Characterization of the hNek5 and hNek10 proteins in the DNA damage response context: a functional and interatomic approach

Author(s)

Priscila Ferreira Papa

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2015-07-08

Examining board members

Jörg Kobarg; Jenifer Saffi; Guido Lenz; Adriana Franco Paes Leme; Aline Mara dos Santos

• Advisor: Jörg Kobarg

Abstract

The Nek (NIMA related kinases) family of serine/threonine protein kinases is similar to its homolog NIMA (never in mitosis, gene A), a highly conserved and critical protein for the cell cycle progression in Aspergillus nidulans. The eleven members of the Neks (1-11) in mammals share biological functions grouped by the analysis of their interactomes in a functional triad, (1) mitosis/centrossome (2) primary cilia/ciliopathies (3) DNA damage response, in a review published by our group (Article I). Among them, hNek5 and hNek10 are least characterized as their biological roles. Therefore, a yeast-two hybrid screening identified Nek5 interactors related to mitochondria processes, such as BCLAF1, Cox11 and MTX2, and to DNA damage, BCLAF1 and TOPII?. Confocal microscopy images and subcellular fractionation indicated hNek5 mitochondrial localization and, by apoptosis assay and cell viability, its effects in cellular death resistance (Article II). Also, in response to genotoxic agents, hNek5 may cause effects in cell cycle arrest, replicative stress and deregulation in the activation of crucial proteins for the correct DNA damage response (Article III). hNek10 interactome was accessed by two approaches: (1) yeast-two hybrid screening indicating 12 interactors and (2) Mass Spectrometry identifying 117 and 58 interacting partners with or without DNA damage induction, respectively, associated to several biological processes, such as mitotic cell cycle, cell proliferation, genic expression, RNA processing. Among the interacting proteins, hNek10 interacted with SMC1 and SMC3, members of the cohesin complex, by immunoprecipitation assay and hNek10 depletion caused multinucleation, nuclear multilobulation, cell accumulation in G1/S, ?-H2AX foci and cell death increased levels, suggesting that hNek10 depletion may sensitize cells to internal genotoxic effects (Article IV). Taken together, the functional and interactomic results presented here for hNek5 and hNek10 characterized or corroborated their role in biological processes already related to other hNeks, especially in DNA damage response (AU)

• FAPESP's process: 10/15262-2 - Cloning and expression of human protein kinase Nek10 structural and functional studies aimed at molecular and cellular
• Grantee: Priscila Ferreira Papa
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)