Abstract
Crosstalk between the SUMO and ubiquitin pathways has recently been reported. However, no approach currently exists to determine the interrelationship between these modifications. Here, we report an optimized immunoaffinity method that permits the study of both protein ubiquitylation and SUMOylation from a single sample. This method enables the unprecedented identification of 10,388 SUMO sites in HEK293 cells. The sequential use of SUMO and ubiquitin remnant immunoaffinity purification facilitates the dynamic profiling of SUMOylated and ubiquitylated proteins in HEK293 cells treated with the proteasome inhibitor MG132. Quantitative proteomic analyses reveals crosstalk between substrates that control protein degradation, and highlights co-regulation of SUMOylation and ubiquitylation levels on deubiquitinase enzymes and the SUMOylation of proteasome subunits. The SUMOylation of the proteasome affects its recruitment to promyelocytic leukemia protein (PML) nuclear bodies, and PML lacking the SUMO interacting motif fails to colocalize with SUMOylated proteasome further demonstrating that this motif is required for PML catabolism.
Highlights
Crosstalk between the small ubiquitin related modifier (SUMO) and ubiquitin pathways has recently been reported
The optimized SUMOylated peptide immunopurification protocol provides an efficient approach to obtain unambiguous identification of SUMOylation sites on protein substrates. This approach enabled the proteome-wide identification of 10,388 SUMO sites on 3,556 proteins in HEK293 cells
Half of these sites were identified in recent large-scale SUMO proteome analyses that used different cell lines and stimuli
Summary
Crosstalk between the SUMO and ubiquitin pathways has recently been reported. no approach currently exists to determine the interrelationship between these modifications. The combination of lysine labelling with the overexpression of a wild-type (WT) like mutant has been reported[21] While these approaches have been designed to enrich SUMOylated peptides from complex cell extracts, they cannot be used alone to uncover the prevalence and significance of crosstalk between UBL modifiers. We developed a combined immunoaffinity enrichment strategy that enables the identification of UBL-modified proteins and applied this method to examine crosstalk between SUMOylation and ubiquitylation in the context of protein degradation Using this approach, we found several interplay between SUMO and ubiquitin including the co-regulation of SUMOylation and Ubiquitylation levels on deubiquitinase enzymes and the SUMOylation of the proteasome for its recruitment to promyelocytic leukemia protein (PML) nuclear bodies (NBs)
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