Ubc9 fusion–directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation

Abstract

Although small ubiquitin-like modifier (SUMO) is conjugated to proteins involved in diverse cellular processes, the functional analysis of SUMOylated proteins is often hampered by low levels of specific SUMOylated proteins in the cell. Here we describe a SUMO-conjugating enzyme (Ubc9) fusion-directed SUMOylation (UFDS) system, which allows efficient and selective in vivo SUMOylation of proteins. Although SUMOylation of overexpressed p53 and STAT1 was difficult to detect in HEK293 cells, up to 40% of p53 and STAT1 were conjugated with endogenous SUMO when fused to Ubc9. We verified the specificity of UFDS using SUMOylation-site mutants and showed that the method is not dependent on SUMO ligases. Using UFDS we demonstrated that SUMOylation of STAT1 inhibits its phosphorylation at Tyr701 and discovered p53 multi-SUMOylation in vivo. We propose that UFDS will be useful for the analysis of function of SUMOylation in protein interactions, subcellular localization as well as enzymatic activity.

Identification of SUMOylated proteins in neuroblastoma cells after treatment with hydrogen peroxide or ascorbate

“…As would be expected the majority of the proteins are nuclear proteins and for the greater part these proteins have already been identified or proteins with similar functions have already been identified in screens for SUMOylated proteins (12,13,22). Cells were treated with hydrogen peroxide, inducing oxidative stress, as evidenced by the presence of topoisomerase II in extracts of these cells.…”

Sgs1's roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6

“…We decided to use a recent approach to generate a constitutively SUMOylated form of Sgs1 to investigate its effect on joint molecule accumulation. The approach involved fusion of the SUMO E2-conjugating enzyme Ubc9 to target proteins (Jakobs et al 2007;Almedawar et al 2012). Fusion of Ubc9 to the C terminus of Sgs1 (Sgs1-Ubc9) generated an allele of Sgs1 that was constitutively SUMOylated (Supplemental Fig.…”

Phosphorylation–acetylation switch in the regulation of STAT1 signaling

“…Similar to acetylation, sumoylation of STAT1 antagonizes its phosphorylation at Y701 and subsequent signaling (Figure 4). Mechanistically, this could be due to sterical hindrance of kinases due to the bulky SUMO adduct [83,84]. STAT1 S727 phosphorylation occurs on chromatin-bound STAT1 and facilitates inhibitory modification of STAT1 with SUMO1 [13].…”

“…Such observations have already been taken into account for sumoylation [96,97]. For example, the inhibitory role of STAT1 sumoylation could only be detected upon forced modification of STAT1 with SUMO1 [83,84]., Equally, cytokines evoke tyrosine phosphorylation and nuclear translocation of only a portion of the cellular STAT1 pool [10]. Accordingly, acetylation of a single molecule could impose latency on STAT1 oligomers via recruitment of the highly active TCP45.…”