Aurora B SUMOylation Is Restricted to Centromeres in Early Mitosis and Requires RANBP2

Abstract

Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis.

Nuclear transport proteins: structure, function, and disease relevance

“…233,234 For example, the kinase Aurora B, a key regulator of mitosis, is SUMOylated at the centromere in early mitosis by SUMO ligases including the RanBP2 complex. 235 Interestingly, NPC also binds to deSUMOylase. The major de-SOMOylating enzyme SENP2 localizes to NPCs by binding to Nup153 and is critical for the de-SUMOylation of ribosomal precursors and their subsequent nuclear export.…”

Distinct Mitotic Functions of Nucleolar and Spindle-Associated Protein 1 (NuSAP1) Are Controlled by Two Consensus SUMOylation Sites

“…To assess the significance of NuSAP1-RANBP2 PLA interactions, we analyzed NuSAP1 in cultures lacking RANBP2 after siRNA-mediated knockdown compared to control cultures, interfered with neutral siRNA targeting the firefly luciferase gene, GL2 [37,38]. Cultures were synchronized using STLC, an inhibitor of kinesin KIF11, to arrest cells in prophase, then released to progress into mitosis and collected via mechanical shake-off to obtain cell populations enriched in late prometaphase and metaphase stages, when NuSAP1-RANBP2 PLA products peak (Figure 1).…”

“…SUMOylation is a highly dynamic modification, which usually concerns a small fraction of target proteins, often with a high turnover and a restricted subcellular localization, which make its detection difficult under physiological conditions. The intramolecular PLA technique has been developed to circumvent these issues and has been successfully employed in several studies to visualize in situ ligation products formed by any given protein and SUMO peptides attached to it, avoiding any alteration of the original ratio of target protein to modifying SUMO peptides, but amplifying their detection [37,38,44,45]. Using this technique, we visualized PLA products formed by endogenous NuSAP1 and SUMO 2/3 peptides.…”

“…After nuclear envelope breakdown, RANBP2 and RANGAP1-SUMO co-localize at the spindle microtubules and at microtubule-attached kinetochores [30,31] and can regulate the SUMO-conjugated state of proteins therein. Several studies have shown that RANBP2 silencing is detrimental to proper mitotic progression and chromosome segregation [30,[32][33][34], which reflects, at least in part, its activity in regulation of SUMOylated mitotic factors at their site of action [25,[35][36][37][38].…”