Evaluating the Role of the COP9 Signalosome and Neddylation during Cytokinesis and in Response to DNA Damage

Abstract

The covalent attachment of the ubiquitin-like protein NEDD8 via lysine residues on target proteins, termed neddylation, regulates the activity and stability of numerous proteins, particularly through regulating the activity of a key family of cellular enzymes known as the cullin E3 ubiquitin ligases. Neddylation is implicated in cell cycle regulation and DNA repair; however, the exact role and mechanism(s) are unclear. Here, two neddylation-regulated processes were investigated: control of cell division and response to DNA damage. The role of neddylation in cell division was evaluated using the neddylation inhibitor MLN4924, and by monitoring the localization of NEDD8, the subunits of the deneddylase COP9 signalosome (CSN) and cullin proteins during mitosis. Human HeLa cervical cancer cells treated with MLN4924 exhibited delayed physical separation of the daughter cells (abscission) and resulted in the appearance of multinucleated cells. Furthermore, treatment of mitotic cells with MLN4924 resulted in the earlier accumulation of the cytokinesis protein MKLP1 to the midbody. These results could provide a possible explanation for the ability of MLN4924 to increase the proportion of cells with >4N DNA content. The role of neddylation was and the CSN in response to DNA damage induced by ultraviolet laser irradiation was investigated using live-cell microscopy of DNA repair factors and CSN subunits. Laser-induced DNA damage in human U-2 OS osteosarcoma cells expressing fluorescently-tagged CSN3 and CSN4 subunits indicated that these CSN subunits accumulated in the nucleus following DNA damage, consistent with a possible role in the DNA damage response (DDR). Collectively, these findings indicate that neddylation and the CSN are linked to cytokinesis and the DDR.