The function of NEK5 kinase in normal and cancer cells: from the identification of physiological substrates to the development of new inhibitors

Author(s)

Camila de Castro Ferezin

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2021-04-23

Examining board members

Jörg Kobarg; Leonardo dos Reis Silveira; Munira Muhammad Abdel Baqui; Ana Carolina Migliorini Figueira; Andréia Machado Leopoldino

• Advisor: Jörg Kobarg

Abstract

The NEKs (Nima Related Kinases) are a family of eleven protein kinases that regulate important cellular functions. Because they control essential events, such as mitosis, DNA Replication and Repair and also functions related to cell-metabolism such as apoptosis, mitochondrial functions and cytoskeleton organization, NEKs are often mutated or altered in cancer cells, thus being potential therapeutic targets for tumors malignancies. Among the NEK family, NEK5 is one of the least characterized family members, with previous studies pointing to its relationship with DNA Damage Response and mitochondrial functions. The participation of NEK5 in prostate and breast cancer have already been described elsewhere, although the mechanisms underlying these alterations are still unclear. The main goal of this work is to understand and describe the functions regulated by NEK5 in normal and cancer cells, and to achieve that, we followed two different approaches. At first, we studied the involvement of NEK5 with mitochondrial homeostasis and its role in mtDNA maintenance. Here, we showed that the overexpression of wild-type NEK5 (NEK5WT) and its inactive version (NEK5K33A - kinase-dead) alters the mitochondrial mass and membrane potential, and it is involved with mtDNA integrity in a kinase-dependent manner. We have also shown that NEK5 interacts with LonP1 participating in the LonP1-TFAM signaling axis and this interaction might be related to mtDNA Repair since NEK5-LonP1 interaction increases upon oxidative damage. In the second moment of this work, in order to understand the role of NEK5 in malignant transformations, we explored the network of protein interactions, identifying possible partners interactions and signaling pathways regulated by NEK5 in breast cancer model. Using Proteomics techniques based on Mass Spectrometry, we built a network of interactions with potential direct partners and signaling pathways affected by the expression of NEK5 in epithelial breast cells. We demonstrated that the overexpression of NEK5 leads to an increase in colony formation and an increase in breast acini in 3D cell culture (Matrigel), also leading to morphological alteration of the acini. In this work, we also explored NEK5-regulated signaling pathways that are dependent on and independent of its kinase function, using the expression of mutants such as kinase-dead and kinase-domain, and demonstrated possible biological processes regulated by NEK5 and its kinase activity. In general, the data generated in this work contributed to our understanding of the NEK5 kinase biology and will serve as a basis for future investigations (AU)

• FAPESP's process: 16/10530-5 - The funtion of the protein kinase Nek5 in normal and cancer cells: from the identification of physiological substrates to inhibitor design
• Grantee: Camila de Castro Ferezin
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)