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-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 modeling and in vitro assays, we provide evidence that Bora wraps around the N-terminal lobe of AURKA to position its phospho-Ser112 near AURKA's T-loop, mimicking T-loop phosphorylation. Additionally, Bora transiently interacts with the αC 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 laevis egg extracts. Our results reveal critical molecular details of mitotic kinase activation, which could lead to the development of pathway-specific inhibitors.