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Phosphorylation of SUMO-1 Occurs in Vivo and Is Conserved through Evolution

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Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany, and Organelle Architecture and Dynamics, Max Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany
* To whom correspondence should be addressed. Prof. Dr. Matthias Mann, Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany. Tel, +49 89-8578-2557; fax, +49 89-8578-2219; e-mail, [email protected]
†Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry.
‡Organelle Architecture and Dynamics, Max Planck Institute for Biochemistry.
Cite this: J. Proteome Res. 2008, 7, 9, 4050–4057
Publication Date (Web):August 16, 2008
https://doi.org/10.1021/pr800368m
Copyright © 2008 American Chemical Society

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    Abstract

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    Protein dynamics is regulated by an elaborate interplay between different post-translational modifications. Ubiquitin and ubiquitin-like proteins (Ubls) are small proteins that are covalently conjugated to target proteins with important functional consequences. One such modifier is SUMO, which mainly modifies nuclear proteins. SUMO contains a unique N-terminal arm not present in ubiquitin and other Ubls, which functions in the formation of SUMO polymers. Here, we unambiguously show that serine 2 of the endogenous SUMO-1 N-terminal protrusion is phosphorylated in vivo using very high mass accuracy mass spectrometry at both the MS and the MS/MS level and complementary fragmentation techniques. Strikingly, we detected the same phosphorylation in yeast, Drosophila and human cells, suggesting an evolutionary conserved function for this modification. The nearly identical human SUMO-2 and SUMO-3 isoforms differ in serine 2; thus, only SUMO-3 could be phosphorylated at this position. Our finding that SUMO can be modified may point to an additional level of complexity through modifying a protein-modifier.

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    Supplementary Figure 1, gel region around free SUMO-1 or Smt3 (approximately 5−20 kDa) was digested in gel and analyzed by LC-MS/MS; Supplementary Figure 2, MS/MS fragmentation spectrum of the human SUMO-1 peptide Acetyl-pSDQEAKPSTEDLGDK. Precursor ion mass was measured in the orbitrap analyzer (m/z 871.3618 (2+); mass deviation, −0.06 ppm) and the peptide was fragmented by CID and acquired in the LTQ mass spectrometer; Supplementary Figure 3, MS/MS fragmentation spectrum of the human SUMO-1 peptide Acetyl-pSDQEAKPSTEDLGDKK. Precursor ion mass was measured in the orbitrap analyzer (m/z 935.4093 (2+); mass deviation, −0.19 ppm) and the peptide was fragmented by CID and acquired in the LTQ mass spectrometer; Supplementary Figure 4, MS/MS fragmentation spectrum of the human SUMO-1 peptide Acetyl-pSDQEAKPSTEDLGDKK. Precursor ion mass was measured in the orbitrap analyzer (m/z 623.9420 (3+); mass deviation, 0.05 ppm) and the peptide was fragmented by CID and acquired in the LTQ mass spectrometer; Supplementary Figure 5, MS/MS fragmentation spectrum of the yeast Smt3 peptide pSDSEVNQEAK. Precursor ion mass was measured in the orbitrap analyzer (m/z 593.7348 (2+); mass deviation, −0.47 ppm) and the peptide was fragmented by CID and acquired in the LTQ mass spectrometer; Supplementary Figure 6, MS/MS fragmentation spectrum of the yeast Smt3 peptide Acetyl-pSDSEVNQEAKPEVKPEVK. Precursor ion mass was measured in the orbitrap analyzer (m/z 1067.9988 (2+); mass deviation, −0.52 ppm) and the peptide was fragmented by CID and acquired in the LTQ mass spectrometer; Supplementary Figure 7, MS/MS fragmentation spectrum of the Drosophila Smt3 peptide Acetyl- pSDEKKGGETEHINLK. Precursor ion mass was measured in the orbitrap analyzer (m/z 602.9472 (3+); mass deviation, −1.76 ppm) and the MS/MS spectra were acquired in the LTQ mass spectrometer; Supplementary Figure 8, His6-SUMO-2 MS/MS data from Matic et al. (10) were analyzed with MaxQuant. MS/MS fragmentation spectrum of the human SUMO-1 peptide Acetyl-pSDQEAKPSTEDLGDKKEGEYIK. Precursor ion mass was measured in the orbitrap analyzer (m/z 863.7250 (2+); mass deviation, 0.44 ppm) and the peptide was fragmented by CID and acquired in the LTQ mass spectrometer. Note that the multistage activation was not enabled and that the main fragmentation ion is the neutral loss of the precursor ion. The enlarged left and right regions of the MS/MS spectrum show the presence of other fragment ions. This material is available free of charge via the Internet at http://pubs.acs.org.

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