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.
Highlights
This is suggestive of increased conjugation of endogenous proteins with His FLAG-tag (HF)-ubiquitin Serine65Aspartate phosphomimetic mutant (UbS65D) compared to His FLAG tagged ubiquitin wild-type (HF-ubiquitin wild-type (UbWT)) in HEK293T cells, and/or that the phosphomimetic is more resistant to deubiquitylation, consistent with previous observations in yeast cells [23, 18]
Given our observations that ‘hybrid’ chains consisting of phospho-Ser65-ubiquitin and small ubiquitin-related modifier 2 (SUMO2) may be physiologically relevant, we investigated if unanchored polyubiquitin chains, that is, covalent assemblies of multiple ubiquitins in a substrate-free form that regulate a range of different biological pathways, represent overlooked substrates of modification by phosphorylation [35,36,37,38,39, 6, 40]
His FLAG-tagged human recombinant UbWT and UbS65D mutant were created and employed to purify protein targets modified by covalent attachment ofubiquitin or phospho-Ser65-(poly)ubiquitin in HEK293T cells by the denaturing immobilised metal affinity chromatography (IMAC) protocol
Summary
Ubiquitin is a small (8.6 kDa) 76-residue regulatory protein which is found ‘ubiquitously’ in the cytoplasm and nucleus of eukaryotic cells. Lys63-linked polyubiquitin chains function as scaffolds to assemble signalling complexes including activation of the transcription factor NF-κB, involved in inflammatory and immune response
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