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...
| Autor: | |
|---|---|
| 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 |
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oai:diposit.ub.edu:2445/36327 |
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España |
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| dc.title.none.fl_str_mv |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis |
| title |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis |
| spellingShingle |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis Bertran Domingo, M. Teresa Fosforilació Proteïnes quinases Mitosi Phosphorylation Protein kinases Mitosis |
| title_short |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis |
| title_full |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis |
| title_fullStr |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis |
| title_full_unstemmed |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis |
| title_sort |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosis |
| dc.creator.none.fl_str_mv |
Bertran Domingo, M. Teresa |
| author |
Bertran Domingo, M. Teresa |
| author_facet |
Bertran Domingo, M. Teresa |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
Roig Amorós, Joan Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Farmàcia) |
| dc.subject.none.fl_str_mv |
Fosforilació Proteïnes quinases Mitosi Phosphorylation Protein kinases Mitosis |
| topic |
Fosforilació Proteïnes quinases Mitosi Phosphorylation Protein kinases Mitosis |
| description |
[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. |
| publishDate |
2012 |
| dc.date.none.fl_str_mv |
2012 |
| dc.type.none.fl_str_mv |
info:eu-repo/semantics/doctoralThesis info:eu-repo/semantics/publishedVersion |
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doctoralThesis |
| status_str |
publishedVersion |
| dc.identifier.none.fl_str_mv |
https://hdl.handle.net/2445/36327 http://hdl.handle.net/10803/96415 |
| url |
https://hdl.handle.net/2445/36327 http://hdl.handle.net/10803/96415 |
| dc.language.none.fl_str_mv |
Inglés |
| language_invalid_str_mv |
Inglés |
| dc.rights.none.fl_str_mv |
(c) Bertran Domingo, 2012 info:eu-repo/semantics/openAccess |
| rights_invalid_str_mv |
(c) Bertran Domingo, 2012 |
| eu_rights_str_mv |
openAccess |
| dc.format.none.fl_str_mv |
application/pdf |
| dc.publisher.none.fl_str_mv |
Universitat de Barcelona |
| publisher.none.fl_str_mv |
Universitat de Barcelona |
| dc.source.none.fl_str_mv |
Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Farmàcia) reponame:Dipòsit Digital de la UB instname:Universidad de Barcelona |
| instname_str |
Universidad de Barcelona |
| reponame_str |
Dipòsit Digital de la UB |
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Dipòsit Digital de la UB |
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|
| repository.mail.fl_str_mv |
|
| _version_ |
1869422090994581504 |
| spelling |
Study of the phosphorylation and activation of the protein kinase NEK9 during mitosisBertran Domingo, M. TeresaFosforilacióProteïnes quinasesMitosiPhosphorylationProtein kinasesMitosis[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.[cat] Nek9 és una quinasa de la família de les NIMA que és fosforilada en mitosis i una petita part d’aquesta és activada als centrosomes mitjançant un mecanisme complex desconegut fins a ara. Nek6 i Nek7 són dues proteïnes de la mateixa família, que s’uneixen a la cua de Nek9 i que són fosforilades i activades directament per la fosforilació de Nek9, formant d’aquesta manera una casset de quinases mitòtiques. Una vegada actives, Nek6 i Nek7 fosforilen la quinesina Eg5 al residu Ser1033, residu que s’ha descrit que és important per una correcta progressió de la mitosis. En aquest treball hem descrit que Nek9 és activada mitjançant un mecanisme de dos passos, en el qual primer és fosforilada per Cdk1, que permet la unió de Plk1 mitjançant el seu domini PBD i posteriorment és fosforilada per Plk1 a diferents llocs, incloent el llaç d’activació a la Thr210. Demostrem que les fosforilacions tant per part de Cdk1 i Plk1 són necessàries per l’activació de Nek9 in vivo. Resultats del nostre grup demostren que Nek9 activa i Nek6 indueixen la separació dels centrosomes en profase de manera dependent d’Eg5, i també que Nek9 activa i Nek6 poden recuperar la separació dels centrosomes causade2013-11-28s per una downregulació de Plk1, però no la produïda per la downregulació d’Eg5. Aquest treball demostra que l’activació de Nek9 per part de Plk1 és necessària per la fosforilació d’Eg5 a la Ser1044, i que aquesta fosforilació és necessària per la separació dels centrosomes en profase i pel reclutament d’Eg5 als centrosomes. Durant els últims anys hem inhibit Nek9 utilitzant diversos enfocs, però per tal d’obtenir una inhibició de la quinasa de forma aguda vàrem decidir seguir un “chemical genetic approach”. Aquesta estratègia consisteix en fer mutacions funcionalment silencioses en la butxaca d’unió de l’ATP (gatekeeper residue) que la sensititza per la inhibició mitjançant un anàleg d’ATP que no inhibeix la forma salvatge de la quinasa. Fins al moment, hem identificat el residu gatekeeper de Nek9 i hem pogut demostrar que aquest mutant actua com un mutant sensitiu a l’anàleg d’ATP in vitro.Universitat de BarcelonaRoig Amorós, JoanUniversitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Farmàcia)2012info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/36327http://hdl.handle.net/10803/96415Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Farmàcia)reponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaInglés(c) Bertran Domingo, 2012info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/363272026-05-27T06:46:51Z |
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15,301603 |