The NIMA-family kinases Nek9/Nercc1, Nek6 and Nek7 form a signalling module required for mitotic spindle assembly. Nek9, the upstream kinase, is activated during prophase at centrosomes although the details of this have remained elusive. We now identify Plk1 as Nek9 direct activator and propose a two-step activation mechanism that involves Nek9 sequential phosphorylation by CDK1 and Plk1. Furthermore, we show that Plk1 controls prophase centrosome separation through the activation of Nek9 and ultimately the phosphorylation of the mitotic kinesin Eg5 at Ser1033, a Nek6/7 site that together with the CDK1 site Thr926 we establish contributes to the accumulation of Eg5 at centrosomes and is necessary for subsequent centrosome separation and timely mitosis. Our results provide a basis to understand signalling downstream of Plk1 and shed light on the role of Eg5, Plk1 and the NIMA-family kinases in the control of centrosome separation and normal mitotic progression.

The accumulation of γ-tubulin at the centrosomes during maturation is a key mechanism that ensures the formation of two dense microtubule (MT) asters in cells entering mitosis, defining spindle pole positioning and ensuring the faithful outcome of cell division. Centrosomal γ-tubulin recruitment depends on the adaptor protein NEDD1/GCP-WD and is controlled by the kinase Plk1. Surprisingly, and although Plk1 binds and phosphorylates NEDD1 at multiple sites, the mechanism by which this kinase promotes the centrosomal recruitment of γ-tubulin has remained elusive. Using Xenopus egg extracts and mammalian cells, we now show that it involves Nek9, a NIMA-family kinase required for normal mitotic progression and spindle organization. Nek9 phosphorylates NEDD1 on Ser377 driving its recruitment and thereby that of γ-tubulin to the centrosome in mitotic cells. This role of Nek9 requires its activation by Plk1-dependent phosphorylation but is independent from the downstream related kinases Nek6 and Nek7. Our data contribute to understand the mechanism by which Plk1 promotes the recruitment of γ-tubulin to the centrosome in dividing cells and position Nek9 as a key regulator of centrosome maturation.

The NIMA family protein kinases Nek9/Nercc1 and the highly similar Nek6 and Nek7 form a signaling module activated in mitosis, when they are involved in the control of spindle organization and function. Here we report that Nek9, the module upstream kinase, binds to DYNLL/LC8, a highly conserved protein originally described as a component of the dynein complex. LC8 is a dimer that interacts with different proteins and has been suggested to act as a dimerization hub promoting the organization and oligomerization of partially disorganized partners. We find that the interaction of LC8 with Nek9 depends on a (K/R)XTQT motif adjacent to the Nek9 C-terminal coiled coil motif, results in Nek9 multimerization, and increases the rate of Nek9 autoactivation. LC8 binding to Nek9 is regulated by Nek9 activity through the autophosphorylation of Ser 944 , a residue immediately N-terminal to the (K/R)XTQT motif. Remarkably, LC8 binding interferes with the interaction of Nek9 with its downstream partner Nek6 as well as with Nek6 activation, thus controlling both processes. Our work sheds light into the control of signal transduction through the module formed by Nek9 and Nek6/7 and uncovers a novel manner in which LC8 can regulate partner physiology by interfering with protein complex formation. We suggest that this and other LC8 functions can be specifically regulated by partner phosphorylation.

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