Fig 2: AURKAT288V supports AURKA function in maintaining the mitotic state in CSF-arrested Xenopus oocytes.a In the ovary, oocytes are arrested in prophase of meiosis I. At the time of ovulation and upon hormone stimulation (progesterone), oocytes re-enter into meiosis and reach the second meiotic division where they arrest in metaphase II, awaiting fertilization (CSF arrest). Upon Gwl immunodepletion (?Gwl in red), the extracts exit the “mitotic state”. Complementation of the extract with recombinant hyperactive GwlK72M kinase (+GwlK72M in green) restores the mitotic state, if the extracts contain a functional Bora, AURKA, Plk1 pathway. GVBD: germinal vesicle breakdown. The green and red area represents the high and low CyclinB-Cdk1 (MPF) activity. b Network representing the pathways controlling Cyclin-Cdk activity and maintaining the “mitotic state” in CSF-arrested Xenopus egg extracts. c Flow chart of the experimental approach used to test the functionality of AURKA mutants in maintaining the “mitotic state” in CSF-arrested Xenopus oocytes extracts. Endogenous AURKA and Gwl were sequentially immunodepleted from CSF egg extracts and then complemented with recombinant human AURKAWT, catalytically dead AURKAD274N, or downregulated mimic AURKAT288V and hyperactive GwlK72M kinase (K72M) (top panel). CSF extracts non-depleted or sequentially depleted of AURKA (?AURKA) and then Greatwall (?Gwl) were separated by SDS-PAGE and analyzed by Western blot using Xenopus Gwl and AURKA antibodies. Asterisks denote non-specific bands. d CSF egg extracts sequentially depleted of AURKA (?AURKA) and then Gwl (?Gwl) were supplemented at time 0 with recombinant AURKAWT, or catalytically dead AURKAD274N or AURKAT288V in the presence of hyperactive GwlK72M. A fraction of the extracts was collected at the indicated time-points (0, 20, 40, 60, 90 min) and analyzed by Western blot using specific antibodies to monitor the levels of huGreatwall, Hu/Xe AURKA, Plk1, Cdc25, as well as the phosphorylation of Plx1 on T201 (pPlk1), PP1 phosphatase on T320 (pPP1), and Cdk on Y15 (pTyr). The green and red area represents the high and low CyclinB-Cdk1 (MPF) activity (“mitotic state”).

Fig 3: S112 residue and Tpx2-like motifs are essential for Bora function during the mammalian cell cycle.a Schematic for the structure–function analysis of Bora during the mammalian cell cycle. Endogenous Bora located on chromosome (Chr) XIII was C-terminally tagged with Neongreen (NG) and the Auxin-Inducible degron (AID) in DLD1 cells constitutively expressing the Tir1 subunit of the SCFTIR1 E3-ligase. A wild-type or a mutant copy of untagged Bora driven by the doxycycline promoter (pTRE3G) was inserted on Chr XIX of the same cells using CRISPR/Cas9 genome editing. Doxycycline promotes expression of untagged wild-type or mutant Bora, while Auxin induces SCFTIR1-mediated proteasome degradation of Bora-NG-AID resulting in a Bora knockdown. b Workflow used to follow the progression of the cells through the cell cycle. Expression of BoraWT or the indicated mutants were induced with doxycycline during the second thymidine block. Bora-NG-AID was then depleted at the time of release from DTB using Auxin. Cells were collected 10 and 13 h after DTB release for Western blot and FACS analyses. c Histogram plots showing the cell cycle distributions (percentage of cells in G1, S, and G2/M) for DLD1-Bora-NG-AID cell lines, expressing either BoraWT or Bora[S41A S112A S137A] and treated as outlined in b. AS: Asynchronous cells; Ctrl: non-treated; A: Auxin-treated cells; A/D: Doxycyclin + Auxin-treated cells. d Box plots indicate median (middle line) and min max (Wiskers) showing the quantified rescue index (RI) for each DLD1 Bora-NG-AID cell line expressing BoraWT (in dark green) or the indicated mutants: Bora[S41A S112A S137A] (in red), BoraS41A (in blue), BoraS112A (in orange), BoraS137A (in green), Bora?35 (in magenta), Bora[F25D Y31D] (in pink), and Bora[F103D F104D] (in purple) assessed 10 (first column) and 13 (second column) hours after DTB release; n indicates the number of replicates. e Western blot analysis of protein lysates from a DLD1-Bora-NG-AID cell line expressing BoraWT or the indicated mutants 10 h after DTB release as depicted in b, c, and d. Bora, pT210-Plk1, and panPlk1 antibodies were used for these analyses (from top to bottom). (-) Asynchronous cells.

Fig 4: Bora is an intrinsically disordered protein that binds AURKA.a NMR analysis of the binding interaction between non-phosphorylated 15N-labeled Bora (15N-Bora) and unlabeled AURKA and Plk1. On the left, superimposed 1H-15N HSQC spectra of 15N-Bora alone in black with 15N-Bora in red in the presence of two moles equivalents [1:2] of Plk1 KD. On the right, 15N-Bora alone in black with 15N-Bora in blue in the presence of two moles equivalents [1:2] of AURKA. b NMR analysis of the binding interaction between CyclinA2-Cdk2 phosphorylated 15N-Bora and unlabeled AURKA and Plk1. On the left, superimposed 1H-15N HSQC spectra of phosphorylated 15N-Bora alone in black with phosphorylated 15N-Bora in red in the presence of two moles equivalents [1:2] of Plk1 KD. On the right, phosphorylated 15N-Bora alone in black with phosphorylated 15N-Bora in blue in the presence of two moles equivalents [1:2] of AURKA. c Pull-down experiments between immobilized biotinylated AVI-tagged Bora1–224 (bAVI-Bora1–224) or biotinylated AVI-tagged pBora1–224 and AURKA (left panel), Plk1 KD (middle panel), or a mix of AURKA and Plk1 KD (right panel). d, e SPR binding analysis of immobilized Bora1–224 (d) and pBora1–224 (e) to AURKAWT. Kd values represent mean values, n = 2. Representative profiles shown are from one experiment. See Supplementary Fig. 2d, e, respectively, for sensogram traces.

Fig 5: Bora contains Tpx2-like motifs and a unique phosphosite required for its activation function on AURKA in vitro.a Schematic of Bora1–224 highlighting ten S/T-P consensus sites that are phosphorylatable by the proline-directed kinases Cyclin-Cdk or ERK. Residue position of the phospho-sites are indicated and highlighted by the letter P (above). Also highlighted are two Cyclin-binding motifs (Cy, violet) and two Tpx2-like motifs 1 and 2 (green). The boundaries of the different Bora fragments analyzed in b–d, are indicated. The most essential phospho-site S112 is highlighted by a large P. b Western blot analysis of 2-step kinase reactions carried out with the indicated Bora fragments phosphorylated (+) or not (-) by the ERK kinase (step 1) in the presence of Plk1K82R KD substrate and AURKAWT or AURKAT288V (step 2). Blots were probed with antibodies to Bora, AURKA, and phosphoT210 Plk1 or pan Plk1 as indicated (from top to bottom). c Alignments of Bora motifs M1 and M2 with Tpx2 motifs responsible for binding to AURKA. Essential residue numbers in Tpx2 responsible for binding AURKA are highlighted in red below the alignments. The corresponding residue numbers in Bora are highlighted in red above the alignments. Also shown is a phospho-motif M3 in Bora not conserved in Tpx2. d Western blot analysis of kinase reactions carried out with the indicated Bora18–120 mutants (S27A and T52A plus/minus mutations within motifs M1 or M2) phosphorylated (+) or not (-) by the ERK kinase (step 1) in the presence of Plk1K82R KD and AURKAWT or AURKAT288V (step 2). Blots were probed with antibodies to Bora, AURKA, and phosphoT210 Plk1 or pan Plk1 as indicated (from top to bottom). Note that the Bora18–120 [S27A T52A F103D F104D] mutant was not recognized by our Bora antibody but Coomassie staining of the step1 reaction (bottom panel of step 1) revealed the presence of the protein at the expected size.