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|Title:||Regulation of the human Nek8/NPHP9 protein during cell cycle progression and ciliogenesis|
|Presented at:||University of Leicester|
|Abstract:||The primary cilium, once considered an evolutionary vestige, has re-emerged as an essential organelle for the normal functioning of a wide variety of cellular processes. It is now clear that it has important roles in the control of cell proliferation and signalling during development, with defects in primary cilia being a major cause of many human diseases. These disorders, collectively referred to as ciliopathies, include the cystic kidney diseases that are characterized by aberrant cell proliferation and cyst formation. Mutations in the NIMA-related kinase, Nek8, are associated with cystic kidney disease in both humans and mice, with Nek8 being the candidate NPHP9 gene in the human juvenile cystic kidney disease, nephronophthisis. Previous localisation studies have shown that Nek8 localises to centrosomes and cilia in dividing and ciliated cells, respectively, strengthening the ciliary hypothesis of cystic kidney disease. However, the role of Nek8 in ciliogenesis remains to be defined and no substrates for this kinase have yet been identified. In this thesis, I present data from a series of biochemical and cell biology experiments aimed at investigating the regulation and function of Nek8 with respect to cell cycle progression and ciliogenesis. I first of all show that localization of Nek8 to centrosomes and cilia is dependent upon both its kinase activity and its C-terminal non-catalytic RCC1 domain. Interestingly, Nek8 was capable of phosphorylating the RCC1 domain, which in isolation also localized to centrosomes and cilia. This leads us to propose that centrosome recruitment is mediated by the RCC1 domain, but requires a conformational change in the full-length protein that is promoted by autophosphorylation. I then established conditions required for measurement of Nek8 catalytic activity. Besides confirming the predicted activity of catalytic site mutants, this revealed that the three NPHP9-associated point mutants did not exhibit loss of activity. However, as one of these mutants, H425Y, failed to localize correctly, we predict that this mutation, which lies in the RCC1 domain, alters the RCC1 conformation such that it disrupts its centrosome targeting motif. Importantly, I also show that serum starvation induces proteasomal degradation of Nek8, specifically in cell lines in which serum starvation induces quiescence and ciliogenesis. Strikingly, serum starvation also induces Nek8 kinase activity, whilst maintained expression of Nek8 appears to suppress ciliogenesis. Taken together, these findings not only reveal important mechanisms through which Nek8 activity and localization are regulated, but suggest that Nek8 itself may have both positive and negative activities in the process of ciliogenesis.|
|Rights:||Copyright © the author, 2012.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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