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Originally published In Press as doi:10.1074/mcp.M800025-MCP200 on June 18, 2008.
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Molecular & Cellular Proteomics 7:2107-2122, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

The Ubiquitin-Proteasome System Is a Key Component of the SUMO-2/3 Cycle*,S

Joost Schimmel{ddagger},§, Katja M. Larsen§,¶, Ivan Matic§,||, Martijn van Hagen{ddagger}, Jürgen Cox||, Matthias Mann||, Jens S. Andersen,** and Alfred C. O. Vertegaal{ddagger},{ddagger} {ddagger}

From the {ddagger} Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands, Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark, and || Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Many proteins are regulated by a variety of post-translational modifications, and orchestration of these modifications is frequently required for full control of activity. Currently little is known about the combinatorial activity of different post-translational modifications. Here we show that extensive cross-talk exists between sumoylation and ubiquitination. We found that a subset of SUMO-2-conjugated proteins is subsequently ubiquitinated and degraded by the proteasome. In a screen for preferential SUMO-1 or SUMO-2 target proteins, we found that ubiquitin accumulated in purified SUMO-2 conjugates but not in SUMO-1 conjugates. Upon inhibition of the proteasome, the amount of ubiquitin in purified SUMO-2 conjugates increased. In addition, we found that endogenous SUMO-2/3 conjugates, but not endogenous SUMO-1 conjugates, accumulated in response to proteasome inhibitors. Quantitative proteomics experiments enabled the identification of 73 SUMO-2-conjugated proteins that accumulated in cells treated with proteasome inhibitors. Cross-talk between SUMO-2/3 and the ubiquitin-proteasome system controls many target proteins that regulate all aspects of nucleic acid metabolism. Surprisingly the relative abundance of 40 SUMO-2-conjugated proteins was reduced by proteasome inhibitors possibly because of a lack of recycled SUMO-2. We conclude that SUMO-2/3 conjugation and the ubiquitin-proteasome system are tightly integrated and act in a cooperative manner.

The ubiquitin-proteasome system plays a key role in virtually all cellular processes by tightly regulating the degradation of a large set of proteins (1). Proteins are targeted for degradation by lysine 48-linked polyubiquitin chains that are covalently conjugated to lysines in target proteins. Ubiquitination furthermore regulates target proteins in a degradation-independent manner, e.g. monoubiquitination is important for endocytosis (2). A significant part of the human genome encodes components of the ubiquitin-proteasome system, including E1,1 E2, and hundreds of E3 enzymes that mediate the conjugation of target proteins to ubiquitin and ubiquitin proteases that remove ubiquitins from target proteins.

The ubiquitin family comprises ubiquitin-like proteins NEDD8, ISG15, SUMO-1, -2, -3, FAT10, FUB1, UBL5, URM1, ATG8, and ATG12 (3, 4). These proteins share the three-dimensional structure of ubiquitin and are also conjugated to target proteins. Like ubiquitination, sumoylation is essential for eukaryotic life (5). The largest functional group of SUMO targets are transcription factors (6), and in general, sumoylation inhibits their transcriptional activity (7). Sumoylation also regulates other cellular processes including DNA repair, RNA metabolism, protein transport, translation, and replication (8–10). Whereas mature SUMO-2 and SUMO-3 are nearly identical (~95% identity), they differ significantly from SUMO-1 (~50% identity). Previously we have shown that SUMO-1 and SUMO-2 are conjugated to preferential sets of target proteins (6). Furthermore SUMO-2 and SUMO-3 contain an internal sumoylation site that is used for SUMO chain formation in vivo (11, 12).

Sumoylation is not linked to the degradation of target proteins, although some exceptions have been reported. The SUMO-accepting lysine 160 in PML and in the oncogenic PML-retinoic acid receptor {alpha} protein is required for the degradation of this fusion protein upon arsenic trioxide treatment (13). Furthermore it has been published that sumoylation might be important for the degradation of DNA topoisomerase IIβ in response to a catalytic inhibitor (14).

Different kinds of cross-talk between ubiquitination and sumoylation have been reported recently (15). SUMO and ubiquitin were shown to counteract each other by competing for the same acceptor lysine in I{kappa}B{alpha} (16). NF-{kappa}B signaling is furthermore affected by the ubiquitination and sumoylation of NF-{kappa}B essential modulator (NEMO)/I{kappa}B kinase {gamma}, a structural component of the I{kappa}B kinase complex (17). In this case, SUMO-1 and ubiquitin do not directly compete for the same acceptor lysine but are conjugated in a sequential manner in response to genotoxic stress. Proliferating cell nuclear antigen is also modified by SUMO and ubiquitin on the same acceptor lysine (Lys-164) (15, 18). Sumoylation enables the interaction between proliferating cell nuclear antigen and the helicase Srs2, whereas monoubiquitination enables translesion synthesis by Pol{eta}, a DNA damage-tolerant polymerase, and polyubiquitination is needed for DNA repair.

In a screen for preferential SUMO-1 and preferential SUMO-2 conjugates, we found that ubiquitin specifically co-enriched with SUMO-2. The amount of ubiquitin in SUMO-2-purified fractions significantly increased upon inhibition of the proteasome. Quantitative proteomics enabled us to study the cross-talk between sumoylation and the ubiquitin-proteasome system at the target protein level. We show here that the conjugation of a large set of target proteins to SUMO-2 is tightly connected to the ubiquitin-proteasome system and conclude that the ubiquitin-proteasome system is a key component of the SUMO-2/3 cycle in cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfections—
HeLa cells stably expressing His6-SUMO-1 or His6-SUMO-2 were described previously (6). HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 100 units/ml penicillin and streptomycin (Invitrogen). Stable isotope labeling was carried out essentially as described previously (6, 19) using [12C6,14N4]arginine (referred to as Arg0), [13C6,14N4]arginine (referred to as Arg6), [13C6,15N4]arginine (referred to as Arg10), [12C614N2]lysine (referred to as Lys0), [2H4,12C6,14N2]lysine (referred to as Lys4), or [13C6,15N2]lysine (referred to as Lys8) as indicated. Transfections were carried out using 25-kDa linear polyethyleneimine (Brunschwig-Chemie) essentially as described before (20).

Plasmids, Mutagenesis, Antibodies, Protein Electrophoresis, and Immunoblotting—
The plasmids encoding His6-ubiquitin wild type or 7KR and the plasmid encoding wild-type His6-SUMO-2 were described previously (21, 22). The His6-SUMO-2 K11R, E13A, K32R, K32R,K34R,K41R,K44R, and allKR plasmids were generated by site-directed mutagenesis using the QuikChange II kit according to the instructions of the manufacturer (Stratagene). Mutants were confirmed by DNA sequencing.

The amino acid sequence of the mature protein that we refer to as SUMO-2 is MSEEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGG (12). Peptide antibody AV-SM23-0100 against SUMO-2/3 was generated in rabbit using the peptide MEDEDTIDVFQQQTG (Eurogentec) (6, 22). Peptide antibody 1607 against SART1 was also generated in rabbit by Eurogentec using peptides CSLSIEETNKLRAKLGLKPLEV and CNLDEEKQQQDFSASSTT as described previously (6). Monoclonal antibodies 21C7 against SUMO-1 and 19C7 against RanGAP1 were obtained from Zymed Laboratories Inc.. Monoclonal antibodies HIS-1 against polyhistidine, R-3902 against hnRNP M, and M-7931 against MCM-7 were obtained from Sigma. Monoclonal antibody SC-8017 against ubiquitin and polyclonal antibody SC-8152 against PIAS1 were obtained from Santa Cruz Biotechnology. Monoclonal antibody ab8060 against SAFB was obtained from Abcam. This antibody also recognizes SAFB2. Secondary antibodies used were anti-rabbit HRP and anti-mouse HRP (1:5000; Pierce) and anti-goat HRP (1:5000; Sigma).

Protein samples were size-fractionated on Novex 4–12% Bis-Tris gradient gels using MOPS buffer (Invitrogen). For immunoblotting experiments, size-fractionated proteins were subsequently transferred onto Hybond-C Extra membranes (Amersham Biosciences) using a submarine system (Invitrogen). The membranes were incubated with specific antibodies as indicated. Bound antibodies were detected via chemiluminescence with ECL Plus (Amersham Biosciences).

Purification of His6-SUMO- and His6-Ubiquitin-conjugated Proteins—
His6-SUMO conjugates and His6-ubiquitin conjugates were purified essentially as described previously (23). Cells were scraped in ice-cold PBS. Two small aliquots of each sample were lysed in lithium dodecyl sulfate protein sample buffer (Invitrogen) as input control or in 8 M urea, 100 mM Na2HPO4/NaH2PO4, 10 mM Tris/HCl, pH 7.0 to determine the protein concentration. The remaining cells were solubilized in lysis buffer (6 M guanidinium-HCl, 100 mM Na2HPO4/NaH2PO4, 10 mM Tris/HCl, pH 8.0, 20 mM imidazole, 10 mM β mercaptoethanol) and sonicated to reduce the viscosity. His6-SUMO conjugates or His6-ubiquitin conjugates were enriched on nickel-nitrilotriacetic acid-agarose beads (Qiagen) and washed using wash Buffers A–D (Buffer A: 6 M guanidinium-HCl, 100 mM NaH2PO4/Na2HPO4, 10 mM Tris/HCl, pH 8.0, 0.2% Triton-X-100; Buffer B: 8 M urea, 100 mM NaH2PO4/Na2HPO4, 10 mM Tris/HCl, pH 8.0, 0.2% Triton-X-100; Buffer C: 8 M urea, 100 mM NaH2PO4/Na2HPO4, 10 mM Tris/HCl, pH 6.3, 0.2% Triton-X-100; Buffer D: 8 M urea, 100 mM NaH2PO4/Na2HPO4, 10 mM Tris/HCl, pH 6.3, 0.1% Triton-X-100). These wash buffers also contained 10 mM β mercaptoethanol. Samples were eluted in 6.4 M urea, 80 mM NaH2PO4/Na2HPO4, 8 mM Tris/HCl, pH 7.0, 200 mM imidazole.

For the experiment described in Fig. 1, A–C, a previously described method was used (6). For the experiments described in Figs. 3, A and B, and 6, A and B, His6-SUMO conjugates were immunoprecipitated using monoclonal antibody HIS-1 (Sigma) as described previously (23). For the experiment described in Fig. 7A HeLa cells were transfected with the His6-ubiquitin 7KR encoding plasmid. Cells were lysed in 6 M guanidinium-HCl, 100 mM NaH2PO4/Na2HPO4, 10 mM Tris/HCl, pH 8.0, and proteins were digested with endopeptidase Lys-C. His6-ubiquitin conjugates were subsequently purified on Talon beads (BD Biosciences), washed two times with lysis buffer and four times with 8 M urea, 100 mM NaH2PO4/Na2HPO4, 10 mM Tris/HCl, pH 8.0, and eluted in 6.4 M urea, 80 mM NaH2PO4/Na2HPO4, 8 mM Tris/HCl, pH 7.0, 200 mM imidazole. His6-ubiquitin and conjugated peptides were digested with trypsin in solution and identified by mass spectrometry.


Figure 1
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  		FIG. 1. SUMO-2 conjugates are enriched for ubiquitin. A, a quantitative proteomics strategy to identify SUMO-1 and SUMO-2 conjugates. B, HeLa cells were labeled with Lys0, HeLaHis6-SUMO-1 cells were labeled with Lys4, and HeLaHis6-SUMO-2 cells were labeled with Lys8. Equal amounts of nuclear lysates from the three different populations were mixed, and proteins conjugated to His6-SUMO were purified. The SUMO-enriched fraction was separated by SDS-PAGE, proteins were visualized by Coomassie staining, the gel lane was cut in slices, and the proteins present in these slices were digested by Lys-C and identified by mass spectrometry. Peptide mass spectra were quantified to identify proteins potentially conjugated to SUMO-1 and/or SUMO-2. C, the His6-SUMO-2-purified fraction is specifically enriched for ubiquitin. Two different ubiquitin peptides (aa MQIFVK and TITLEVEPSDTIENVK) were found to be enriched in His6-SUMO-2 conjugates but not in His6-SUMO-1 conjugates in the top part of the gel lane. The peptide mass spectra of the ubiquitin peptide TITLEVEPSDTIENVK is shown. D–F, the proteasome inhibitor MG132 increases the amount of ubiquitin in His6-SUMO-2 conjugates. HeLa cells, HeLaHis6-SUMO-1 cells, and HeLaHis6-SUMO-2 cells were treated for 1 or 3 h with MG132 or were treated with DMSO for 3 h. Whole cell lysates were prepared, size-separated by SDS-PAGE, and transferred to a membrane. Total protein was visualized by Ponceau S staining (D), and the membrane was probed using antibody SC-8017 to detect ubiquitin (E). His6-SUMO conjugates from whole cell lysates were purified, size-separated by SDS-PAGE, transferred to a membrane, and probed to detect ubiquitin (F). G–I, the proteasome inhibitor MG132 increases the amount of SUMO-2/3 in His6-ubiquitin conjugates. HeLa cells were transfected with a plasmid that encodes His6-ubiquitin or with an empty control plasmid, and cells were subsequently treated with MG132 or DMSO for 3 h. Whole cell lysates were prepared, size-separated by SDS-PAGE, and transferred to a membrane. Total protein was visualized by Ponceau S staining (G), and the membrane was probed using antibody AV-SM23-0100 to detect SUMO-2/3 (H). His6-ubiquitin conjugates were purified from whole cell lysates, size-separated by SDS-PAGE, transferred to a membrane, and probed to detect SUMO-2/3 (I).

 

Figure 3
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  		FIG. 3. Dynamic alterations in the SUMO-2-conjugated proteome in response to the proteasome inhibitor MG132. A, a quantitative proteomics strategy to study the effect of MG132 on the SUMO-2-conjugated proteome. HeLaHis6-SUMO-2 cells were labeled with Arg0 and Lys0 and treated with DMSO for 3 h, and a second pool of HeLaHis6-SUMO-2 cells was labeled with Arg10 and Lys8 and treated with MG132 for 3 h. Equal amounts of whole cell lysates from the two different populations were mixed, and proteins conjugated to His6-SUMO-2 were purified, digested by trypsin, and identified by mass spectrometry. Peptide mass spectra were quantified to identify MG132-induced changes in the SUMO-2-conjugated proteome. B, in total 847 proteins were identified by at least two unique peptides, including 73 proteins that were preferentially enriched upon MG132 treatment and 40 proteins that were significantly reduced in response to MG132. The natural logarithm of the SILAC ratio for each protein is depicted to visualize up-regulated and down-regulated proteins.

 

Figure 6
Figure 6
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  		FIG. 6. SUMO chains accumulate in cells treated with MG132. A and B, a quantitative proteomics experiment was performed to identify changes in SUMO chains induced by MG132. HeLaHis6-SUMO-2 cells were labeled with Arg0 and Lys0 and treated with DMSO for 3 h, and a second set of HeLaHis6-SUMO-2 cells was labeled with Arg10 and Lys8 and treated with MG132 for 3 h. Equal amounts of whole cell lysates from the two different populations were mixed, and proteins conjugated to His6-SUMO-2 were purified, digested by trypsin in solution, and identified by mass spectrometry. A, MS spectrum of a tryptic peptide consisting of aa 59–92 of SUMO-2 and aa 8–20 of another molecule of SUMO-2 (m/z 1338.1246 (4+); mass deviation, –1.21 ppm). B, MS spectrum of a tryptic peptide consisting of aa 59–92 of SUMO-2 and aa 8–21 of SUMO-3 (m/z 1366.6353 (4+); mass deviation, –1.65 ppm). C–G, HeLa cells were transfected with plasmids that encode His6-tagged forms of wild type (w.t.) or K11R or E13A SUMO-2 mutants that are reduced for SUMO chain formation. Cells were treated with MG132 or DMSO for 5 h, and His6-SUMO-2 conjugates were purified from equal amounts of whole cell lysates. Purified fractions were size-separated by SDS-PAGE, transferred to membranes, and probed using antibodies to detect SUMO-2/3 (C), ubiquitin (D) hnRNP M (E), MCM-7 (F), or PIAS1 (G).

 

Figure 7
Figure 7
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  		FIG. 7. SUMO-2 is ubiquitinated in cells. A, HeLa cells were transfected with a plasmid that encodes a His6-tagged lysine-deficient ubiquitin mutant. Cells were lysed, and His6-ubiquitin conjugates were purified and digested by trypsin. The digest was analyzed by mass spectrometry, and a peptide was identified that corresponds to ubiquitinated (ub) SUMO-2/3. The MS/MS fragmentation spectrum of this tryptic peptide consisting of aa 21–34 of SUMO-2 and the diglycine fragment of ubiquitin attached to lysine 32 of SUMO-2 is shown. This tryptic peptide is identical to a tryptic peptide consisting of aa 22–35 of SUMO-3 and the diglycine fragment of ubiquitin attached to lysine 33 of SUMO-3. Precursor ion mass was measured in the orbitrap mass spectrometer (m/z 530.6264 (3+); mass deviation, –1.12 ppm), and the peptide was fragmented and acquired in the LTQ mass spectrometer (Mascot score, 49.73; Mascot delta score, 3.28; posterior error probability, 1.765 x 10–20). B–F, His6-SUMO-2 plasmids were generated that encode K32R or K32R,K34R,K41R,K44R (4KR) mutants. HeLa cells were transfected with plasmids that encode His6-tagged forms of wild type (w.t.) or K32R or 4KR SUMO-2 mutants. Cells were treated with MG132 or DMSO for 5 h, and His6-SUMO-2 conjugates were purified from equal amounts of whole cell lysates. Purified fractions were size-separated by SDS-PAGE, transferred to membranes, and probed using antibodies to detect SUMO-2/3 (B), ubiquitin (C), hnRNP M (D), MCM-7 (E), or PIAS1 (F).

 
Mass Spectrometry and Data Analysis—
Mass spectrometric analysis was performed by nanoscale LC-MS/MS using a linear ion trap (LTQ) FT-ICR mass spectrometer (Thermo-Fisher Scientific, Bremen, Germany). Eluates were analyzed by one-dimensional gel electrophoresis. Gel lanes were cut in slices and subjected to in-gel digestion with Lys-C. The resulting peptides were extracted, concentrated, and then loaded onto a fused silica capillary with a 75-µm inner diameter and an 8-µm tip opening (New Objective, Woburn, MA) filled with Reprosil 3-µm reverse phase material (Dr. Maisch, Ammerbuch, Germany). Peptides were eluted with a 140-min linear gradient of 95% buffer A (0.5% acetic acid in H2O) to 50% buffer B (80% acetonitrile, 0.5% acetic acid in H2O). The LTQ-FT-ICR instrument was operated in the data-dependent mode to acquire high resolution precursor ion spectra (from m/z 300 to 1500, R = 50,000, and ion accumulation to a target value of 5,000,000) in the ICR cell. The three most intense ions were sequentially isolated for accurate mass measurements by selected ion monitoring scans (10-Da mass window, R = 50,000, and a target accumulation value of 90,000). The ions were simultaneously fragmented in the linear ion trap with a normalized collision energy setting of 27% and a target value of 10,000.

Peak list-generating software was DTA supercharge (release date, April 20, 2006). The combined peak list was searched in the International Protein Index database (release date, April 21, 2006; total of 66,279 sequences) using the Mascot program (Matrix Science, London, UK). The enzyme specificity was set to Lys-C, allowing for cleavage N-terminal of proline and between aspartic acid and proline. Cysteine carbamidomethylation was selected as a fixed modification, and methionine oxidation, protein N-acetylation, lysine-d4, and [13C6,15N2]lysine were searched as variable modifications. LTQ-FT-ICR data were searched with a peptide mass tolerance of 10 ppm and a fragment mass tolerance of 0.6 Da. Iterative calibration algorithms on the basis of identified peptides resulted in an average absolute peptide mass accuracy of better than 1 ppm. A maximum of one missed cleavage was allowed. Stringent criteria were required for protein identification based on the LTQ-FT-ICR data: at least two matching peptides per protein, a mass accuracy within 3 ppm, a Mascot score for individual ions of better than 20, and a delta score of better than 5.

Protein ratios were calculated for each peptide, and peptide ratios were averaged for all quantified peptides sequenced for each protein. MSQuant, an in-house-developed software program was used to extract information from the Mascot HTML database search files and to manually validate the certainty in peptide identification and in peptide abundance ratio. The quantitation was based on relative intensities from combined scans. The program is available as open source from SourceForge, Inc.

For the experiments described in Figs. 3, A and B, and 6, A and B, mass spectrometric analysis was performed by nanoscale LC-MS/MS using an LTQ-Orbitrap mass spectrometer, (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark) and coupled to an Agilent 1200 nano-HPLC system (Agilent Technologies) fitted with an in-house-made 75-µm reverse phase C18 column as described previously (24). In-solution digestion was performed essentially as before (25). The resulting peptides were desalted on reverse phase C18 stop and go extraction (STAGE) tips (26). Peptides were eluted with a 140-min linear gradient of 98% solvent A (0.5% acetic acid in H2O) to 50% solvent B (80% acetonitrile, 0.5% acetic acid in H2O).

Data were acquired in the data-dependent mode: full scan spectra (m/z 300–2000, R = 60,000, and ion accumulation to a target value of 1,000,000) were acquired in the orbitrap. The 10 most intense ions were fragmented and recorded in the ion trap as described before (24). Raw files were processed with our in-house quantitative proteomics software MaxQuant (version 1.0.7.5) that performs peak list generation, SILAC-based quantitation, false discovery rate determination, peptide to protein assembly, and data filtration essentially as described previously (27, 28). The quantitation was based on relative intensities from combined scans. The data were searched against a target/decoy human International Protein Index database (version 3.24) supplemented with frequently observed contaminants (total of 66,948 forward) using Mascot (Matrix Science, version 2.1.04). The enzyme specificity was set to trypsin, allowing for cleavage N-terminal of proline and between aspartic acid and proline. Cysteine carbamidomethylation was selected as a fixed modification, and methionine oxidation and protein N-acetylation and deamidation of asparagine and glutamine were searched as variable modifications. Spectra determined to be heavy labeled in the presearch MaxQuant detection of SILAC pairs were searched with the fixed modifications Arg10 and Lys8; for MS/MS spectra with a SILAC state not determinable before the database search Arg10 and Lys8 were taken as variable modifications. Initial maximum allowed mass deviation was set to 7 ppm for peptide masses and 0.5 ppm for MS/MS peaks. The minimum peptide length was set to 6 amino acids, and a maximum of three missed cleavages and three labeled amino acids were allowed. Two proteins were grouped together if the peptide sequence set of one protein was equal to or a subset of the set of the second protein.

A false discovery rate of 1% at both the protein and peptide level was used. Peptide posterior error probabilities were calculated by deriving, with Bayes’ theorem, the probability of a false identification for a top scoring peptide from Mascot score and peptide sequence length-dependent histograms. Protein group posterior error probabilities were calculated by multiplying the posterior error probabilities of the contained peptide sequences. Each distinct peptide sequence contributed only one factor, the posterior error probability of the MS/MS spectrum for a given peptide sequence with the best posterior error probability value. All MS/MS spectra associated with a peptide sequence, which might consist of resequencing events on the same peak, sequencing on different isotopic peaks in the same isotope pattern, sequencing of different charge states, or different SILAC or modification states, were used to calculate the protein false discovery rate only with one single peptide posterior error probability. Protein groups were then sorted by the posterior error probability, and a given false discovery rate was ensured by terminating a list of proteins so that a given percentage of reverse proteins were contained. The used false discovery rate of 1% ensured that at most 1% of the proteins were wrongly identified. Peptides that lost all proteins contained within a group after this procedure were also removed from the peptide list. In addition to the protein false discovery rate threshold, proteins were considered identified by at least two unique sequence peptides quantified with at least one quantifiable SILAC pair. No outliers were removed because of the use of median instead of average values.

Significance of protein ratios was calculated in two different ways. Significance A was calculated by first estimating the variance of the distribution of all protein ratios in a non-parametric way and then reporting the error function for the z score corresponding to the given ratio. A robust and asymmetrical estimate of the standard deviation was obtained by calculating the 15.87, 50, and 84.13 percentiles r–1, r0, and r1, which correspond to 1 {sigma} in each direction from the average. r1 – r0 was defined as the right- and r0 – r–1 was defined as left-sided robust standard deviations. In case of normally distributed data, r1 – r0 and r0 – r–1 would be equal to each other (conventional definition of standard deviation). The distance of a ratio r > r0 from the main distribution is measured in terms of the right standard deviation as follows.

Formula 1(EQ. 1)

An analogous calculation is defined for r < r0. Significance A is the value of the complementary error function for z above, which for a normal distribution corresponds to the probability of obtaining a value this large or larger by chance. Significance B was calculated using the same strategy, but in addition it is based on the dependence of the distribution on the summed protein intensity. We consider a protein as up-regulated if its significance B was below 0.001 and the ratio was higher than 1, down-regulated with significance B below 0.001 and ratio lower than 1, and not regulated if significance B was higher than 0.001.

Raw mass spectrometric files are stored at Tranche, a public repository for sharing scientific data. From the Tranche Website files can be downloaded with the following hash: 51ebd8yrJijxprwSrYIms0w9M4ZhR6rwulAmeLgyGs4WgqWQ4e8vcMgaqspsuaRcEwBJ4l1pFV6ZlLZBVLdEMWQJO5oAAAAAAAAFjA==.

To address the reproducibility of the data, independent control immunoblotting experiments were performed (Fig. 4). We used antibodies directed against six different proteins identified by mass spectrometry and confirmed increases and decreases in sumoylation of these proteins upon inhibition of the proteasome, showing the reproducibility of the data.


Figure 4
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  		FIG. 4. Dynamic alterations in the SUMO-2-conjugated proteome in response to the proteasome inhibitor MG132 detected by immunoblotting. A–F, HeLa cells and HeLaHis6-SUMO-2 cells were treated with MG132 or DMSO for 3 h, and His6-SUMO-2 conjugates were purified from equal amounts of whole cell lysates. His6-SUMO-2 conjugates or equal amounts of whole cell extracts were size-separated by SDS-PAGE, transferred to membranes, and probed using antibodies to detect hnRNP M (A), MCM-7 (B), PIAS1 (C), RanGAP1 (D), SAFB (E), or SART1 (F). Note that the PIAS levels are slightly higher in the HeLaHis6-SUMO-2 cells compared with the HeLa cells.

 

   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ubiquitin Co-purifies Preferentially with SUMO-2 Conjugates—
The quantitative proteomics experiment that we described previously to show that SUMO-1 and SUMO-2 are conjugated to preferential sets of target proteins (6) was repeated using lysine encoding instead of arginine encoding (Fig. 1A). Control HeLa cells were labeled with Lys0, HeLa cells stably expressing His6-SUMO-1 were labeled with Lys4, and HeLa cells stably expressing His6-SUMO-2 were labeled with Lys8. Cells were harvested, and nuclear lysates from the three different populations were mixed in a 1:1:1 ratio. His6-SUMO conjugates were subsequently purified and separated on a one-dimensional gel (Fig. 1B). The gel lane was cut in slices, and proteins were in-gel-digested by Lys-C and analyzed by mass spectrometry. Interestingly we identified two different ubiquitin peptides from the top slices of the gel lane that were preferentially enriched in the Lys8-encoded form, indicating that endogenous ubiquitin co-purified with large His6-SUMO-2 conjugates but not with large His6-SUMO-1 conjugates (Fig. 1C and supplemental Fig. S1). The amount of ubiquitin that co-purified with His6-SUMO-2 conjugates significantly increased upon inhibition of the proteasome (Fig. 1, D–F). The reverse experiment showed that endogenous SUMO-2/3 co-purified with His6-ubiquitin conjugates (Fig. 1, G–I).

Endogenous SUMO-2/3 Conjugates Accumulate in Cells Treated with Proteasome Inhibitors—
To study the effect of proteasome inhibition on endogenous SUMO-1 and endogenous SUMO-2/3, HeLa cells were treated with MG132 or epoxomicin for up to 8 h (Fig. 2). These inhibitors caused rapid accumulation of ubiquitin in cells (Fig. 2, A and E) and in addition caused the accumulation of SUMO-2/3 conjugates albeit with slower kinetics (Fig. 2, C and G). Simultaneously the amount of non-conjugated SUMO-2/3 was significantly reduced by these inhibitors (Fig. 2M). The total amount of SUMO-1 conjugates in cells was not affected by MG132 or epoxomicin treatments, although the pattern of SUMO-1 conjugates slightly changed (Fig. 2, B and F). Control DMSO treatments did not affect ubiquitination or sumoylation (Fig. 2, I–K).


Figure 2
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  		FIG. 2. Endogenous SUMO-2/3 conjugates accumulate in cells treated with proteasome inhibitors. A–M, HeLa cells were treated for the indicated periods of time with the proteasome inhibitors MG132 (A–D and M) or epoxomicin (E–H and M) or with DMSO (I–L and M). Whole cell extracts of HeLa cells were separated by SDS-PAGE, transferred to membranes, stained with Ponceau S to visualize total protein (D, H, and L), and probed using antibody SC-8017 to detect ubiquitin (A, E, and I), antibody 21C7 to detect SUMO-1 (B, F, and J), or antibody AV-SM23-0100 to detect SUMO-2/3 (C, G, K, and M).

 
Identification of SUMO-2 Target Proteins That Are Affected by the Proteasome Inhibitor MG132—
To identify individual SUMO-2 target proteins that are sensitive to proteasome inhibition, quantitative proteomics was used (Fig. 3A). Two pools of HeLaHis6-SUMO-2 cells were used for this experiment, the first pool of cells was labeled with Arg0 and Lys0 and treated with DMSO, and the second pool of cells was labeled with Arg10 and Lys8 and treated with MG132. It is important to note that this strategy was optimal for identifying SUMO-2 conjugates that were sensitive to proteasome inhibition, but it is likely that contaminating non-sumoylated proteins were co-purified. Especially the group of purified proteins with an unaltered ratio might contain a significant percentage of contaminants. Cells were harvested, and whole cell lysates were mixed in a 1:1 ratio. His6-SUMO-2 conjugates were subsequently purified, digested in solution with trypsin, and analyzed by mass spectrometry. A summary of the results is depicted in Fig. 3B. 847 proteins were identified by at least two unique peptides (supplemental Table 1 protein sheet), and in total 7643 peptides were identified by mass spectrometry (supplemental Table 1 peptide sheet). Interestingly two different subsets of MG132-sensitive SUMO-2 target proteins were identified; 73 target proteins showed a significant increase in sumoylation upon inhibition of the proteasome (supplemental Table 3), and 40 target proteins showed a significant decrease in sumoylation in response to MG132 (supplemental Table 4). The MG132-mediated increase in SUMO-2 conjugation was consistent with the immunoblot experiments (Fig. 2), whereas the MG132-mediated decrease in SUMO-2 conjugation of other target proteins was unexpected. The decrease in sumoylation of these target proteins might potentially be explained by a reduction in free SUMO-2 (Fig. 2M).

The largest functional group of MG132-regulated SUMO-2-conjugated proteins controls nucleic acid metabolism (supplemental Fig. S2). This group constitutes 47% of all MG132-up-regulated targets and 40% of all MG132-down-regulated targets and includes DNA repair factors, replication factors, helicases, basal transcription machinery components, transcription factors, chromatin modifiers, and RNA binding and processing factors (supplemental Fig. S2). In addition, both MG132-sensitive subsets contain a variety of other proteins, implicating that cross-talk between SUMO-2 and ubiquitin has a broad impact on cellular processes.

To confirm our findings independently, His6-SUMO-2 conjugates were purified from DMSO or MG132-treated cells, and immunoblotting experiments were carried out (Fig. 4). These experiments confirmed the accumulation of SUMO-2-conjugated forms of hnRNP M, MCM-7, and PIAS1 upon proteasome inhibition (Fig. 4, A–C) and a decrease of SUMO-2-conjugated forms of SAFB and SART1 (Fig. 4, E and F). We noticed a slight decrease in SUMO-2 conjugation of RanGAP1 in this experiment (Fig. 4D). Strikingly the total pools of these proteins were not affected by MG132, indicating that the ubiquitin-proteasome system specifically regulates SUMO-2-conjugated forms of these proteins (Fig. 4, A–F, inputs).

The most obvious explanation for our results would be that a subset of SUMO-2/3 target proteins is subsequently ubiquitinated and degraded by the proteasome. Inhibition of the proteasome system could then lead to proteins accumulating in the sumoylated and ubiquitinated form. To study the ubiquitination status of hnRNP M, MCM-7, PIAS1, SAFB, and SART1, similar experiments were carried out using His6-ubiquitin instead of His6-SUMO-2 (Fig. 5). As expected, hnRNP M, MCM-7, and PIAS1 were indeed ubiquitinated in an MG132-sensitive manner. PIAS1 (29) was also reported previously to be ubiquitinated. In contrast, ubiquitinated forms of SAFB or SART1 could not be detected in these experiments.


Figure 5
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  		FIG. 5. hnRNP M, MCM7, and PIAS1 are conjugated to ubiquitin. A–E, HeLa cells were transfected with a plasmid that encodes His6-ubiquitin or with an empty control plasmid, and cells were treated with MG132 or DMSO for 3 h. His6-ubiquitin conjugates were subsequently purified from equal amounts of whole cell lysates. His6-ubiquitin conjugates or equal amounts of whole cell extracts were size-separated by SDS-PAGE, transferred to membranes, and probed using antibodies to detect hnRNP M (A), MCM-7 (B), PIAS1 (C), SAFB (D), or SART1 (E).

 
SUMO-2/3 Chains Accumulate in Cells Treated with MG132—
Previously we have shown that SUMO-2 and SUMO-3 are able to multimerize in cells in a ubiquitin-like manner (11). To investigate whether SUMO-2/3 chains accumulated in cells treated with proteasome inhibitors, we searched for tryptic SUMO-SUMO peptides in purified His6-SUMO-2 conjugates from our quantitative proteomics experiments. Interestingly both SUMO-2-SUMO-2 and SUMO-2-SUMO-3 peptides were shown to accumulate upon MG132 treatment (Fig. 6, A and B).

To determine whether these SUMO polymers are functionally important for the processing of SUMO-2 targets by the proteasome, plasmids were generated that encode SUMO-2 mutants reduced for chain formation. HeLa cells were transiently transfected and treated with DMSO or MG132, and proteins conjugated to wild-type or mutant SUMOs were purified and analyzed by immunoblotting. The SUMO-2 mutants accumulated in MG132-treated cells similarly to wild-type SUMO-2 (Fig. 6C), and ubiquitin still co-purified with the SUMO-2 mutants (Fig. 6D). Furthermore both wild-type and mutant SUMO-2-conjugated forms of hnRNP M, MCM-7, and PIAS1 accumulated upon inhibition of the proteasome (Fig. 6, E–G). We conclude that SUMO chain formation is not required for the processing of SUMO-2 targets by the proteasome.

Ubiquitination of SUMO-2/3—
The accumulation of ubiquitin in SUMO-2 conjugates could potentially be explained by the formation of mixed SUMO-2/ubiquitin chains and also by the conjugation of SUMO-2 and ubiquitin to independent lysines in target proteins. To test the formation of mixed chains, His6-ubiquitin was purified from cells, digested, and analyzed by mass spectrometry to investigate the ubiquitination of endogenous SUMO-2/3. A high quality MS/MS spectrum was obtained showing the ubiquitination of endogenous SUMO-2 on lysine 32 or the ubiquitination of endogenous SUMO-3 on lysine 33 (Fig. 7A).

To determine whether mixed chains are functionally important for the processing of SUMO-2 targets by the proteasome, a plasmid was generated that encoded a SUMO-2 K32R mutant, and a second mutant was generated that lacked other adjacent lysines 34, 41, and 44 (designated 4KR). HeLa cells were transiently transfected and treated with DMSO or MG132, and proteins conjugated to wild-type or mutant SUMOs were purified and analyzed by immunoblotting. The SUMO-2 mutants accumulated in MG132-treated cells similarly to wild-type SUMO-2 (Fig. 7B), and ubiquitin co-purified efficiently with the SUMO-2 mutants (Fig. 7C). Both wild-type and mutant SUMO-2-conjugated forms of hnRNP M, MCM-7, and PIAS1 all accumulated upon inhibition of the proteasome (Fig. 7, D–F). Moreover a lysine-deficient SUMO-2 mutant accumulated in MG132-treated cells similarly to wild-type SUMO-2, and both wild-type and mutant SUMO-2-conjugated forms of hnRNP M, MCM-7, and PIAS1 all accumulated upon inhibition of the proteasome (Fig. 8, A–E) We conclude that SUMO-2/ubiquitin mixed chain formation and SUMO-SUMO chain formation are not required for the processing of SUMO-2 targets by the proteasome. In principle, it is still possible that these SUMO-2 mutants form residual polymers with endogenous SUMO-2/3. However, dimers consisting of the lysine-deficient SUMO-2 mutant and endogenous SUMO-2/3 could not be detected by immunoblotting (Fig. 8A, lanes 5 and 6). Our data are compatible with consecutive sumoylation and ubiquitination of a significant subset of SUMO-2 target proteins via independent lysines in these target proteins (Fig. 9). Upon ubiquitination, SUMO-2/3-conjugated proteins are degraded by the proteasome, enabling the recycling of SUMO-2/3.


Figure 8
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  		FIG. 8. A lysine-deficient SUMO-2 mutant is sensitive to proteasome inhibition. A–E, a His6-SUMO-2 plasmid was generated that encodes a lysine-deficient mutant (allKR). HeLa cells were transfected with plasmids that encode His6-tagged forms of wild type (w.t.) or the allKR SUMO-2 mutant. Cells were treated with MG132 or DMSO for 5 h, and His6-SUMO-2 conjugates were purified from equal amounts of whole cell lysates. Purified fractions were size-separated by SDS-PAGE, transferred to membranes, and probed using antibodies to detect SUMO-2/3 (A), ubiquitin (B), hnRNP M (C), MCM-7 (D), or PIAS1 (E).

 

Figure 9
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  		FIG. 9. The ubiquitin-proteasome system is a component of the SUMO-2/3 cycle. Our data indicate that the turnover of a subset of SUMO-2/3 (S2/3) conjugates is regulated by the ubiquitin-proteasome system. Target proteins that are conjugated to a single monomer of SUMO-2 or SUMO-3 or to multiple monomers can subsequently be ubiquitinated and degraded by the proteasome. This process enables the recycling of SUMOs to provide sufficient amounts of free SUMOs for new rounds of conjugation. In addition, mixed SUMO/ubiquitin (Ubi) chains and SUMO-SUMO chains are formed.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Ubiquitination and sumoylation are generally considered to be independent protein modifications. We have shown here that extensive cross-talk exists between ubiquitin and SUMO-2/3 conjugation of target proteins (Fig. 9). This cross-talk regulates selected SUMO-2/3 targets either directly or indirectly. A substantial percentage of SUMO-2/3 conjugates is directly regulated by subsequent ubiquitination and processing by the proteasome. A second subset of target proteins is indirectly dependent on the ubiquitin-proteasome system to provide a sufficiently large pool of free recycled SUMO-2/3 for subsequent rounds of conjugation.

The recent identification of the SLX5/8 complex in yeast and RNF4 in mammals (also known as small nuclear RING finger) provides mechanistic insight in the ubiquitination of sumoylated proteins (30–35). These proteins contain both a RING domain and SUMO interaction motifs and act as ubiquitin E3 ligases that are targeted to sumoylated proteins via these SUMO interaction motifs (31–33, 35–37). SLX5 and SLX8 are required for maintenance of the genome (31–35, 38–42). The identification of a large subset of nucleic acid-binding proteins that are sumoylated in a proteasome inhibitor-sensitive manner provides an important framework for further detailed analysis of the regulation of nucleic acid metabolism by SUMO-2/3 and the ubiquitin-proteasome system.

The precise roles of SUMO-SUMO chains as shown in Fig. 6, A and B, and of mixed SUMO-2/3 ubiquitin chains as shown in Fig. 7A is currently unclear. Because mutants that lack acceptor lysines still accumulate in cells treated with proteasome inhibitors and ubiquitin still co-purified similarly to wild-type SUMO forms, these chains are not required for targeting the majority of SUMO-2/3 targets to the proteasome. Nevertheless it is possible that certain proteins are targeted to the proteasome via these chains (36, 37). Alternatively these chains could regulate degradation-independent processes.

The conjugation of another subset of SUMO-2 target proteins was unaffected by the inhibition of the proteasome, including RanGAP1. Interestingly a very large fraction of RanGAP1 exists in a sumoylated form mainly conjugated to SUMO-1 (6). It seems likely that sumoylated forms of RanGAP1 are very stable and have a very slow turnover compared with most other known target proteins. The half-life of the sumoylated forms of a subset of target proteins is regulated by ubiquitination and degradation by the proteasome as shown here but also via SUMO proteases (Sentrin-specific proteases) (43). Sumoylated RanGAP1 is localized at the cytoplasmic surface of the nuclear pore complex (44, 45); this might positively contribute to its stability because the majority of SUMO proteases are nuclear proteins (43).

Although the sumoylated forms of hnRNP M, MCM-7, and PIAS1 and the ubiquitinated forms of these proteins are strongly stabilized by MG132, the total pools of these proteins were unaffected by this inhibitor. It is therefore likely that only a small fraction of these proteins are modified within the 3-h time frame of the experiment.

Transiently sumoylated proteins with short half-lives of the sumoylated forms because of the activity of SUMO proteases are likely to show the strongest decrease in sumoylation upon inhibition of the proteasome because of insufficient amounts of free SUMO-2/3. Therefore, the relative reduction in sumoylation of SUMO-2/3 target proteins upon inhibition of the proteasome is likely to reflect the relative instability of the sumoylated forms of these proteins.

Currently it is unclear why some SUMO-2/3 targets are subsequently ubiquitinated and degraded, whereas sumoylated forms of other SUMO-2/3 targets such as SART1 and SAFB are not subjected to detectable amounts of ubiquitination. Most likely, RNF4 plays a critical role and binds preferentially to a subset of SUMO-2/3 target proteins. Alternatively the localization of RNF4 in the cell might limit its activity to a subset of SUMO-2/3 target proteins in a manner similar to the SUMO proteases (43). It is interesting to note that RNF4 specifically localizes to PML bodies (46), sites that are significantly enriched in SUMOs (22, 47). These nuclear bodies have also been shown to contain ubiquitin and proteasomal proteins (48), suggesting that the ubiquitination and degradation of sumoylated proteins can occur in PML bodies (36, 37).

The total levels of SUMO-1 were not affected by inhibition of the proteasome (Fig. 2) in contrast to a previous publication (49); nevertheless SUMO-1 accumulated in purified SUMO-2 in a manner similar to ubiquitin. Currently the role of SUMO-1 attached to SUMO-2 conjugates is unclear. Previously we have shown that SUMO-1 can directly be attached to lysine 11 of SUMO-2 (11). These mixed SUMO chains are unlikely to play a role in targeting proteins to the proteasome because SUMO-2 K11R or E13A mutants still accumulated upon proteasome inhibition, and ubiquitin efficiently co-purified with these mutants (Fig. 6).

In summary, we have shown here that the ubiquitin-proteasome system is an important component of the cellular SUMO-2/3 cycle that is required for the processing of a subset of SUMO-2/3 targets and the recycling of SUMO-2/3. The identification of other components of this pathway and the detailed dissection of the regulated target proteins will be an important step toward uncovering the connection between SUMO-2/3 and the ubiquitin-proteasome system in detail.


  FOOTNOTES
 
Received, January 17, 2008

Published, June 18, 2008

Published, MCP Papers in Press, June 18, 2008, DOI 10.1074/mcp.M800025-MCP200

1 The abbreviations used are: E1, SUMO-activating enzyme; E2, SUMO protein carrier protein; E3, SUMO ligase; aa, amino acids; Arg0, [12C6,14N4]arginine; Arg6, [13C6,14N4]arginine; Arg10, [13C6,15N4]arginine; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; hnRNP M, heterogeneous nuclear ribonucleoprotein M; LTQ, linear quadrupole ion trap; Lys0, [12C6,14N2]lysine; Lys4, [2H4,12C6,14N2]lysine; Lys8, [13C6,15N2]lysine; MCM-7, minichromosome maintenance protein 7; PIAS, protein inhibitor of activated signal transducer and activator of transcription; PML, promyelocytic leukemia protein; RanGAP1, Ran GTPase-activating protein 1; RNF4, RING finger protein 4; SAFB, scaffold attachment factor B; SART, squamous cell carcinoma antigen recognized by T-cells; SILAC, stable isotope labeling by amino acids in cell culture; SUMO, small ubiquitin-like modifier; HRP, horseradish peroxidase. Back

* This work was supported by the Netherlands Organisation for Scientific Research (NWO) (to A. C. O. V.) as part of the Innovational Research Incentives Scheme, by a generous grant from the Danish National Research Foundation (to J. S. A. and M. M.), and by a grant from The European Community (RUBICON, VI Framework) (to M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

§ These authors contributed equally to this work. Back

** To whom correspondence may be addressed. Tel.: 45-6550-2365; E-mail: jens.andersen{at}bmb.sdu.dk

{ddagger}{ddagger} To whom correspondence may be addressed: Dept. of Molecular Cell Biology, Leiden University Medical Center, Postal zone S1-P, P. O. box 9600, 2300 RC Leiden, the Netherlands. Tel.: 31-71-5269621; Fax: 31-71-5268270; E-mail: vertegaal{at}lumc.nl


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