Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif.
Reversible protein modification by small ubiquitin-like modifiers (SUMOs) is critical for eukaryotic life. Mass spectrometry-based proteomics has proven effective at identifying hundreds of potential SUMO target proteins. However, direct identification of SUMO acceptor lysines in complex samples by mass spectrometry is still very challenging. We have developed a generic method for the identification of SUMO acceptor lysines in target proteins. We have identified 103 SUMO-2 acceptor lysines in endogenous target proteins. Of these acceptor lysines, 76 are situated in the SUMOylation consensus site [VILMFPC]KxE. Interestingly, eight sites fit the inverted SUMOylation consensus motif [ED]xK[VILFP]. In addition, we found direct mass spectrometric evidence for crosstalk between SUMOylation and phosphorylation with a preferred spacer between the SUMOylated lysine and the phosphorylated serine of four residues. In 16 proteins we identified a hydrophobic cluster SUMOylation motif (HCSM). SUMO conjugation of RanGAP1 and ZBTB1 via HCSMs is remarkably efficient.
Matic
Ivan
I
Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried D-82152, Germany.
Schimmel
Joost
J
Hendriks
Ivo A
IA
van Santen
Maria A
MA
van de Rijke
Frans
F
van Dam
Hans
H
Gnad
Florian
F
Mann
Matthias
M
Vertegaal
Alfred C O
AC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014162:Transfection
D020690:GTPase-Activating Proteins; C499062:NOP58 protein, human; D009687:Nuclear Proteins; C092317:RANGAP1 protein, human; D020718:Ribonucleoproteins, Small Nucleolar; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D008239:Lysine