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Characterisation of the Nek6 and Nek7 mitotic protein kinases

By Laura O'Regan


Entry into mitosis results in a dramatic reorganization of the cellular architecture to allow for segregation of duplicated DNA to nascent daughter cells. This complex biomechanical feat is orchestrated by members of the Cyclin-dependent, Aurora, Polo-like and NIMA-related kinase families. Four NIMA-related kinase proteins are implicated in regulation of mitotic progression, Nek2, Nek9, Nek6 and Nek7. Nek6 and Nek7 are closely related in sequence, encode little more than a catalytic domain and have been implicated in a mitotic NIMA-related kinase cascade downstream of Nek9. Functional data on Nek9 has implicated it in regulation of mitotic spindle architecture and thus Nek6 and Nek7 are also thought likely to function in spindle organization. However, functional data validating such a role is sparse and the roles of Nek6 and Nek7 remain poorly defined. In this thesis I set out to carry out a detailed functional analysis of Nek6 and Nek7, focusing on their proposed roles in mitotic progression. We show that expression of Nek6 and Nek7 mutants whose kinase activity is compromised results in mitotic arrest leading to apoptosis. However, whilst mutants with no activity cause an arrest at metaphase with fragile mitotic spindles, hypomorphic mutants, which retain intermediate levels of activity, result in an arrest in late mitosis. Nek6 and Nek7 interact with γ-tubulin, and interference with Nek6 or Nek7 disturbs the centrosomal localization of γ-tubulin. Nek6 localizes to the microtubules of the mitotic spindle and RNAi depletion of Nek6 leads to destabilization of the spindle microtubules. Thus, Nek6 and Nek7 may function during metaphase to regulate microtubule nucleation both from the spindle poles and from within the spindle itself. Furthermore, we identified the spindle components, Hsp70 and β-tubulin as Nek6 substrates, providing a possible mechanism by which Nek6 may achieve such a role. Finally, we also identified Cortactin A, a regulator of actin dynamics, as a Nek6 substrate. Cortactin A and Nek6 localize to the cleavage furrow of late mitotic cells raising the possibility that Nek6 may be involved in the regulation of membrane dynamics during cytokinesis. Together, the functional data suggests that Nek6 and Nek7 may regulate multiple events during mitosis and the identification of Nek6 substrates provides possible mechanisms by which they might achieve these functions

Publisher: University of Leicester
Year: 2008
OAI identifier:

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