期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 285, 期 25, 页码 19324-19329出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110.106955
关键词
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资金
- Academy of Finland
- Sigrid Juselius Foundation
- Finnish Cancer Organizations
- Abo Akademi University
- Turku Graduate School of Biomedical Sciences
- Magnus Ehrnrooth Foundation
- Stiftelsens for Abo Akademi forskningsinstitut
Small ubiquitin-like modifier ( SUMO) is conjugated to its substrates via an enzymatic cascade consisting of three enzymes, E1, E2, and E3. The active site of the E2 enzyme, Ubc9, recognizes the substrate through binding to a consensus tetrapeptide Psi KXE. However, recent proteomics studies suggested that a considerable part of sumoylation occurs on non-consensus sites. Current unbiased sumoylation site identification techniques typically require high stoichiometry in vitro sumoylation, mass spectrometry, and complex data analysis. To facilitate in vivo analysis, we have designed a mass spectrometric method based on an engineered human SUMO-1 construct that creates a signature tag on SUMO substrates. This construct enables affinity purification by covalent binding to cysteine residues in LysC/trypsin-cleaved peptides and site identification by diglycyl lysine tagging of sumoylation sites. As a proof of concept, site-specific and substrate-unbiased in vivo sumoylation analysis of HeLa cells was performed. We identified 14 sumoylation sites, including well known sites, such as Lys(524) of RanGAP1, and novel non-consensus sites. Only 3 of the 14 sites matched consensus sites, supporting the emerging view that non-consensus sumoylation is a common event in live cells. Six of the non-consensus sites had a nearby SUMO interaction motif (SIM), which emphasizes the role of SIM in non-consensus sumoylation. Nevertheless, the lack of near by SIM residues among the remaining non-consensus sites indicates that there are also other specificity determinants of non-consensus sumoylation. The method we have developed proved to be a useful tool for sumoylation studies and will facilitate identification of novel SUMO substrates containing both consensus and non-consensus sites.
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