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.
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Acknowledgements
We thank A. Münster-Kühnel (Hannover Medical School) for the expression plasmids pEGFP-C1-PIASxß and the pGADT7-InhSTAT3 (PIAS3), F. Melchior (University of Göttingen) for the pGST-RanBPΔFG, B. Valdez (Baylor College of Medicine) for the pCMV-mGBP (PIAS1), D. Wotton (University of Virginia) for the pFLAG-PC2, R. Bernards (Netherlands Cancer Institute) for the pRc/CMV-HA-Ubc9, J.E. Darnell (Rockefeller University) for the pRc/CMV-STAT1α, B. Vogelstein (Johns Hopkins Oncology Center) for the pC53-SN3 and the company ProQinase (Freiburg) for the JAK3 kinase. We also thank A. Kotlyarov and E. Hitti (Hannover Medical School) for helpful discussions, and T. Binz (Hannover Medical School) for critical reading of the manuscript. This work was supported by the Hannover Medical School Hochschulinterne Leistungsförderung program (to R.N.).
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A.J. performed the experiments, J.K. performed the experiments and analyzed the data, F.H. performed the experiments, M.F. and B.K. contributed reagents and materials, M.G. discussed experiments and wrote the paper, R.N. conceived and designed the experiments, analyzed the data and wrote the paper.
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M.F. is an employee of MediGene AG and is an owner of stock options of MediGene AG.
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Jakobs, A., Koehnke, J., Himstedt, F. et al. Ubc9 fusion–directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation. Nat Methods 4, 245–250 (2007). https://doi.org/10.1038/nmeth1006
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DOI: https://doi.org/10.1038/nmeth1006
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