SUMO assay with peptide arrays on solid support: insights into SUMO target sites.
The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif psiKxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the psiKxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.
Schwamborn
Klaus
K
Pepscan Therapeutics BV, Zuidersluisweg 2, 8243 RC Lelystad, the Netherlands. k.schwamborne@pepscan.com
Knipscheer
Puck
P
van Dijk
Evert
E
van Dijk
Willem J
WJ
Sixma
Titia K
TK
Meloen
Rob H
RH
Langedijk
Johannes P M
JP
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016384:Consensus Sequence; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D011499:Protein Processing, Post-Translational; D025841:Small Ubiquitin-Related Modifier Proteins / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity
D020690:GTPase-Activating Proteins; D019151:Peptide Library; D010455:Peptides; D025841:Small Ubiquitin-Related Modifier Proteins