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.

[1]  M. Mann,et al.  Is Proteomics the New Genomics? , 2007, Cell.

[2]  U. Landegren,et al.  Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. , 2008, Methods.

[3]  F. Melchior,et al.  Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association , 1998, The Journal of cell biology.

[4]  Steven P. Gygi,et al.  A Proteomic Strategy for Gaining Insights into Protein Sumoylation in Yeast*S , 2005, Molecular & Cellular Proteomics.

[5]  L. Sistonen,et al.  PDSM, a motif for phosphorylation-dependent SUMO modification. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Ivan Dikic,et al.  Atypical ubiquitin chains: new molecular signals , 2008, EMBO reports.

[7]  M. Mann,et al.  PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites , 2007, Genome Biology.

[8]  Hiroshi Suzuki,et al.  Repression of PML Nuclear Body-Associated Transcription by Oxidative Stress-Activated Bach2 , 2004, Molecular and Cellular Biology.

[9]  Henning Urlaub,et al.  “ChopNSpice,” a Mass Spectrometric Approach That Allows Identification of Endogenous Small Ubiquitin-like Modifier-conjugated Peptides , 2009, Molecular & Cellular Proteomics.

[10]  Xuedong Liu,et al.  A Method of Mapping Protein Sumoylation Sites by Mass Spectrometry Using a Modified Small Ubiquitin-like Modifier 1 (SUMO-1) and a Computational Program*S , 2005, Molecular & Cellular Proteomics.

[11]  M. Mann,et al.  The Ubiquitin-Proteasome System Is a Key Component of the SUMO-2/3 Cycle*S , 2008, Molecular & Cellular Proteomics.

[12]  M. Mann,et al.  Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle. , 2008, Molecular cell.

[13]  J. Licht,et al.  Sequence-specific DNA Binding and Transcriptional Regulation by the Promyelocytic Leukemia Zinc Finger Protein* , 1997, The Journal of Biological Chemistry.

[14]  Steven P Gygi,et al.  A proteomics approach to understanding protein ubiquitination , 2003, Nature Biotechnology.

[15]  J Wade Harper,et al.  Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases. , 2009, Molecular cell.

[16]  M. Mann,et al.  In Vivo Identification of Human Small Ubiquitin-like Modifier Polymerization Sites by High Accuracy Mass Spectrometry and an in Vitro to in Vivo Strategy*S , 2008, Molecular & Cellular Proteomics.

[17]  D. Leprince,et al.  The LAZ3/BCL6 oncogene encodes a sequence-specific transcriptional inhibitor: a novel function for the BTB/POZ domain as an autonomous repressing domain. , 1995, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[18]  Zeinab Anvarian,et al.  Identification of a New Site of Sumoylation on Tel (ETV6) Uncovers a PIAS-Dependent Mode of Regulating Tel Function , 2008, Molecular and Cellular Biology.

[19]  S. Jentsch,et al.  Principles of ubiquitin and SUMO modifications in DNA repair , 2009, Nature.

[20]  M. Mann,et al.  Higher-energy C-trap dissociation for peptide modification analysis , 2007, Nature Methods.

[21]  M. Dasso,et al.  Modification in reverse: the SUMO proteases. , 2007, Trends in biochemical sciences.

[22]  S. Elledge,et al.  BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3 , 2003, Nature.

[23]  M. Mann,et al.  Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks , 2006, Cell.

[24]  V. Wilson,et al.  Ubiquitin proteolytic system: focus on SUMO , 2008, Expert review of proteomics.

[25]  Y. Lam,et al.  A Proteomic Screen for Nucleolar SUMO Targets Shows SUMOylation Modulates the Function of Nop5/Nop58 , 2010, Molecular cell.

[26]  U. Landegren,et al.  Direct observation of individual endogenous protein complexes in situ by proximity ligation , 2006, Nature Methods.

[27]  Jürgen Cox,et al.  A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics , 2009, Nature Protocols.

[28]  K. Kamiya,et al.  Transcriptional repressor ZF5 identifies a new conserved domain in zinc finger proteins. , 1993, Nucleic acids research.

[29]  D. Koh,et al.  A Novel POK Family Transcription Factor, ZBTB5, Represses Transcription of p21CIP1 Gene* , 2009, The Journal of Biological Chemistry.

[30]  Patrick G. A. Pedrioli,et al.  Using mass spectrometry to identify ubiquitin and ubiquitin‐like protein conjugation sites , 2009, Proteomics.

[31]  M. Mann,et al.  Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast , 2008, Nature.

[32]  G. Gill,et al.  Something about SUMO inhibits transcription. , 2005, Current opinion in genetics & development.

[33]  K. Resing,et al.  Mapping protein post-translational modifications with mass spectrometry , 2007, Nature Methods.

[34]  C. Lima,et al.  A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 Ubc9 , 2009, Nature Structural &Molecular Biology.

[35]  G. Blobel,et al.  SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex , 1998, The Journal of cell biology.

[36]  M. Mann,et al.  In-gel digestion for mass spectrometric characterization of proteins and proteomes , 2006, Nature Protocols.

[37]  F. Melchior,et al.  Concepts in sumoylation: a decade on , 2007, Nature Reviews Molecular Cell Biology.

[38]  Erik Meulmeester,et al.  Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25. , 2008, Molecular cell.

[39]  J. Eriksson,et al.  In Vivo Identification of Sumoylation Sites by a Signature Tag and Cysteine-targeted Affinity Purification* , 2010, The Journal of Biological Chemistry.

[40]  Matthias Mann,et al.  A Proteomic Study of SUMO-2 Target Proteins* , 2004, Journal of Biological Chemistry.

[41]  M. Mann,et al.  Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions , 2009, Science.

[42]  R. Hay,et al.  SUMO-specific proteases: a twist in the tail. , 2007, Trends in cell biology.

[43]  M. Mann,et al.  Distinct and Overlapping Sets of SUMO-1 and SUMO-2 Target Proteins Revealed by Quantitative Proteomics*S , 2006, Molecular & Cellular Proteomics.

[44]  L. Zon,et al.  SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. , 2002, Molecular cell.

[45]  M. MacCoss,et al.  Quantitative Profiling of Ubiquitylated Proteins Reveals Proteasome Substrates and the Substrate Repertoire Influenced by the Rpn10 Receptor Pathway*S , 2007, Molecular & Cellular Proteomics.

[46]  Chae-Ok Yun,et al.  ZBTB2, a Novel Master Regulator of the p53 Pathway* , 2009, The Journal of Biological Chemistry.

[47]  Brian Raught,et al.  Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software , 2006, Nature Methods.

[48]  Christopher D. Lima,et al.  Structural Basis for E2-Mediated SUMO Conjugation Revealed by a Complex between Ubiquitin-Conjugating Enzyme Ubc9 and RanGAP1 , 2002, Cell.

[49]  A. Sharrocks,et al.  An extended consensus motif enhances the specificity of substrate modification by SUMO , 2006, The EMBO journal.

[50]  A. Deelder,et al.  Positively charged amino acids flanking a sumoylation consensus tetramer on the 110kDa tri-snRNP component SART1 enhance sumoylation efficiency. , 2010, Journal of proteomics.

[51]  Matthias Mann,et al.  A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed* , 2009, Molecular & Cellular Proteomics.

[52]  M. Mann,et al.  Global and site-specific quantitative phosphoproteomics: principles and applications. , 2009, Annual review of pharmacology and toxicology.

[53]  G. Barton,et al.  System-Wide Changes to SUMO Modifications in Response to Heat Shock , 2009, Science Signaling.

[54]  M. Mann,et al.  Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips , 2007, Nature Protocols.

[55]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[56]  Tharan Srikumar,et al.  Global map of SUMO function revealed by protein-protein interaction and genetic networks. , 2009, Molecular cell.

[57]  J. Yates,et al.  Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. , 2006, Journal of proteome research.