ausblenden:
Schlagwörter:
*Amino Acid Motifs
Amino Acid Sequence
GTPase-Activating Proteins/metabolism
HeLa Cells
Humans
Hydrophobic and Hydrophilic Interactions
Lysine
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Nuclear Proteins/metabolism
Phosphorylation
*Protein Processing, Post-Translational
Proteomics/methods
Ribonucleoproteins, Small Nucleolar/metabolism
Small Ubiquitin-Related Modifier Proteins/genetics/*metabolism
Tandem Mass Spectrometry
Transfection
Zusammenfassung:
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.
