Uncovering global SUMOylation signaling networks in a site-specific manner

Abstract

SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution MS, we have studied global SUMOylation in human cells in a site-specific manner, identifying a total of >4,300 SUMOylation sites in >1,600 proteins. To our knowledge, this is the first time that >1,000 SUMOylation sites have been identified under standard growth conditions. We quantitatively studied SUMOylation dynamics in response to SUMO protease inhibition, proteasome inhibition and heat shock. Many SUMOylated lysines have previously been reported to be ubiquitinated, acetylated or methylated, thus indicating cross-talk between SUMO and other post-translational modifications. We identified 70 phosphorylation and four acetylation events in proximity to SUMOylation sites, and we provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, precursor-mRNA splicing and ribosome assembly.

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Figure 1: A strategy for mapping SUMO-2–acceptor lysines in endogenous proteins.
Figure 2: Overview of MS results.
Figure 3: SUMO is extensively involved in PTM cross-talk.
Figure 4: New insight into the SUMOylation consensus motif.
Figure 5: SUMOylation is a predominantly nuclear event and is involved in many biological processes and protein complexes.
Figure 6: SUMO modifies highly interconnected functional networks of proteins.

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Acknowledgements

The authors are grateful for support from the European Research Council, grant 310913 (A.C.O.V.), the Netherlands Organization for Scientific Research (NWO), grants 70058425, 93511037 and 70059006 (A.C.O.V.), and the Max-Planck Society for the Advancement of Science (M.M.). We would like to acknowledge J. Cox for his help with MaxQuant and K. Sharma for helpful discussions. We would like to acknowledge our colleagues A.G. Jochemsen and D. Baker (both at Leiden University Medical Center) for reagents and helpful discussions.

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Authors

Contributions

A.C.O.V. and I.A.H. conceived the biochemical methodology and designed the experiments. I.A.H. optimized the biochemical methodology and prepared all MS samples. M.M. supervised the initial MS experiments and the optimization of the MS configuration performed by R.C.J.D. and I.A.H.; B.Y. and I.A.H. performed further optimization of the MS configuration. B.Y. and R.C.J.D. operated Q-Exactive machines. I.A.H. processed the MS data and performed bioinformatics analysis. I.A.H. and M.V.V. prepared biochemical samples and performed immunoblotting experiments. A.C.O.V. conceived the project. A.C.O.V. and M.M. supervised the project. I.A.H. and A.C.O.V. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Alfred C O Vertegaal.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 SUMOylation of proteins changes drastically upon heat shock, MG-132 treatment and PR-619 treatment.

(a) Schematic overview of all SUMOylation proteins identified to be differentially regulated after PR-619 treatment, using Label Free Quantification (LFQ).(b) As (a), but for MG-132 treatment.(c) As (a), but for heat shock.(d) Principle Component Analysis on all 17 biological replicates, revealing correlation between all samples.(e) Pearson correlation (R) analysis between all biological replicates. Pearson correlation is shown for both proteins (LFQ) and sites. Internal refers to correlation between same-condition samples, and external refers to correlation between different-condition samples. Error bars represent standard deviation. Average R values for internal correlation are shown below each condition.(f) Hierarchical Cluster analysis on all biological replicates. Samples are grouped based on correlation. Protein LFQ intensities were Z-scored prior to clustering. Green is indicative of upregulation of SUMOylation, and red is indicative of downregulation of SUMOylation.

Supplementary Figure 2 Validation of the lysine-deficient SUMO-2 mutant and confirmation of new SUMO-2 targets.

(a) Immunoblot analysis (IB) of total lysates and His10-pulldown samples (PD) from HeLa cells expressing His10-SUMO-2 wild-type (WT) or lysine-deficient (K0) mutant, which were mock treated, or treated with heat shock, PR-619 or MG-132. Ponceau-S staining on total lysates is shown as a loading control. The experiment shown was replicated in biological duplicate.(b) Immunoblot analysis of total lysates and His10-pulldown samples from HeLa cells or HeLa cells expressing His10-SUMO-2. The experiment shown was performed in biological duplicate.(c) Similar to (b), but additionally using either mock treated or PR-619 treated cells. PR-619 LFQ ratios are indicated below their respective antibodies. The experiment shown was performed in biological duplicate.

Supplementary Figure 3 A comparison of SUMOylated proteins and sites identified in this work to proteins and sites previously identified in other SUMOylation studies.

(a) Schematic overview of SUMOylated proteins identified in this study, as compared to previously published studies. From Golebiowski et al.1, control SUMOylated proteins and heat shock SUMOylated proteins were classed separately. From Bruderer et al.2, all SUMOylation proteins and molecular-weight corrected SUMOylated proteins were classed separately. From Becker et al.3, SUMO-2 modified proteins and all SUMO-1 and SUMO-2 modified proteins were classed separately. From Tammsalu et al.4, proteins SUMOylated after heat shock were compared with proteins SUMOylated after heat shock in this study, and separately with proteins identified under all other conditions. (b) Schematic overview of SUMOylation sites identified in this study, as compared to Matic et al.5, Schimmel et al.6, and all known SUMOylation sites from PhosphoSitePlus (PSP). Sites identified after heat shock by Tammsalu et al. were compared to sites identified after heat shock in this study, as well as sites detected under standard growth conditions and sites detected after MG-132 or PR-619 treatment in this study.

Supplementary Figure 4 SUMOylation occurs on phylogenetically highly conserved proteins.

(a) Phylogenetic conservation analysis (within orthologues) of SUMOylation, as compared to other major PTMs ubiquitylation, acetylation, methylation and phosphorylation, as well as all human proteins (total). The conservation line descends by average percentage of conservation from human to 62 Ensembl-annotated eukaryotic organisms. The analysis was performed within orthologues, and only genes that have an orthologue were considered on a per-organism basis. P values for significance of difference between all datasets are indicated, and were determined by two-tailed paired Student’s t test.(b) Phylogenetic conservation analysis outside of orthologues. Synonymous to (a), with the exception that genes lacking an orthologue were considered to be 0% conserved on a per-organism basis. P values for significance of difference between all datasets are indicated, and were determined by two-tailed paired Student’s t test.

Supplementary Figure 5 The glutamate in the SUMOylation consensus motif of RanGAP1 is required for efficient SUMOylation, and Tel contains an inverted ExK SUMOylation site at Lys302.

(a) Immunoblot analysis (IB) of total lysates from HeLa cells or HeLa cells expressing His10-SUMO-2 which were transfected with wild-type (WT) HA-RanGAP1, HA-RanGAP1 E526D, or a SUMOylation-deficient RanGAP1 (ΔGL). On the left side, “S2” indicates SUMO-modified RanGAP1, and asterisks indicate non-specific bands. Ponceau-S staining is shown as a loading control. The experiment shown was replicated in biological duplicate.(b) Similar to (a), but using U2-OS cells. The experiment shown was replicated in biological duplicate.(c) Immunoblot analysis of His10-pulldown samples (PD) from HeLa cells or HeLa cells expressing His10-SUMO-2 which were transfected with the indicated HA-Tel constructs. On the left side, “S2” indicates SUMO-modified Tel, and asterisks indicate non-specific bands. SART1 is shown as a pulldown control, and Ponceau-S staining is shown as a pulldown and loading control. HA-Tel-K11R was used to monitor SUMOylation of K300, because Tel harbors a dominant major SUMOylation site at lysine-117. The experiment shown was replicated in biological triplicate.(d) Total lysates corresponding to (c), analyzed by SDS-PAGE and immunoblotting. Ponceau-S staining is shown as a loading control. The experiment shown was replicated in biological triplicate.

Supplementary Figure 6 SUMO and other PTMs compete for modification of lysines in a set of highly SUMOylated and functionally interconnected proteins.

(a) STRING network analysis of all proteins where SUMOylation and acetylation occur on at least one of the same lysines. STRING interaction confidence was set at 0.7 or greater, and the size and color of individual proteins corresponds to the amount of SUMOylation sites per protein.(b) As (a), but with SUMOylation and ubiquitylation occurring on at least one of the same lysines.(c) As (a), but with SUMOylation, acetylation and ubiquitylation occurring on at least one of the same lysines.(d) Schematic overview of the amount of SUMOylation sites identified per protein in total, or within the core STRING network (Fig. 6A), or within the three networks corresponding to (a), (b), and (c). Sites shared with the respective other PTMs are displayed in red. Additionally, the total amount of modification sites per protein for ubiquitylation and acetylation are shown. (e) Overview of STRING analyses depicted in (a), (b), and (c). “Enrichment” is a ratio derived from the observed amount of interactions divided by the expected amount of interactions. “Connected” refers to the percentage of input proteins connected to the core cluster. P values for all individual analyses are < 1E-15.(f) Overview of relative network score. This score was computed through multiplication of the interaction enrichment ratio, protein network connectivity, and the average STRING confidence of all interactions.

Supplementary Figure 7 Six interconnected networks of proteins modified by SUMO.

MCODE clusters 4-9 corresponding to Fig. 6A. The size and color of the individual proteins corresponds to the amount of SUMOylation sites identified in the protein.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Note (PDF 8694 kb)

Supplementary Data Set 1

A compilation of all uncropped images corresponding to all scans of gels, membranes and films displayed throughout this manuscript (PDF 12582 kb)

Supplementary Table 1

A complete list of all (4,361) identified SUMO sites and all (5,339) identified SUMOylated peptides (XLSX 4085 kb)

Supplementary Table 2

A complete list of all identified SUMOylated proteins and a list of putative SUMOylated proteins identified by only non-SUMOylated peptides (XLSX 1878 kb)

Supplementary Table 3

Label-free quantification of SUMOylated proteins in response to MG-132, PR-619 or heat shock (XLSX 451 kb)

Supplementary Table 4

An overlap matrix of all SUMOylated proteins identified in this work, as compared to SUMOylated proteins previously identified in other studies (XLSX 635 kb)

Supplementary Table 5

An overlap matrix of all identified SUMO sites, along with previously identified SUMO sites, and all known ubiquitination, acetylation and lysine-methylation sites (XLSX 7289 kb)

Supplementary Table 6

A list of all SUMOylated enzymes involved in phosphorylation, ubiquitination, methylation and acetylation (XLSX 25 kb)

Supplementary Table 7

A list of SUMOylated peptides exclusively or nonexclusively detected in the presence of acetylation or phosphorylation (XLSX 53 kb)

Supplementary Table 8

A complete term enrichment analysis on all SUMOylated proteins, as compared to the human proteome (XLSX 604 kb)

Supplementary Table 9

A fully annotated list of all SUMOylated proteins (XLSX 2257 kb)

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Hendriks, I., D'Souza, R., Yang, B. et al. Uncovering global SUMOylation signaling networks in a site-specific manner. Nat Struct Mol Biol 21, 927–936 (2014). https://doi.org/10.1038/nsmb.2890

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