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|Title:||The mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization|
|Presented at:||University of Leicester|
|Abstract:||Mitosis is a tightly coordinated process that is regulated by many proteins to ensure the correct segregation of chromosomes and the production of two genetically identical daughter cells. Incorrect mitotic spindle assembly and the incorrect segregation of chromosomes often results in aneuploidy, which gives rise to very severe consequences such as tumorigenesis and cancer. The mitotic kinases, Nek6, Nek7 and Nek9 play a pivotal role within mitotic spindle assembly and their dysregulation has been associated with several cancers and solid tumour formation. Previous work has demonstrated that Nek7 exists in an autoinhibitory conformation as a result of the aromatic side chain of tyrosine-97 protruding into the active site and forming a hydrogen-bond with leucine-180 of the aspartate-leucine-glycine (DLG) motif (which is aspartate-phenylalanine-glycine (DFG) motif in most kinases). Mutation of tyrosine-97 to an alanine (Y97A) or phenylalanine (Y97F) residue results in a hyperactive kinase. The addition of the non-catalytic C- terminal domain of Nek9 relieves the autoinhibition of wild-type Nek7, but the hyperactive Nek7 mutant overrides the effect of the Nek9 C-terminal domain (CTD). This study focuses on mapping the site of the Nek7-Nek9 interaction and key residues involved through the use of GST co-precipitation assays and confirming the binding site using X-ray crystallography. The minimal binding region of Nek9810-828 binds at a novel allosteric site on the C-lobe of Nek7Y97F, but it alone is not sufficient to activate Nek7. Nek7Y97F crystallised as a back-to-back dimer, forming interactions mainly via its N‐lobes. The self-association of the Nek9-CTD via its coiled-coil domain is necessary for the activation of Nek7. Here we propose a novel mechanism for Nek7 activation where Nek9 oligomerises via its coiled-coil domain and binds to oligomers of Nek7, which can subsequently form back-to-back dimers and autoactivate to enhance their kinase activity.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Leicester Theses|
Theses, Dept. of Biochemistry
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