In vivo identification of sumoylation sites by a signature tag and cysteine-targeted affinity purification

A1 Originalartikel i en vetenskaplig tidskrift (referentgranskad)


Interna författare/redaktörer


Publikationens författare: Blomster, Imanishi, Siimes, Kastu, Morrice, Eriksson, Sistonen
Förläggare: American Society for Biochemistry and Molecular Biology, Inc.
Publiceringsår: 2010
Tidskrift: Journal of Biological Chemistry
Tidskriftsakronym: J Biol Chem
Volym: 285
Nummer: 25
Artikelns första sida, sidnummer: 19324
Artikelns sista sida, sidnummer: 19329
ISSN: 1083-351X
eISSN: 1067-8816


Abstrakt

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 PsiKXE. 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 nearby 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.

Senast uppdaterad 2019-18-09 vid 06:31