Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis
[eng] Nek9 is a NIMA-related kinase that is phosphorylated in mitosis and a small pool of it (5%) is activated at the centrosomes by a complex mechanism which has remained elusive until now. Nek6 and Nek7 bind to the C-terminal tail of Nek9 and are directly phosphorylated and activated by Nek9, thus...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2012 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/36327 |
| Acceso en línea: | https://hdl.handle.net/2445/36327 http://hdl.handle.net/10803/96415 |
| Access Level: | acceso abierto |
| Palabra clave: | Fosforilació Proteïnes quinases Mitosi Phosphorylation Protein kinases Mitosis |
| Sumario: | [eng] Nek9 is a NIMA-related kinase that is phosphorylated in mitosis and a small pool of it (5%) is activated at the centrosomes by a complex mechanism which has remained elusive until now. Nek6 and Nek7 bind to the C-terminal tail of Nek9 and are directly phosphorylated and activated by Nek9, thus forming with Nek9 a signalling cassette of mitotic kinases. Once active, Nek6 and Nek7 phosphorylate the kinesin Eg5 at the Ser1033, a residue that has been shown to be important for mitotic progression. We have described that Nek9 is activated by a two-step mechanism, first by the phosphorylation of Cdk1, which leads to the binding of Plk1 through its PBD domain and further phosphorylation of Plk1 at different sites, including the activation loop at the Thr210. We show that both phosphorylation by Cdk1 and Plk1 are necessary for the activation of Nek9 in vivo. Results in our group demonstrate that active Nek9 and Nek6 induce centrosome separation in an Eg5-dependent manner, and also that active Nek9 and Nek6 can rescue Plk1 but not Eg5 downregulation in prophase centrosome separation. This work has demonstrated that Nek9 activation by Plk1 is necessary for the phosphorylation of the kinesin Eg5 at the Ser1033, and that this phosphorylation is necessary for prophase centrosome separation and Eg5 recruitment. During the last years we have inhibited Nek9 using different approaches, but in order to have an acute inhibition of the kinase we decided to take a chemical genetic approach. As a general overview, the strategy consists on doing a functionally silent mutation in the ATP binding pocket (gatekeeper residue) of the target kinase that sensitizes it to inhibition by an ATP analog and that does not inhibit wild type kinases. So far I have identified the gatekeeper residue of Nek9 and have been able to find a mutant that acts as an analog sensitive mutant in vitro. |
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