Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

The evolutionarily conserved, intrinsically disordered protein Bora is critical for initiating the activation of mitotic kinases. Once phosphorylated at Ser112 by Cyclin A-Cdk1 kinase, phospho-(p)Bora activates unphosphorylated Aurora A kinase (AURKA), directing it towards Polo-like kinase 1 (Plk1), thus promoting Cyclin B-Cdk1 activation and mitotic entry. Here, by combining structural modelling and in vitro assays, we provide evidence that Bora wraps around the N-terminal lobe of AURKA to position its phospho-Ser112 near AURKAs T-loop, mimicking T-loop phosphorylation. Additionally, Bora transiently interacts with the Alpha C helix of the Plk1 kinase domain through a conserved motif, guiding AURKA activity towards the Plk1 T-loop, which is otherwise impervious to phosphorylation by AURKA. We highlight the importance of this motif for Bora function in vitro and during mitotic entry in Xenopus egg extracts. Our results reveal critical molecular details of mitotic kinase activation, which could lead to the development of pathway-specific inhibitors. HighlightsO_LIMITOKINAC: a versatile approach that reconstitutes Bora and AURKA-dependent Plk1 T-loop phosphorylation in E. coli. C_LIO_LIAlphaFold 3 structural modeling of the phospho-Bora - AURKA complex tested by MITOKINAC using 39 Bora variants. C_LIO_LIPhospho-Bora wraps around the N-terminal lobe of AURKA to position its essential phospho-Serine 112 in the activation loop of AURKA. C_LIO_LIpBora delivers the AURKA activity to the Plk1 T-loop, which is otherwise impervious to AURKA phosphorylation. C_LI