Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

Abstract

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis.

An in vivo genome‐wide shRNA screen identifies BCL6 as a targetable biomarker of paclitaxel resistance in breast cancer

“…Although not yet studied in the context of drug response, knockdown of COPS4 in MDA-MB-231 cells was recently shown to increase apoptosis [49]. This is reminiscent of some of our findings with resistance screen hit BCL6.…”

“…COPS4 is part of the multisubunit COP9 that plays roles in DNA repair and cell cycle regulation in part through modulating the activity of E3 ubiquitin ligases [49]. Although not yet studied in the context of drug response, knockdown of COPS4 in MDA‐MB‐231 cells was recently shown to increase apoptosis [49]. This is reminiscent of some of our findings with resistance screen hit BCL6.…”

“…Although not studied in the context of breast cancer, UGP2 expression (both high and low) has been implicated in the progression of other cancers [47,48], and our data suggest a potential uncharacterized role for UGP2 in breast cancer in the context of treatment. COPS4 is part of the multisubunit COP9 that plays roles in DNA repair and cell cycle regulation in part through modulating the activity of E3 ubiquitin ligases [49]. Although not yet studied in the context of drug response, knockdown of COPS4 in MDA‐MB‐231 cells was recently shown to increase apoptosis [49].…”

Interaction between NSMCE4A and GPS1 links the SMC5/6 complex to the COP9 signalosome

“…One of the main functions of CRLs is to regulate the DNA damage response (DDR), and CSN regulates CRLs activity at sites of DNA damage [19][20][21][22][23][24][25][26]. Additionally, the abrogation of CSN function has been shown to affect chromosome segregation during mitosis and meiosis [27][28][29][30][31].…”