Characterization of NLP, a novel centrosomal substrate of the NEK2 kinase

The centrosome is a non-membraneous organelle composed of two centrioles surrounded by pericentrolar material. The primary function of the centrosome is to act as the dominant microtubule organizing centre in animal cells. It therefore contributes to formation of both the interphase cytoskeleton and bipolar mitotic spindle. Both centrosome structure and microtubule organization are controlled in a cell cycle dependant manner by protein phosphorylation. The centrosomal kinase Nek2 regulates centrosome organization, mitotic progression and bipolar spindle assembly. However, the only core centrosomal substrate of this kinase so far identified is C-Napl, a structural protein required for centriolar cohesion. The aims of this project were therefore to isolate a Xenopus laevis homologue of C-Napl in order to study its function using Xenopus based cell free assays and identify and characterize novel centrosomal substrates of Nek2. By database screening, we identified a Xenopus protein of high similarity to C-Napl called rootletin, while using the yeast two hybrid system we identified the Xenopus Nip protein as a novel substrate of Nek2. Human Nip is a recently characterized centrosomal protein involved in microtubule organization and which is regulated by another centrosomal kinase, Plkl. Antibodies were raised to Xenopus Nip and used to confirm subcellular localization to the centrosome in Xenopus cells. Further localization and expression studies revealed that Nip is a mother centriole specific protein that is displaced from the centrosome, but not degraded, during mitosis. These data suggest that Nip is involved in interphasic microtubule anchorage. By transfection into Xenopus and human cells, we found that although Nek2 and Plkl phosphorylate Nip at distinct sites, they can both trigger Nip displacement from the centrosome at the onset of mitosis. Finally, data were obtained raising the possibility that Nek2 may act as a novel priming kinase for recruitment of Plkl to its substrate Nip