Activator mimicry by PP6 regulatory subunits directs Aurora A dephosphorylation in mitosis.

Aurora A kinase is a conserved regulator of mitotic spindle assembly whose activity must be precisely balanced to ensure genome stability. Its activation at centrosomes and spindle microtubules is promoted by CEP192, TACC3 and TPX2, and counteracted by protein phosphatase 6 (PP6), which dephosphorylates the Aurora A T-loop. PP6 forms three holoenzymes defined by regulatory subunits PPP6R1-R3, yet how these confer substrate recognition and regulation is poorly understood. Here, we identify a phenylalanine-rich motif (A-box), present in PPP6R2 and PPP6R3 but absent from PPP6R1, that binds a surface on Aurora A overlapping those used by CEP192, TPX2 and TACC3. Mutation of A-box phenylalanine residues disrupts Aurora A binding and T-loop dephosphorylation, elevating spindle-associated Aurora A activity, and enlarging mitotic spindles. PPP6R2 additionally contains a unique PLK1-docking site downstream of its A-box, explaining how PLK1 competes with Aurora A for PPP6R2 and thereby suppresses PP6 activity towards Aurora A. Together, these findings show that PP6 regulatory subunits mimic Aurora A activators to direct dephosphorylation, and reveal how competing interactions regulate PP6 in mitosis.