Cell cycle regulation of the centrosomal kinase Nek2 and its substrate C-Nap1
thesisposted on 15.12.2014, 10:32 by Rebecca. Hames
The centrosome is the major microtubule organising centre in animal cells. In mitosis, it forms the two poles of the mitotic spindle upon which chromosomes are segregated. During cell cycle progression the centrosome undergoes a series of major structural and functional transitions that are regulated in part by phosphorylation. The aims of this thesis have been to investigate the regulation and function of the centrosomal kinase Nek2, which has previously been implicated in regulating centrosome cohesion at G2/M. Nck2 kinase activity is regulated by a number of mechanisms, including autophosphorylation and interaction with protein phosphatase 1. Here, we have identified two further mechanisms of regulation: (i) the expression of human Nek2 as two alternative splice variants, Nek2A and Nek2B; (ii) the specific destruction of Nek2A by the proteasome in early mitosis. This destruction follows ubiquitylation by the APC/C-Cdc20 complex and depends on a novel destruction motif which is highly related to the extended D box present in cyclin A. Previous work has indicated that Nek2 may regulate centrosome cohesion through phosphorylation of the core centrosomal protein C-Nap1. Here we show that C-Nap1 interacts with and is phosphorylated by both Nek2A and Nek2B at two distinct domains. Furthermore, both Nek2 and C-Nap1 associate with microtubules, suggesting that they may move along microtubules in a dynein dependent manner. Finally, using C-Nap1 as a bait in a yeast two hybrid interaction screen, the novel centrosomal protein BPC-1 was isolated BPC-1 also interacted with Nek2 and may inhibit the centrosome cohesion function of Nek2. This work substantially advances our understanding of Nek2 regulation and brings us closer to understanding how centrosome cohesion is regulated through specific phosphorylation of components such as C-Nap1.