Modification of small ubiquitin-related modifier 2 (SUMO2) by phosphoubiquitin in HEK293T cells

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.

Automated summary

The study revealed a previously unappreciated complexity in ubiquitin modification, involving SUMO2 modification and phosphorylation of unanchored polyubiquitin chains.