Ser 326 in vivo and Figure 3—figure supplement 3. The phosphorylation status of RAD9 under various genotoxic stresses.