Generating biomarkers of human Nek8 kinase activity

Nek8 is a member of the human NIMA-related serine/threonine protein kinase family that has roles in cell cycle progression, primary cilia function and the DNA damage response. Mutations in the human nek8 gene cause Nephronophthisis (NPHP), an autosomal recessive polycystic kidney disease in which defects in primary cilia lead to a broad range of often severe symptoms that affect many organs of the body. In the kidneys, NPHP causes the development of cortico-medullary cysts that impair kidney function and ultimately lead to kidney failure. Nek8 localises to the primary cilium where it interacts with Inversin and PC-2, products of genes that are also mutated in inherited cystic kidney diseases. Nek8 also plays a role in the intra-S phase DNA damage checkpoint where it contributes to cell cycle arrest by inhibiting CDK2 activity to allow time for repair of stalled replication forks. The purpose of this study was to generate novel biomarkers of Nek8 kinase activity that could be used to shed light on its role at the primary cilium and in the replication stress response. Our aim was therefore to identify phosphosites of Nek8 in its potential substrate proteins, Inversin and PC-2, and to generate phospho-specific antibodies against these sites. We chose three sites in the Inversin N-terminus for phospho-specific antibody generation and showed that these were capable of detecting purified, phosphorylated Inversin upon incubation with Nek8. Second, we characterised a phospho-Nek8 antibody that recognises a phosphorylation site in the activation loop of the kinase. This antibody was capable of detecting autophosphorylated Nek8 by Western blot and immunofluorescence microscopy as confirmed using two, newly identified chemical inhibitors of Nek8. Localisation studies with this antibody revealed novel data on the presence of active Nek8 at centrosomes, cilia and sites of DNA damage. Finally, we found that Nek8 inhibition was associated with generation of enlarged multinucleated cells and accumulation of DNA damage foci. Together, these data support a role for Nek8 in linking ciliary signalling pathways and the DNA damage response, while the phospho-specific antibodies represent a new set of tools that can be used to explore Nek8 function in normal and pathological states.