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Updated: Nov 3, 2025

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
Published on: January 29, 2018
Modification of small ubiquitin-related modifier 2 (SUMO2) by phosphoubiquitin in HEK293T cells
Julius T Dongdem1,2, Simon P Dawson1, Robert Layfield1
1School of Life Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, UK.
Abstract:
Additional complexity in the post-translational modification of proteins by ubiquitin is achieved by ubiquitin phosphorylation, for example within PINK1-parkin mediated mitophagy. We performed a preliminary proteomic analysis to identify proteins differentially modified by ubiquitin in HEK293T, compared to phosphomimetic ubiquitin (Ser65Asp), and identified small ubiquitin-related modifier 2 (SUMO2) as a candidate. By transfecting SUMO2 and its C-terminal-GG deletion mutant, along with phosphomimetic ubiquitin, we confirm that ubiquitin modifies SUMO2, rather than vice versa. Further investigations revealed that transfected SUMO2 can also be conjugated by endogenous phospho-Ser65-(poly)ubiquitin in HEK293T cells, pointing to a previously unappreciated level of complexity in SUMO2 modification, and that unanchored (substrate-free) polyubiquitin chains may also be subject to phosphorylation.
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