Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level.
The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin substrates were identified. Lysine residues targeted by the ubiquitin-ligase system show no unique sequence feature. Surface accessible lysine residues located in ordered secondary regions, surrounded by smaller and positively charged amino acids are preferred sites of ubiquitylation. Lysine ubiquitylation shows promiscuity at the site level, as evidenced by low evolutionary conservation of ubiquitylation sites across eukaryotic species. Among lysine modifications a significant overlap (20%) between ubiquitylation and acetylation at site level highlights extensive competitive crosstalk among these modifications. This site-specific crosstalk is not prevalent among cell cycle ubiquitylations. Between SUMOylation and ubiquitylation the preferred interaction is through mixed-chain conjugation. Overall these data provide novel insights into the site-specific selection and regulatory function of lysine ubiquitylation.
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, DK-2200 Copenhagen.
Jannie M R
JM
Danielsen
Kathrine B
KB
Sylvestersen
Simon
S
Bekker-Jensen
Damian
D
Szklarczyk
Jon W
JW
Poulsen
Heiko
H
Horn
Lars J
LJ
Jensen
Niels
N
Mailand
Michael L
ML
Nielsen
D000107:Acetylation; D000595:Amino Acid Sequence; D002460:Cell Line; D017124:Conserved Sequence; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D058207:Sumoylation; D025801:Ubiquitin / Q000378:metabolism; D057149:Ubiquitinated Proteins / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
D025801:Ubiquitin; D057149:Ubiquitinated Proteins; D008239:Lysine