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The Nek2 protein kinase: its phosphorylation, activation and inhibition.

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posted on 2010-03-15, 11:11 authored by Donna-Marie Cheary
Nek2 is a cell cycle-regulated protein kinase localised to the centrosome. Nek2 is involved in regulating mitotic events and centrosomal activity through phosphorylation of various substrates including C-Nap1, Rootletin and PP1. Nek2 is overexpressed in cancer cell lines and primary tumours. Overexpression of active Nek2 is characterised by the premature splitting of centrosomes and altered microtubule nucleation. Activation of Nek2 is dependent upon dimerisation and autophosphorylation. Using mass spectrometry, 13 sites of autophosphorylation have been identified in the Nek2 kinase. Individual phosphorylation sites were mutated and the effect that this had on Nek2 activity in vitro and in vivo was characterised. Phosphomimiking mutation of T170 or S171 created hyperactive Nek2A kinases. However, mutation of the C-terminal autophosphorylation sites had no effect upon Nek2A activity. The proposal that the C-terminal domain of Nek2 acts as an autoinhibitory domain was investigated although the removal of C-terminal residues did not create a hyperactive Nek2 kinase. An investigation into the role of a novel αT-helix found at the N-terminal end of the T-loop of an inactive Nek2 crystal structure, found that mutation of αT-helix residues resulted in loss of Nek2A kinase activity. Phospho specific antiserum directed against the Nek2 T175 autophosphorylation site was purified and characterised. This reagent should prove to be a valuable tool for detection of active Nek2 kinase in tumours as well as cells in culture. Furthermore, the intramolecular interactions of C-Nap1 and Nek2 were investigated to determine how centrosome cohesion is regulated. Protein binding assays revealed that C-Nap1 can form hetero- and homo-dimers and that the ability of C-Nap1 to heterodimerise is compromised as a consequence of phosphorylation by Nek2. Finally, the consequences of Cdk1 inhibition upon Nek2A activity and centrosome cohesion were investigated. Inhibition of Cdk1 in cells induced a substantial G_2/M arrest accompanied by premature centriole disengagement resulting in multipolar spindle formation observed in cells released from Cdk1 inhibition.

History

Supervisor(s)

Fry, A.

Date of award

2009-10-23

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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