Article pubs.acs.org/jpr
Regulation of SUMO2 Target Proteins by the Proteasome in Human Cells Exposed to Replication Stress Sara Bursomanno,† Joanna F. McGouran,‡,§ Benedikt M. Kessler,‡ Ian D. Hickson,† and Ying Liu*,† †
Center for Chromosome Stability, and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Panum Institute, Blegdamsvej 3b, DK-2200 Copenhagen, Denmark ‡ Ubiquitin Proteolysis Group, TDI Mass Spectrometry Laboratory, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Headington, OX3 7FZ Oxford, UK S Supporting Information *
ABSTRACT: In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2Q87R tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome inhibitor. We identified subgroups of putative SUMO2 targets that were either degraded or stabilized by EPOX upon SUMO2 conjugation in response to replication stress. Interestingly, the subgroup of proteins degraded upon SUMO2 conjugation was enriched in proteins playing roles in DNA damage repair and replication, while the proteins stabilized upon SUMOylation were mainly involved in chromatin maintenance. In addition, we identified 43 SUMOylation sites in target proteins, of which 17 are located in the proximity of phosphorylated residues. Considering that DNA replication stress is a major source of genome instability, which is suggested to drive tumorigenesis and possibly aging, our data will facilitate future functional studies in the fields of DNA metabolism and cancer biology. KEYWORDS: DNA replication stress, epoxomicin, mass spectrometry, proteolysis, SUMOylation consensus sites
■
termini, and they are often referred to as SUMO2/3.4 In contrast, SUMO2/3 share only 45% protein sequence homology with SUMO1 and are considered distinct proteins. Consistent with their structural differences, SUMO1 and SUMO2/3 are found conjugated to different sets of proteins and in response to different cellular conditions. It has been shown that conjugated-SUMO2/3 levels are increased in response to cellular stress, such as heat shock or replication stress, whereas SUMO1 is constitutively found attached to target proteins, and its conjugation status is not affected by cellular stresses.8,9 DNA damage can arise following exposure of cells to exogenous agents such as ultraviolet light or ionizing radiation.10 However, a major source of DNA damage occurs endogenously during the process of DNA replication. For instance, replication forks can encounter lesions in the template or bound proteins that interfere with fork progression.11 This perturbation of replication can cause deletions or gene rearrangements, particularly at sites in the genome that are intrinsically fragile. Treatment of cells with replication
INTRODUCTION The small ubiquitin-like modifier (SUMO) has been shown to regulate protein function in various ways.1 Along with structural similarity, SUMO shares with ubiquitin the typical three-step conjugation pathway, which leads to the formation of a covalent bond between a carboxy-terminal glycine of SUMO and a lysine in the target protein.2 SUMO is first bound to an E1 SUMO-activating enzyme and subsequently transferred to a protein substrate by an E2 SUMO-conjugating enzyme (ubiquitin-conjugating enzyme E2I; UBE21). The action of the relatively low number of E3 ligases, as compared to ubiquitin E3 ligases, is not always required for the faithful attachment of SUMO to the target protein but seems to increase the specificity of the modification.3,4 The reversible nature of SUMO modification is tightly regulated in vivo by SUMO-specific proteases such as SENP1−3 and SENP5−7, which deconjugate the modifier from the target protein.2,5,6 Four different isoforms of SUMO have been described in human cells, SUMO1−4. SUMO1−3 are expressed in all cell types, while SUMO4 is mostly expressed in the kidney, lymph nodes, and spleen, and its function remains to be clarified.7 The protein sequences of SUMO2 and SUMO3 are highly similar as they only differ in three amino acidic residues at their N© 2015 American Chemical Society
Received: September 23, 2014 Published: March 7, 2015 1687
DOI: 10.1021/pr500997p J. Proteome Res. 2015, 14, 1687−1699
Article
Journal of Proteome Research
proteins that are either degraded or stabilized by the proteasome once they are SUMOylated by SUMO2 under replication stress conditions. We have also identified a panel of potential SUMO2 conjugation sites, among which more than one-third do not conform to known consensus motifs but instead have at least one phosphorylation site in the vicinity of the SUMOylated residue.
perturbing agents, such as low doses of the DNA polymerase inhibitor aphidicolin (APH) that slow, but do not arrest, replication forks, can induce breakage of so-called common fragile sites (CFSs).12−14 Strong evidence exists that CFSs are a primary target for oncogene-induced DNA damage in preneoplastic lesions, which suggests that CFS instability could be a key player during tumorigenesis.15 We previously identified a group of proteins with an increased level of conjugation to SUMO2 in cells subjected to DNA replication stress.9 We were therefore intrigued to understand how protein function might be altered upon SUMO2 conjugation. Previous studies by other groups have indicated that the regulation of a protein’s activity by SUMOylation is exercised in several ways including through altering subcellular localization, providing a surface for interaction with downstream proteins, or blocking the interaction sites of target proteins.1,16 It has been reported that a cross-talk also exists between SUMOylation and ubiquitination.17−19 Although a direct role in targeting proteins for degradation has not been demonstrated, it was proposed that SUMOylation can act as a signal for the recruitment of SUMO-dependent ubiquitin ligases (STUBLs), which catalyze the ubiquitination of target protein and its consequent proteasome-mediated degradation.20 Interestingly, following treatment of cells with proteasome inhibitors such as MG132 or epoxomicin (EPOX), many SUMOylated proteins are stabilized, while others show a reduction in the level of their SUMOylated form.21 Considering that we observed only a modest enhancement of SUMO2 conjugation in a relatively small subset of proteins,9 we hypothesized that some of the SUMOylated proteins might be degraded or de-SUMOylated very quickly after their SUMOylation in cells undergoing DNA replication stress. To fully understand the regulatory role of SUMO2 conjugation, it is essential to identify the locations of the modified lysine residues in the target proteins. Although mass spectrometry (MS) is the most suitable tool for this specific identification, a practical restriction exists due to a 32-aminoacid long branched peptide of SUMO2 remaining attached to the target peptide after tryptic digestion, which thus prevents peptide identification using standard MS database search algorithms. Several strategies have been developed to convert some of the residues at the C-terminus of SUMO2 to trypsin recognition sites, which thus promotes the formation of shorter and less complex branched SUMO-peptides.22 For example, an arginine residue inserted at position 87 or 90 of SUMO2 (Q87R or T90R) can be cleaved by trypsin, which consequently would generate a five- or two-amino-acid-long SUMO fragment attached to the target peptide. Previous studies have demonstrated that both SUMO2Q87R and SUMO2T90R can conjugate to target proteins in a similar manner to that of wild type SUMO2, which has allowed the identification of some SUMO2 conjugation sites.23,24 On the basis of the above findings, we hypothesized that a subgroup of proteins that are SUMOylated by SUMO2 in response to replication stress might be degraded via the ubiquitin mediated proteolytic system. To identify this subgroup of proteins, we adopted a SILAC-based quantitative proteomic approach in U2OS cells treated with replication inhibitors in the presence or absence of the proteasome inhibitor EPOX. To facilitate the identification of SUMOylation sites in the target proteins, we utilized a U2OS cell line stably expressing SUMO2Q87R tagged with StrepHA at its Nterminus. Our data indicate the existence of subgroups of
■
MATERIALS AND METHODS
Cloning and Stable Cell Line Generation
The U2OS-StrepHA-SUMO2 cell line was generated as described previously.9 To generate a mutation at amino acidic position 87 of SUMO2, a Gln-coding codon was replaced by an Arg-coding codon using the pcDNA-StrepHA-SUMO2 plasmid as template. The mutation was generated by QuickChange sitedirected mutagenesis (Stratagene) using oligonucleotides 5′ACAATTGATGTGTTCCGACAGCAGACGGGAGGT-3′ and 5′-ACCTCCGTCTGCTGTCGGAACACATCAATTGT3′. The mutated construct was verified by sequencing and transfected into U2OS cells using FuGENE 6 transfection reagent (Promega). G418 resistant clones expressing StrepHASUMO2Q87R were selected. A clone with StrepHA-SUMO2Q87R expression comparable with the endogenous SUMO2 was chosen for further analysis. Cell Culture Conditions and Drug Treatments
All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml), and streptomycin (100 μg/ mL). Where required, G418 (600 μg/mL) was added to the medium to maintain the selection of cells expressing Strep-HASUMO2, or StrepHA-SUMO2Q87R. To enrich for cells in a perturbed S phase, an asynchronously growing cell population was first synchronized at the G1/S boundary by incubation in medium containing 3 mM HU for 18 h. Cells were then rinsed twice with PBS and released in medium without the drug for 7 h. To induce a perturbed S-phase, the HU-treated cells were released into drug-free medium and subsequently were incubated in medium containing 0.4 μM APH for 8 h. In all cases, the cells were then harvested and subjected to flow cytometry analysis, or pull-down assays. For the analysis of the role of ubiquitin-proteasome system in the turnover of SUMOylated proteins, cells were treated as above, except that EPOX (final concentration of 0.5 μM) or DMSO was added to the medium for the last 6 h of each treatment. In all cases, the cells were then harvested and subjected to flow cytometry analysis or pull-down assays. Pull-Down of Proteins Conjugated to StrepHA-SUMO2 or StrepHA-SUMO2Q87R
U2OS cells stably expressing StrepHA-SUMO2 or StrepHASUMO2Q87R were treated as described above and harvested using SUMO lysis buffer (modified RIPA buffer: 20 mM TrisHCl, pH 7.5, 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% SDS, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Roche), and 20 mM NEM (SigmaAldrich). Protein concentration was determined using BCA protein assay kit (Thermo Scientific), and 2.0−2.5 mg of protein was incubated with Strep-tactin (IBA) resin for 2 h at 4 °C with rotation (10 rpm). The purified proteins were washed four times with SUMO-lysis buffer with high salt (1 M NaCl) and once in SUMO lysis buffer. Proteins were eluted by boiling 1688
DOI: 10.1021/pr500997p J. Proteome Res. 2015, 14, 1687−1699
Article
Journal of Proteome Research
Figure 1. Regulation of SUMOylation by the proteasome. (A) Human U2OS cells were treated with 0.5 μM EPOX, or DMSO for 6 h and harvested. Thirty micrograms of protein cell extract was separated on 4−12% SDS-PAGE and blotted with a SUMO2 antibody. Unconjugated SUMO2 is indicated with an arrow. Actin was used as loading control. (B) Schematic representation of treatments used to induce replication stress and inhibition of the proteasome. (C) Cell cycle profiles of the six cell populations described in panel B. Percentages of cells in different phases of the cell cycle are shown. (D, E) U2OS-StrepHA-SUMO2 cells were treated as described in panel B. Proteins conjugated to StrepHA-SUMO2 were purified using the Strep-tactin resin, separated on an SDS-PAGE, and immunoblotted with either SETX or BLM antibodies (D) or a PCNA antibody (E). The lower parts of the “Input” blots were immunoblotted with a tubulin antibody to serve as a loading control of the samples. The ∧ symbol denotes the position in the single blot where a protein marker lane was removed. The asterisk denotes endogenous PCNA nonspecifically bound to Streptactin beads. 1689
DOI: 10.1021/pr500997p J. Proteome Res. 2015, 14, 1687−1699
Article
Journal of Proteome Research the protein−beads mixture at 95 °C in SDS sample buffer (4% SDS, 120 mM Tris-HCl).
slices, and proteins were in-gel digested using trypsin (Promega) as described previously.25
Antibodies and Western Blotting Analysis
Mass Spectrometry Analysis
Protein samples were separated on 4−12% Criterion Bis-Tris (Bio Rad) gradient gels using MOPS running buffer. Sizefractionated proteins were subsequently transferred onto PVDF transfer membrane (GE Healthcare) using a semidry-blotting system (Bio Rad). Membranes were blocked using PBS containing 0.1% Tween-20 and 5% milk, and incubated with primary antibodies against the following proteins: SUMO2/3 (1:2000; Abcam ab81371), BLM (1:1000; Abcam ab476), PCNA (1:2000, Santa Cruz sc-56), HA (1:2000; Santa Cruz sc7392), actin (1:6000, Sigma A3853), β-tubulin (1:8000, Sigma T4026). An antibody specific for human SETX was kindly provided by Dr. Domenico Delia (1:1500, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy). Horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (Sigma) were used at the dilution 1:2000. Proteins were visualized using an ECL Plus system (Thermo Scientific).
Mass spectrometric analysis was performed by LC−MS/MS using an LTQ Orbitrap Velos (Thermo) coupled to a nanoUPLC system (Nanoacquity Waters). Peptides were separated on a 25 cm 75 μm inner diameter reverse-phase column and detected as previously described.26 Raw data files were processed with the MaxQuant software (version 1.0.14.3).26 Cysteine carbamidomethylation was considered as a fixed modification, and methionine oxidation, protein N-acetylation, and phosphorylation on serine, threonine, and tyrosine residues were set as variable modifications. A mass addition of 471.2078 (QQTGG signature tag remnant of SUMO2Q87R) on lysine was additionally considered as a variable modification. A merged peak list representing all gel slices generated by MaxQuant was searched against the IPI human database (version.3.80, 86719 entries) and also Mascot (http://www.matrixscience.com/) version 2.3.01, which allowed one missed cleavage and 20 ppm/ 0.5 Da mass deviations in MS and MS/MS. Gene ontology term enrichment analysis was performed using the DAVID bioinformatics resource.27 Network analysis of proteins was performed with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database,28 and proteins were visualized using Cytoscape.29 The MS proteomics data are deposited with the ProteomeXchange Consortium (http:// proteomecentral.proteomexchange.org) via the PRIDE partner repository, with the data set identifier PXD001341.30
Immunofluorescence Analysis
Cells were grown on coverslips for 24 h and subsequently fixed using PTMEF fixation buffer (0.2% Triton X-100, 20 mM PIPES pH 6.8, 1 mM MgCl2, 10 mM EGTA pH 8.0, 4% formaldehyde) for 20 min. Cells were then incubated overnight with blocking solution containing PBS and 3% high purity BSA (Sigma-Aldrich) followed by incubation with anti-HA primary antibody (Santa Cruz, sc-7392; 1:300 dilution). Cells were incubated with a secondary antibody conjugated to Alexa Fluor 488 for 1 h at room temperature. Coverslips with cells were mounted in DAPI-containing mounting medium (Vestashield), and images were acquired on a Nikon Eclipse 80i microscope using a 60× objective.
Manual Validation of Identified Peptides
The MS/MS spectra of all 316 SUMOylated proteins identified in three metabolically labeled forms (a total of 950 peptides) were manually validated using the viewer module of Mascot software. The peptide matches were filtered according to previously described criteria.31 Only a maximum mass deviation smaller than 5 ppm and internal SUMOylated lysines were considered acceptable, as the presence of the tag can lead to inhibition of cleavage. Moreover, Q87R CID spectra were required to show the QQTGG signature fragment ions in the low mass region.
Flow Cytometry
Once treated, the cells were harvested using trypsin, washed twice in PBS, and then resuspended in 1 mL of ice-cold 70% ethanol and incubated at −20 °C overnight. Fixed cells were rinsed twice in PBS and incubated at 37 °C for 30 min with 1 mL of PBS containing 40 μg/mL propidium iodide (SigmaAldrich) and 200 μg/mL RNase A. The DNA content was analyzed by measuring propidium iodide staining using a FACS Calibur (Becton Dickinson). The percentages of cells in G1, S, and G2/M cell cycle phases were calculated using CellQuest software.
■
RESULTS
Proteins Modified by SUMO2 Are Targeted for Proteolytic Degradation
We first examined the global changes of SUMO2 conjugation levels in U2OS cells treated with the proteasome inhibitor EPOX. After 6 h of exposure to this inhibitor, increased levels of global SUMOylation were observed, concomitantly with a small reduction in the pool of unconjugated SUMO2 (Figure 1A). To pursue this further, we investigated the possible role of proteasomal degradation on proteins conjugated to SUMO2 in response to replication stress. For this, we used a U2OS cell line stably expressing SUMO2 tagged with StrepHA at its Nterminus (the U2OS-StrepHA-SUMO2 cell line).9 Western blot analysis was performed to investigate the changes in the SUMO2 SUMOylation status of BLM, SETX, and PCNA. The reasons for studying these three proteins were: (i) these proteins were previously identified as SUMO2 target proteins;9,21,32 (ii) BLM and SETX were reported to be degraded by the ubiquitin-mediated proteasome system;21 and (iii) these three proteins are known to play an important role in DNA replication stress responses.33−35
SILAC Labeling and Enrichment of Proteins Modified by StrepHA-SUMO2Q87R
Cells stably expressing StrepHA-SUMO2Q87R were cultured in DMEM medium containing L-arginine and L-lysine, L-arginineU−13C6 and L-lysine-U-D4, or L-arginine-U−13C6-15N4 and Llysine-U−13C6-15N2 (Cambridge Isotope Laboratories). The light, medium and heavy cell populations were then subjected to the treatments as described in Figure 3, panel A. Cells were lysed using SUMO lysis buffer. Protein concentration was determined using BCA protein assay kit (Thermo Scientific), and an equal amount of total cell extract protein, corresponding to 40 mg from each cell population, was mixed. Strep-tagged SUMOylated proteins were pulled-down following the same procedure as described above. Proteins were eluted by boiling at 95 °C in SDS sample buffer and then separated on a 4−12% SDS gel (Bio-Rad), which was stained using Colloidal Blue Staining Kit (Life Technologies). The gel lane was cut into 10 1690
DOI: 10.1021/pr500997p J. Proteome Res. 2015, 14, 1687−1699
Article
Journal of Proteome Research
Figure 2. Establishment of a U2OS-StrepHA-SUMO2Q87R cell line. (A) Left, an alignment of the C-terminal sequences of Smt3 (yeast), SUMO2 (human), and SUMO2Q87R (human). A Gln residue (Q) in the sequence of SUMO2 is replaced by an Arg residue (R), where an Arginine in the sequence of Smt3 (highlighted in bold) is located. Right, a schematic representation of the procedure to isolate StrepHA-SUMO2Q87R conjugated proteins and to identify the modified peptides by MS. (B) Western blot analysis of U2OS cell lines stably expressing StrepHA-SUMO2 or StrepHASUMO2Q87R. Proteins from the cell extract (30 μg per sample) were separated onto SDS-PAGE and immuno-blotted with SUMO2 (left) or HA (right) antibodies. In each case, parental U2OS cells were used as control. Actin was used as loading control. (C) Immunofluorescence analysis of the U2OS parental cell line, and the U2OS-StrepHA-SUMO2 and U2OS-StrepHA-SUMO2Q87R cells. Cells were immunostained with anti-HA antibody and nuclei detected using DAPI. (D) Cell cycle profiles of U2OS-StrepHA-SUMO2 and U2OS-StrepHA-SUMO2Q87R cells. Cells were either untreated (UNT) or treated with 3 mM of HU for 18 h. Cells treated with HU were either released in drug-free medium for 7 h (HU-release) or released 1 h in drug-free medium and then incubated for 8 h with aphidicolin (0.4 μM) (HU-APH).
To enrich the cells undergoing replication stress, the U2OSStrepHA-SUMO2 cells were first arrested at the G1/S phase
boundary using hydroxyurea (HU; 3 mM for 18 h). The cells were then released into drug-free medium for 1 h before being 1691
DOI: 10.1021/pr500997p J. Proteome Res. 2015, 14, 1687−1699
Article
Journal of Proteome Research
Figure 3. SILAC-based MS to identify SUMOylated proteins with and without the presence of EPOX in U2OS-StrepHA-SUMO2Q87R cells. (A) Schematic representation of the SILAC-based MS approach (top) and the cell treatments (bottom). (B) Purified SUMO-conjugates were separated by SDS-PAGE. Ten gel slices were collected, subjected to tryptic digestion, and analyzed by MS. (C) Cell cycle profiles of each SILAC-labeled U2OS-StrepHA-SUMO2Q87R cell population at the end of each treatment.
exposed, or not, to low-dose APH (0.4 μM) for 8 h. The proteasome inhibitor EPOX or the vehicle control DMSO was added to the cells in the last 6 h of the above treatment (Figure 1B). EPOX was chosen as the proteasome inhibitor because it is known to be more selective than MG132 that can also target other cysteine proteases and Calpain.36,37 The cell cycle profiles of U2OS-StrepHA-SUMO2 cells treated as above indicated that the presence of EPOX did not influence cell cycle progression (Figure 1C). Following the treatments described in Figure 1, panel B, cells from each of the six conditions were lysed, and the protein mixtures were subjected to pull-down assays using Strep-tactin beads. Western blot analysis was subsequently performed using antibodies against BLM, SETX, and PCNA, respectively. We observed an increased amount of SUMO2-conjugated BLM
and SETX in the EPOX-treated cells and a substantial further increase in the HU-release-EPOX or HU-APH-EPOX samples (Figure 1D). This observation is consistent with our previous finding that HU-released cells still undergo DNA replication stress to some degree.9 Notably, particularly in the case of SETX, there was a substantial “smear” of higher molecular weight SUMO2-containing conjugates in the EPOX-treated samples, which likely represent modifications by poly SUMO2 chains, or mixed ubiquitin-SUMO chains. In contrast, the overall level of non-SUMOylated BLM and SETX did not alter following the EPOX treatment, which indicates that the proteolysis of these proteins is specific for the SUMO2conjugated fraction (Figure 1D). Interestingly, while the overall level of SUMO2-conjugated PCNA rose following HU or HUAPH treatments, this was not the case when the proteasome 1692
DOI: 10.1021/pr500997p J. Proteome Res. 2015, 14, 1687−1699
Article
Journal of Proteome Research
Figure 4. Analysis of SUMOylated proteins identified by MS with and without the presence of EPOX in replication stress. (A) Distribution of the SUMO2Q87R-conjugated proteins according to the SILAC ratio H/M, visualized as a line-plot. Those proteins with log2 H/M > 1.0 are highlighted in pink, and proteins with log2 H/M < −1 are highlighted in gray. (B) Validation of SILAC results for BLM and PCNA by Western blot analysis in U2OS StrepHA-SUMO2Q87R cells treated as described in Figure 3. (C) GO term enrichment analysis of proteins whose SUMOylation was influenced by proteolysis using the “biological process” term. Proteins whose SUMOylation is up-regulated in the presence of EPOX are represented in pink, whereas proteins whose SUMOylation is down-regulated in the presence of EPOX are represented in gray. The significance of the enrichment is indicated for each subclassification term on the right. (D) Functional interaction network analysis of SUMOylated proteins upregulated (top panel) or down-regulated (bottom panel) upon EPOX. Protein−protein interaction network analysis was based on the STRING database and represented using Cytoscape software. Node size is representative of the SILAC log2 H/M ratio. Proteins labeled in green are involved in mechanisms of cellular response to DNA damage and DNA repair. Proteins labeled in red are involved in chromatin organization functions.
Establishment of a U2OS-StrepHA-SUMO2Q87R Cell Line
was inhibited. In fact, the level of SUMO2 conjugated PCNA fell slightly in the presence of EPOX. Again, this difference was independent of the overall level of cellular non-SUMOylated PCNA, which was not affected by EPOX treatment (Figure 1E). This phenomenon might be caused by the decrease in the pool of free SUMO2 following EPOX treatment due to the increased conjugation to other proteins, coupled with the fact that a known SUMOylation site in PCNA, Lys164, is the site also targeted for ubiquitin conjugation.38
To enable the identification of SUMO conjugation sites in target proteins, we established a U2OS cell line expressing a mutated form of SUMO2 tagged with StrepHA at its Nterminus. The mutant was obtained by replacing the Gln87 of SUMO2 with an Arg residue. The position of this Arg corresponds to that of an Arg at the C-terminus of Smt3, the homologue of SUMO in Saccharomyces cerevisiae (Figure 2A). By introducing a cleavage site for trypsin, this strategic substitution allowed the shortening of the long SUMO2 branch (32-amino-acid long), with the consequent formation of a 1693
DOI: 10.1021/pr500997p J. Proteome Res. 2015, 14, 1687−1699
Article
Journal of Proteome Research QQTGG peptide attached to the target protein, thus facilitating the identification of SUMO2-modified peptides by MS analysis (Figure 2A). Henceforth, we will refer to this as the U2OSStrepHA-SUMO2Q87R cell line. The Strep-tag was chosen because it has an intrinsically high affinity toward Strep-tactin resin. Therefore, the Strep-tagged proteins can be purified in a single step from crude cell lysate under denaturing conditions. This is an essential requirement for isolating SUMOconjugates, as the activity of SUMO proteases and the noncovalent binding of SUMO are inhibited. Moreover, the high affinity of the Strep-tag allows stringent washing conditions to be applied, which greatly minimizes the nonspecific binding to the Strep-tactin resin. The protein expression and cellular localization of StrepHASUMO2Q87R was subsequently analyzed and compared with that of both the endogenous SUMO2 and the StrepHASUMO2 in U2OS-StrepHA-SUMO2 cell line. The StrepHASUMO2Q87R protein migrated on SDS-PAGE at the same molecular mass as StrepHA-SUMO2 and could be detected using antibodies to SUMO2 and HA (Figure 2B). Also, the StrepHA-SUMO2Q87R protein was found localized in the nucleus similarly to the endogenous SUMO2 and StrepHASUMO2, which demonstrated that the mutation Q87R does not affect the localization of SUMO (Figure 2C). In addition, the cell cycle profiles of U2OS-StrepHA-SUMO2Q87R cells were compared with that of U2OS-StrepHA-SUMO2 cells. The U2OS-StrepHA-SUMO2Q87R cells showed an accumulation in the S phase of the cell cycle following the ‘HU-release’ or ‘HUAPH’ treatments, which was similar to that of the U2OSStrepHA-SUMO2 cells (Figure 2D), thus demonstrating that mutation at position 87 of SUMO2 does not affect cell cycle progression either.
previous study where two subgroups of proteins were also identified when cells propagated with MG132 but without any exogenous stress.21 Through analysis of H/M ratio changes that were either over 2 (in the first group) or below 0.5 (in the latter group), we found 27 proteins in common between our and the previous study, with 19 common proteins in the former group and eight in the latter group (Figure S1, Tables S2 and S3, Supporting Information). Interestingly, in the latter group, we identified many more proteins than that were reported by Schimmel et al.21 This may be due to the fact that the cells in our study were exposed to DNA replication stress or because different proteasome inhibitors were used. The MS data were also consistent with the Western blot analysis of BLM, SETX, and PCNA in the U2OS-StrepHASUMO2 cells shown (Figure 1D,E). In the presence of EPOX, the SUMOylation levels of BLM and SETX increased, with the H/M SILAC ratios of BLM and SETX being 11.7 and 2.3, respectively, whereas the SUMOylation of PCNA decreased, with the H/M SILAC ratio being 0.4. We further validated the SILAC data using Western blot analysis in the non-SILAC labeled U2OS-StrepHA-SUMO2Q87R cells. As expected, a substantial increase in the level of SUMOylated BLM was observed in the cells treated with HU-APH-EPOX condition, while a mild increase of SUMOylated BLM was observed in the HU-APH-DMSO treated condition (Figure 4B, top panel). In contrast to BLM, the level of SUMOylated PCNA decreased slightly in the HU-APH-EPOX condition, while it was increased significantly in the HU-APH-DMSO condition (Figure 4B, middle panel). We next applied a gene ontology (GO) term enrichment analysis with the terms in the “biological process” and “cellular compartment” categories in all of the 316 proteins identified. Interestingly, we found that more than 88% of these proteins were nuclear proteins involved in the regulation of transcription events. We also performed GO analysis with each of the two subgroups of SUMOylated proteins (up- or down-regulated proteins in response to EPOX) (Tables S2 and S3, Supporting Information) using the terms in “biological process” category. Significantly, the up-regulated proteins were found significantly associated with cellular responses to DNA damage and DNA repair, while the down-regulated proteins were significantly enriched in chromatin organization functions (Figure 4C). The most functionally interconnected group of proteins from the two subgroups was then analyzed via the STRING database. A total of 132 interactions between 56 proteins were identified in the group of up-regulated proteins, whereas 263 interactions were identified between 86 proteins in the group of downregulated proteins, which indicates that proteins from each defined subgroup are closely interconnected (Figure 4D).
Quantitative Analysis of SUMO2 Target Proteins Regulated by Proteolytic Degradation
The U2OS-StrepHA-SUMO2Q87R cells were metabolically labeled by growth in medium supplemented with light, medium, or heavy isotope forms of the amino acids Lys and Arg for a quantitative MS analysis.39 The cells were treated and labeled as described in Figure 3, panel A. Once harvested, the cells were lysed under denaturing conditions, and SUMOylated proteins were affinity purified using Strep-tactin resin. Precipitated proteins were subsequently separated by SDSPAGE and subjected to in-gel digestion using trypsin. Peptide fractions were analyzed on a high-resolution quadrupoleOrbitrap MS, and the raw data were analyzed using MaxQuant40 (Figure 3A,B). The cell cycle distributions of cells treated with the HU-APH-DMSO and those treated with HU-APH-EPOX were comparable (69% versus 73% in S phase, respectively) at the time when they were harvested for MS analysis (Figure 3C). In total, 316 putative SUMO2Q87R conjugated proteins were identified. Each protein was detected by at least two unique peptides (Table S1, Supporting Information). According to the SILAC ratio H/M (heavy/medium), two different subsets of proteins were identified: one group with increased SUMO2 conjugation, and one group with decreased SUMO2 conjugation. In detail, 59 proteins showed a significant increase in their SUMOylation levels following inhibition of the proteasome (H/M ratio >2.0), while 106 proteins displayed decreased levels of SUMOylation in response to EPOX (H/M ratio 2.0, such as those in BLM, ESCO2, HNRNPM, and SETX, were exclusively found in a heavylabeled form, which reflects an enrichment of the SUMOylated form of these proteins in response to proteasome inhibition. On the other hand, both of the SUMOylation sites in RLF were identified in the cell population treated with HU-APH-DMSO (medium labeling), which demonstrates that this protein is preferentially SUMOylated at Lys839 and Lys1156 in response to DNA replication stress and may not be affected by
In addition to the core consensus sequence described above, a growing number of SUMOylation sites has been identified in extended patterns, where a consensus motif is accompanied by other elements that could influence the SUMO conjugation. For example, a negatively charged amino acid dependent SUMOylation motif (NDSM) has been described where several acidic residues are clustered within the 10-amino-acid region located downstream of the modified Lys.41 A second extended SUMO consensus site identified is the phosphorylationdependent SUMOylation motif (PDSM) identified by the sequence ΨKxExxSP, where the serine residue is phosphorylated in a proline-dependent manner.42 A third recently identified alternative consensus site is pSUM, ΨKx(pS), which contains a phosphorylated residue at the position where a Glu or an Asp residue is located in the canonical sequence.43 It has been shown that the presence of acidic residues (in NDSM) as well as phosphorylation of the nearby residues (in PDSM and pSUM) plays an important role both in SUMO E3 ligase recognition of the substrates and in determining the efficiency of SUMO conjugation. We subsequently addressed whether, among the SUMO sites identified, any contain an NDSM, a PDSM, or a pSUM motif in their sequences. We observed that there was a stretch of three acidic residues downstream of the consensus motifs in FOSL2 and RSF1 thus resembling the structure of NDSMs (Figure 5C, Table 1). On 1696
DOI: 10.1021/pr500997p J. Proteome Res. 2015, 14, 1687−1699
Article
Journal of Proteome Research
to proteins involved in DNA damage repair and replication processes.53,54 Furthermore, we successfully mapped SUMOylation sites in a portion of the SUMO2 target proteins. In total, 43 SUMOylation sites in 34 different target proteins were identified, with all but six of them not having been described to date to our knowledge.23,24,31 Interestingly, seven sites match the canonical SUMO consensus site, represented by the tetrapeptide ΨKxD/E, and two match the inverted tetrapeptide D/ExKΨ. Moreover, two SUMOylation sites conformed to the NDSM, and 17 SUMOylated peptides were found to contain one or two phosphorylated residues within a 10-amino-acid region upstream or downstream of the modified Lys. Intriguingly, among these 17 peptides, three contained a “SSQ” peptide sequence close to the SUMOylated Lys (Table 1). Within the SSQ sequence, there is the characteristic Sp-Q phosphorylation motif that is known to be targeted specifically by DNA damage-activated protein kinases such as ATM and ATR.55 Although it is not possible at this stage to address whether the presence of phosphorylated residues could stimulate the conjugation of SUMO2 to the nearby Lys, or vice versa, it is likely that a cross-talk exists between the SUMOylation and phosphorylation within these motifs.42,43,56 Taken together, our results suggest the existence of defined subgroups of proteins that are differentially regulated by the proteasome pathway upon SUMO2 conjugation under conditions of replication stress. Considering that both SUMOylation and phosphorylation are important PTMs in regulating the DNA damage repair, replication, and chromosome maintenance process, our data provide a valuable resource for future functional studies in the proteins involved in those processes. For example, it would be most interesting to analyze the function of candidate proteins by mutating the SUMOylated Lys sites identified, particularly those with phosphorylation sites close by. This may ultimately help us with the management of cancer and other age-related diseases.
proteasome. Similarly, both of the SUMOylation sites in SMARCA1 (Lys317 and Lys750) were identified in peptides found in the untreated cell population, which indicates that this protein might only be SUMOylated outside of S phase. Furthermore, not all the SUMO sites identified in any specific protein were found in the same SILAC-labeled form. For example, two SUMO sites were identified in heterogeneous nuclear ribonucleoprotein R (hnRNPR), with Lys110 being found in peptides deriving from HU-APH-DMSO cells, whereas Lys128 was found in HU-APH-EPOX treated cells. It is plausible, therefore, that some proteins are SUMOylated at different sites in response to different cellular stresses.
■
DISCUSSION Replication stress was shown recently to play a central role in the generation of genome instability in cancer cells,44,45 and the cellular response to replication stress is known to be the key driver of early stage tumorigenesis.46 Most interestingly, a new study has demonstrated in mouse models that replication stress is also a potent driver of the aging process in hematopoietic stem cells.47 Studies in yeast have illustrated that SUMOylation is a key modification in response to replication stress as well as during DNA repair processes.48−50 In human cells, studies from several laboratories have shown that SUMO2 conjugation is associated with cellular response to cellular stress, including DNA replication stress.8,9,51 In addition, it has been shown previously that proteins modified by SUMO2/3, but not SUMO1, are targeted for ubiquitin-mediated proteolytic degradation.21 To further dissect the connections between these processes, we have focused on the role of the proteasome in regulating the SUMO2 target proteins in cells treated with DNA replication stress conditions. To identify the proteasome-dependent changes in the SUMOylated proteins levels, we employed a SILAC-based MS analysis using a U2OS cell line stably expressing StrepHAtagged SUMO2Q87R. These cells were subjected to DNA replication stress, and in parallel with or without the proteasome inhibitor epoxomicin to capture short-lived proteins. Our data reveal the existence of two subgroups of proteins: a group of 59 proteins that are degraded by the proteasome upon SUMO2 conjugation (H/M ratio >2.0), and a group of 106 proteins that are stabilized under these conditions (H/M ratio
