(A) HeLa cells were controltransfected or transfected with siRNAs against Bora (siBora-A & −B). Knockdown efficiency was determined by Western blotting for Bora, Plk1 and p38MAPK (a loading control). (B–E) HeLa cells were synchronized by a double-thymidine treatment and transfected with a control siRNA or siBora-A during the 2^nd thymidine arrest. Cells were released into fresh media and Taxol added at 9 hours post-release to prevent mitotic exit. Cell cycle profile was analyzed by FACS (B–C) and by Western blotting (D). Plk1 from selected timepoints was immunopurified and assayed for its kinase activity using casein as a substrate (E). The Plk1 activity in (E) was normalized to the TT0 sample in siControl. Filled symbols, siControl; Open symbols, siBora. Triangles, G1; Squares, G2/M; Circles, M. Knockdown by siBora-B gave a similar phenotype. (F–G) HeLa cells were transfected with siBora-A, synchronized by a single thymidine arrest and harvested 8 hr post-release (T8) (F) or synchronized by a thymidine-nocodazole arrest and harvested by mitotic shake-off (TN0) (G). Cell cycle profile was determined by FACS (F) and indicated proteins assayed by Western blotting of total cell lysates (F–G). Partial synchronization in (F) enriched G2/M cells and generated cells of similar cell cycle distribution between control and Bora knockdown. (H–I) HeLa cells were transfected with GFP or GFP-Bora. Asynchronous (H) or prometaphase (I) cells were collected at 34 and 48 hours post-transfection, respectively. Prometaphase cells were enriched by a thymidine-nocodazole arrest and harvested by mitotic shake-off. Indicated proteins were analyzed by Western blotting.

(A–C) HeLa cells were synchronized by a double-thymidine treatment and transfected with a control siRNA or siRNA against Aurora A (siAurora A) during the 2^nd thymidine arrest. Cells were released into fresh media and Taxol added at 9 hours post-release to prevent mitotic exit. Cell cycle profile was analyzed by FACS (A–B) and by Western blotting (C). Mad2 served as a loading control. Filled symbols, siControl; Open symbols, siAurora A. Triangles, G1; Squares, G2/M; Circles, M. Inhibition of Aurora A by VX680 (30) in G2 cells released from the double-thymidine arrest resulted in similar phenotypes (data not shown). (D, F) HeLa cells were transfected with myc-Aurora A/myc (D) or with GFP-Bora/GFP (F) and synchronized by a single thymidine arrest. Seven (F) or eight and half (D) hours post-release, cells were treated with 1 µM VX-680 or DMSO and harvested one hour later. Indicated proteins were assayed by Western blotting of either total cell lysates or the Plk1 immunoprecipitates. Cell cycle profile was determined by FACS to ensure that any change in the Plk1-T210-P level was not due to an altered cell cycle distribution among samples. (E) HeLa cells were transfected first with a control siRNA or siBora-A and then with myc-Aurora A. Cells were synchronized by a single thymidine arrest and harvested eight hours post-release. Cell cycle profile was determined by FACS and indicated proteins assayed by Western blotting of either total cell lysates or the Plk1 immunoprecipitates. (G) Summary on the regulation of Plk1 by Bora and Aurora A in the cell cycle.

(A, B & D) HeLa S3 cells were synchronized at the G1/S boundary by a double-thymidine arrest (TT), released into fresh media and harvested at indicated times. Cell cycle stages were assayed by FACS and levels of indicated proteins analyzed by Western blotting of total cell lysates (A). p38MAPK served as a loading control. Plk1 was immunopurified from cell lysates and blotted for Plk1 and Bora or assayed for Plk1 kinase activity using casein as a substrate (B). The Bora complex was immunoprecipitated from cell lysates and blotted for Plk1, Plk1-T210-P, Aurora A and Bora (D). Cdk1-Y15-P in (A) represents the inactive Cdk1 phosphorylated on Tyr15. (C) The Bora complex was purified from G2 cells stably expressing the GFP and S-peptide tagged Bora. Eluates of the tandem-affinity purified Bora complexes were separated by SDS–PAGE and visualized by silver staining. In addition to the proteins labeled in this panel, other interacting proteins identified include β-TrCP1, β-TrCP2, Cul1 and Skp1, which are subunits of the β-TrCP/SCF ubiquitin ligase responsible for degradation of Bora in mitosis (21). These proteins were not detected by silver-staining due to their low abundance. The identity of the band between 116 kDa and 200 kDa remains to be determined. Mw, molecular weight markers. (E) Recombinant MBP-Bora and MBP were incubated with in vitro synthesized ³⁵S-Plk1 and then purified by anti-MBP antibody beads. Bora and associated Plk1 were analyzed by SDS-PAGE. CB, Coomassie blue staining. (F) Plk1 complexes immunoprecipitated from TT9 cell lysates were treated with λ-phosphatase (λPPase) and washed. Materials remained on Plk1 beads were blotted for Bora and Plk1.

(A) Plk1 T-loop and Aurora A recognition sequences. (B) Recombinant Plx1 (purified from asynchronous Sf9 cells) and Histone H3 were mixed together and then incubated with Aurora A and MBP-Bora/MBP for 60 min in the presence of radioactive ATP and BI 2536, a Plx1 inhibitor (3). The relative ³²P incorporation into Plx1 and H3 were plotted after normalizing to their respective samples in lane 3. (C & J) Myc-Plk1 and myc-Plk1-K were expressed in 293 cells by transient transfection, immunopurified and incubated with or without recombinant Aurora A and MBP-Bora/MBP. Myc-Plk1/myc-Plk1-K beads were then washed to remove Aurora A and assayed for Plk1 kinase activity or by Western blotting (WB). 293 cells without transfection were used as a negative control in (C). (D–F) Recombinant Plx1 and Plx1-T201D were sequentially incubated with or without λPPase, with EGTA (to stop the λPPase reaction), and then with or without Aurora A and MBP-Bora/MBP. Plx1/Plx1-T201D were then analyzed by Western blotting or immunopurified away from Aurora A to assay for Plx1 kinase activity. Inactive Plx1 (D) and Plx1-T201D (F) were purified from asynchronous Sf9 cells without okadaic acid treatment, whereas active mitotic Plx1 (E) was purified from okadaic-acid-treated mitotic Sf9 cells (7). In a separate experiment in (D), the kinetics of the Plx1 kinase activity was analyzed and plotted after normalizing all the samples to that of −Bora−Aurora A at the 15 min timepoint. Diamonds, +Bora+Aurora A; Squares, −Bora+Aurora A; Circles, +Bora−Aurora A; Triangles, −Bora−Aurora A. (G) Phosphorylation of Plx1-T201D by Aurora A, as described in (B). (H–I) Recombinant MBP-Bora/MBP/GST-PBD/GST were incubated with in vitro synthesized ³⁵S-proteins and then purified with anti-MBP antibody beads or Glutathione Sepharose. Associated proteins were analyzed by SDS-PAGE. CB, Coomassie blue staining.