Chracterization of the protein interaction profile of the human kinase NEK3 and assignment of its functional context

Author(s)

Vanessa Bomfim Cardoso

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2016-10-26

Examining board members

Jörg Kobarg; Marcio Vinicius Bertacine Dias; Celso Eduardo Benedetti; Andrea Balan; Patricia da Silva Melo

• Advisor: Jörg Kobarg

Abstract

NEKs (NIMA-related kinases) are a group of kinase proteins that share high identity of their amino acid sequences with NIMA (Never in mitosis gene A) and are involved in cell cycle regulation. Among mammalian NEKs already studied, frequently found functions include: cilia function, cell cycle progression - especially during checkpoints- and the DNA damage response. Eleven NEKs have been discovered, with all their orthologous present in mice. NEK3 is a 506-amino acid serine/threonine kinase, present in the cytoplasm and nucleus, and its gene is located at chromosome 13, bands q14.2 According to the association of NEK3 to several disease processes, including breast cancer and axonal degeneration, as well frequent mutations in its chromosomal site, and since it has characteristics different from other more studied NEKs, it becomes necessary to further clarify its role, through functional, molecular and structural studies, aiming to map the interaction network in which it is involved. Using the Matchmaker Gold Yeast Two-Hybrid system, a screening for interaction partners was performed and 65 clones were obtained, which cDNAs encode 27 different proteins. The identified candidate interacting proteins are functionally involved in sumoylation, ubiquitination, transcriptional regulation, DNA repair, RNA processing, and the regulation of cell proliferation, invasiveness and metastasis. Some interaction partners for NEK3 are located in the nucleus and plasma membrane but most of them localize to the cytoplasm. One of the cytoplasmatic interactors for NEK3, RhoGDI2, is a regulator of RhoGTPases and inhibits Rac1 and RhoA activation levels. In our pull down assay, NEK3 overexpression increases Rac1-GTP while concomitant overexpression of RhoGDI2 reduces it to non-detectable levels. Our data suggest that NEK3 interaction with RhoGDI2 are important new regulatory elements in Rac1 signaling pathways. Moreover, the role for NEK3 in cell cycle was also investigated. ShRNA constructs for NEK3 were responsible for cytokinesis delay induced by cytoplasmic and DNA bridges, which corroborate with some NEK3 interactors role and supports the implications of the results from this work (AU)

• FAPESP's process: 11/06730-5 - Cloning and expression of the human protein kinase Nek3 for functional, structural, molecular and cellular studies
• Grantee: Vanessa Bomfim Cardoso
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)