Identification of novel NEK9 substrates and functions through the use of genetically engineered mice. Novel roles in the control of the centrosome cycle
[eng] Mitosis is a process that ensure the correct distribution of the chromosomes between the two newly generated cells, is tightly regulated by two main processes, protein degradation controlled by the APC and protein phosphorylation by different mitotic kinases. CDK1 is the master regulator of mi...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2018 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/126003 |
| Acceso en línea: | https://hdl.handle.net/2445/126003 http://hdl.handle.net/10803/663722 |
| Access Level: | acceso abierto |
| Palabra clave: | Mitosi Cromosomes Càncer Mitosis Chromosomes Cancer |
| Sumario: | [eng] Mitosis is a process that ensure the correct distribution of the chromosomes between the two newly generated cells, is tightly regulated by two main processes, protein degradation controlled by the APC and protein phosphorylation by different mitotic kinases. CDK1 is the master regulator of mitosis but in the last decades proteins from the Aurora or Polo or the NIMA family have been shown to play key roles in mitosis. The objective of this thesis is to identify new roles during the cell cycle and more specifically the late phases of mitosis of Nek9, a NIMA-related kinase. We aim to characterize new substrates and functions of the kinase by using different cell lines and genetically modified mice and interfering with Nek9 expression. The centrosome acts as the major microtubule-organizing center (MTOC) of the cell to maintain cytoskeleton in interphase and to organize the bipolar spindle in mitosis, and its duplication cycle is coupled with the cell cycle. When the cell enters mitosis, the duplicated centrosomes separate to the spindle poles and assemble the bipolar mitotic spindle for accurate chromosome separation and to maintain genomic stability. However, centrosome aberrations occur frequently and often lead to abnormal mitotic spindle formation, which can result in abnormal chromosome segregation and as a consequence tumorigenesis, microcephaly or ciliopathies. Nek9 is inactive during interphase and activated at centrosomes and spindle poles during mitosis by a two-step mechanism mediated by Plk1 and CDK1. Once active, Nek9 is able to bind Nek6 and Nek7 and directly phosphorylate these kinases inducing in turn their activation. Our group has shown that Nek6/7 phosphorylates the kinesin Eg5 at Ser1033 in the C-terminal domain, modulating the accumulation of Eg5 at or around centrosomes and their separation during prophase. Nek9 also phosphorylates the adapter NEDD1/GCP-WD, independently of Nek6/7, contributing to its recruitment to the centrosome and in consequence, to the recruitment of the microtubule nucleating complex formed by y-tubulin to the same organelle. Thus, Nek9, Nek7 and Nek6 regulate different aspects of the centrosome machinery during the entry in mitosis and have a role in spindle organization and correct mitotic progression. Here we show that animals with a single Nek9 KO allele are healthy and fertile but intercrosses between them have not resulted in any homozygous null animals among born offspring indicating that the deletion of Nek9 is embryonic lethal. Also embryos obtained from these intercrosses had a higher frequency of mitotic abnormalities that result in death during the first days of development. As Nek9 is important for the proper development of mitosis we checked whether the expression in heterozygosity of Nek9 results in tumors affecting the viability of the animals. Some differences in tumor-free lifespan between heterozygous and wild type animals have been observed, with the appearance of tumors and aneuploidy. In addition, elimination of Nek9 expression lead to the apparition of abnormal mitosis, aneuploidy and multiple centrosomes both in genetically engineered MEFs and human cells, resulting in accumulation of centrobin, a protein mostly associated with the daughter centrioles, in the amplified centrioles. In the present thesis we describe possible new functions and substrates of Nek9 in the centrosome cycle, closely linked to the cell division cycle, after interfering with its expression using different strategies. |
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