In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein. Definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides

Tung Liang Chung, He Hsuan Hsiao, Yuh Ying Yeh, Hui Ling Shia, Yi Ling Chen, Po Huang Liang, Andrew H.J. Wang, Kay Hooi Khoo, Steven Shoei Lung Li

Research output: Contribution to journalArticlepeer-review

47 Citations (Scopus)

Abstract

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif ψKXE/D, where ψ represents a large hydrophobic amino acid, and X represnts any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, El-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.

Original languageEnglish
Pages (from-to)39653-39662
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number38
DOIs
Publication statusPublished - Sep 17 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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