Fig. 5: Bora contains Tpx2-like motifs and a unique phosphosite required for its activation function on AURKA in vitro.

a Schematic of Bora^1–224 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 Plk1^K82R KD substrate and AURKA^WT or AURKA^T288V (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 Bora^18–120 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 Plk1^K82R KD and AURKA^WT or AURKA^T288V (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 Bora^{18–120 [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.