Cell Cycle Studies on the Human Nek3, Nek5 and Nek11 Protein Kinases

The NIMA-related kinases represent a family of serine/threonine protein kinases implicated in cell cycle control and ciliogenesis. The founding member of this family, the NIMA kinase of Aspergillus nidulans, contributes to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organisation and mitotic exit. Mammals contain a family of eleven NIMA related kinases, named Nek1 to Nek11. Of these, to date there is substantial evidence that Nek2, Nek6, Nek7 and Nek9 regulate mitotic spindle formation, while Nek1 and Nek8 are implicated in microtubule organisation in non-dividing cells during ciliogenesis. The common underlying theme between spindle formation and ciliogenesis is the role of microtubules and microtubule organising centres. Hence, the emerging hypothesis is that all Neks may play a role in the organisation of microtubules. However, for several Neks, little if any information is available. In this thesis, I therefore began to examine the properties of three less well researched human Neks, Nek3, Nek5 and Nek11. First of all, antibodies against each of these kinases were generated or characterised. This allowed a broad study of the localisation, expression and activity of these kinases. Intriguingly, Nek3, Nek5 and Nek11 were all localised to centrosomes. Based on these results a more detailed study was performed with human Nek5 for which there is currently no published data. Human Nek5 is a nuclear protein that localises to the centrosomes during interphase, spindle poles during early mitosis, and to basal bodies in mono- ciliated cells. Specifically, Nek5 localised at the proximal ends of centrioles peaking in intensity in early mitosis. Importantly, siRNA-mediated depletion of Nek5 results in premature centrosome splitting and loss of centrosomal γ-tubulin. Hence, Nek5 is uncovered as a potential player in the negative regulation of centrosome separation.