A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

Edited by Aaron Ciechanover, Technion-Israel Institute of Technology, Bat Galim, Haifa, Israel, and approved March 10, 2015 (received for review November 21, 2014)
March 31, 2015
112 (15) 4630-4635


The identification of specific ubiquitin ligase–substrate pairs is crucial for understanding the roles of protein ubiquitination in the regulation of diverse biological processes. Despite the development of various methodologies for substrate identification, it remains challenging to determine ubiquitin ligase substrates. Based on previously described tandem ubiquitin-binding entity(ies) (TUBE), we designed the trypsin-resistant (TR)-TUBE for expression in cells. The coexpression of TR-TUBE with an ubiquitin ligase stabilizes the ubiquitinated substrates by masking the ubiquitin chains. Using a combination of two strategies for enriching ubiquitinated substrates, TR-TUBE and anti–Lys-ε-Gly-Gly antibody, we successfully identified specific ubiquitin ligase–substrate pairs. Our methodology provides an effective means for the identification of ubiquitin ligase substrates and the detection of ubiquitin ligase activity.


The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE–expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.
Posttranslational modification by ubiquitin regulates diverse processes in cells (1, 2). Ubiquitination is catalyzed by three types of enzymes—E1, E2, and E3, with the selectivity for the target protein provided by E3 ubiquitin ligases. Although the human genome encodes more than 600 ubiquitin ligases, many of them remain to be studied (3). The Skp1–Cul1–F-box protein (SCF) complex, one of the best-characterized ubiquitin ligases, is composed of three invariable components (Skp1, Cul1, and Rbx1) and a variable component F-box protein that serves as the substrate recognition module. Among the over 70 F-box proteins found in humans, less than half have been characterized (4).
The identification of substrates for a specific ubiquitin ligase has been challenging despite considerable efforts. To date, the physical interaction between an ubiquitin ligase and its substrates has been exploited as the major approach for substrate identification (57). In these studies, immunoprecipitation followed by MS has been used to isolate ligase–substrate complexes. However, there are several difficulties associated with this approach: Most ligase–substrate interactions are generally too weak and transient to isolate the substrates by immunoprecipitation, and the abundances of relevant in vivo substrates are often low due to proteasomal degradation.
Recently, an antibody that recognizes the ubiquitin remnant motif Lys-ε-Gly-Gly (diGly), which is exposed upon tryptic digestion of ubiquitinated proteins, has been developed for global proteomic applications aimed at identifying ubiquitinated substrates (8, 9). Although a few quantitative proteomics studies have identified a particular ubiquitin ligase substrate using stable isotope labeling utilizing amino acids in cell culture and the anti-diGly antibody (10), these examples required large quantities of samples and advanced techniques.
Tandem ubiquitin-binding entity(ies) (TUBE) based on ubiquitin-associated domains have been developed for isolation of polyubiquitinated proteins from cell extracts (11). Notably, TUBE reagents protect polyubiquitin-conjugated proteins in cell lysates from both proteasomal degradation and deubiquitinating enzymes (DUBs) as efficiently as specific inhibitors of these enzymes (11). In this paper, we applied the TUBE technology to in vivo capture of ubiquitinated proteins. To develop a versatile method for identifying substrates of a specific ubiquitin ligase, we designed a mammalian expression vector encoding a FLAG-tagged trypsin-resistant (TR) TUBE, which protects ubiquitin chains from trypsin digestion under native conditions. Using two enrichment methods, TR-TUBE and the anti-diGly antibody, we succeeded in identifying the target substrates of the uncharacterized F-box protein FBXO21.


Protection of Polyubiquitin Chains on Substrates by TR-TUBE.

Our method is based on stabilization of ubiquitinated substrates in vivo by masking of ubiquitin chains with exogenously expressed TR-TUBE (Fig. 1A). We confirmed that the TR-TUBE can bind to all eight types of ubiquitin chain linkages (Fig. S1A). To examine the effect of overexpressed TR-TUBE on ubiquitin homeostasis and cytotoxicity, we first expressed TR-TUBE or an ubiquitin-binding–deficient TR-TUBE mutant (12) in 293T cells (Fig. S1 B and C). We then detected the cellular levels of free and conjugated ubiquitin by immunoblotting with an ubiquitin-specific antibody, and also analyzed cell death by propidium iodide staining (Fig. 1 B and C). The level of conjugated ubiquitin was increased by 48 h posttransfection, with a concomitant reduction in free ubiquitin (Fig. 1B). By contrast, we detected little accumulation of ubiquitin conjugates in cells expressing the ubiquitin-binding–deficient TR-TUBE mutant or in cells treated with the proteasome inhibitor MG132. The accumulation of ubiquitin conjugates upon TR-TUBE expression did result in some cell death at 48 h, comparable to the levels resulting from long-term treatment with MG132 (Fig. 1C). Although prolonged expression of TR-TUBE did not induce significant cell death, the accumulation of ubiquitin conjugates 72 h posttransfection in cells was reduced relative to the accumulation of ubiquitin conjugates at 48 h (Fig. 1 B and D), suggesting that cells highly expressing TR-TUBE gradually undergo cell death.
Fig. 1.
Protection of polyubiquitin chains on substrates by TR-TUBE. (A) TR-TUBE method for isolation of ubiquitinated substrates. Polyubiquitin chains on substrates are masked by exogenously expressed TR-TUBE, and thereby protected from DUBs and the proteasome. Ubiquitinated proteins are enriched by immunoprecipitation (IP) of TR-TUBE from cells expressing E3 ubiquitin ligase and TR-TUBE. Exogenously expressed proteins are shown in red. Ub, ubiquitin. (B) Accumulation of ubiquitin conjugates in TR-TUBE–expressing cells. 293T cells were transfected with FLAG-TR-TUBE or ubiquitin-binding–deficient FLAG-TR-TUBE mutant plasmid, and the transfected cells were harvested at the indicated times. Cells transfected with HA-empty (emp) vector were treated with 10 μM MG132 for the indicated time before harvesting. Whole-cell lysates (WCLs) were analyzed by immunoblotting using antiubiquitin antibody. (C) Effect of TR-TUBE expression or MG132 treatment on cell viability, as determined by propidium iodide staining. Three independent plates of transfected or MG132 cells were analyzed. Error bars represent means ± SEM. (D) Detection of ubiquitin conjugates and ubiquitinated endogenous p27. Cells (1.3 × 106) were cotransfected with 3.5 μg of FLAG-TR-TUBE and 3.5 μg of HA-empty or HA-Skp2 expression plasmids, and the transfected cells were harvested at the indicated times. WCLs and anti-FLAG immunoprecipitates were analyzed by immunoblotting. The arrow indicates the position of p27. (E) Detection of ubiquitination of endogenous and overexpressed p27. Cells expressing FLAG-ubiquitin or FLAG-TR-TUBE with or without HA-Skp2 and/or HA-p27 were treated with or without MG132, and the cells were harvested at 48 h posttransfection. Anti-FLAG immunoprecipitates were analyzed by immunoblotting. The arrow indicates the position of p27.
To confirm that ubiquitinated substrates were actually included in the high-molecular-weight ubiquitin conjugates, we monitored the ubiquitination levels of p27/CDKN1B, one of the best-characterized ubiquitinated substrates. To this end, we expressed FLAG-tagged TR-TUBE or an ubiquitin-binding–deficient TR-TUBE mutant in cells with or without exogenously expressed Skp2/FBXL1, the F-box protein that recognizes p27 (13). Cell lysates were immunoprecipitated with an anti-FLAG antibody, and both whole-cell lysates and precipitates were analyzed by immunoblotting (Fig. 1D and Fig. S2).
Both ubiquitin conjugates and ubiquitinated p27 were efficiently enriched in the TR-TUBE immunoprecipitates. Although ubiquitin conjugates were detected in TR-TUBE mutant–expressing cells that were treated with proteasome/DUB inhibitors, ubiquitinated p27 was barely detectable, even when cells overexpressing Skp2 were treated with inhibitors (Fig. S2). By contrast, ubiquitinated p27 was detected in lysates of TR-TUBE– and Skp2-overexpressing cells in the absence of inhibitor treatment, suggesting that TR-TUBE both protects the polyubiquitin chains on p27 from DUBs and circumvents proteasome degradation. As shown in Fig. 1D, ubiquitin conjugates and ubiquitinated p27 were detectable in TR-TUBE immunoprecipitates from cells harvested 24 h posttransfection in the absence of exogenous Skp2 expression. Notably, ubiquitinated p27 was present at high levels 48 h posttransfection in cells coexpressing Skp2 and TR-TUBE, suggesting that the ubiquitination of p27 detected in these samples was carried out by SCFSkp2, primarily exogenous Skp2.

Detection of Ubiquitination Activity Using TR-TUBE.

Conventionally, detection of specific ubiquitin ligase activity has been conducted using in vitro reconstitution or overexpression systems consisting of ubiquitin, a substrate, and an ubiquitin ligase in the presence of proteasome inhibitors. Indeed, we detected ubiquitination of p27 by SCFSkp2 in ubiquitin immunoprecipitates only when the substrate was overexpressed (Fig. 1E, lanes 5–8). However, overexpression of Skp2 failed to increase the ubiquitination of p27 to a detectable level. By contrast, in the presence of TR-TUBE, exogenous expression of Skp2 increased ubiquitination of p27 (Fig. 1E, lanes 9–12): Ubiquitinated p27 was detectable even in the absence of exogenously overexpressed p27 or MG132. Because the elevated level of ubiquitin ligase was reflected by an increase in the accumulation of ubiquitinated substrates, the TR-TUBE system appears to be useful for quantitative detection of ubiquitin ligase activity toward a specific substrate.
To suppress ubiquitination by endogenous Skp2, we next expressed the dominant-negative mutant Skp2ΔF, which lacks the F-box domain essential for binding to Cul1, thereby inhibiting formation of the SCF ubiquitin ligase complex. Although expression of the ΔF mutant failed to suppress the accumulation of ubiquitinated p27 in the presence of MG132, the absence of the inhibitor altered accumulation of ubiquitinated substrate but did not necessarily alter activity of the ligand (Fig. 2A). A similar ubiquitination pattern was detected for another Skp2 substrate, CDT1 (14). Next, we examined the ubiquitination activity of the other well-characterized F-box proteins, FBXW7 and FBXW1/βTrCP1 (Fig. 2 BD). The ubiquitination of c-Myc (15), a known substrate of FBXW7, was clearly increased by overexpression of FBXW7 and decreased by overexpression of its dominant-negative mutant (Fig. 2B). Overexpression of FBXW1 also increased the ubiquitination of NFKBIA (16) and PDCD4 (17), known FBXW1 substrates, even in the absence of stimuli (Fig. 2 C and D). Furthermore, we investigated the ubiquitination activity of RING-type E3 MDM2 for p53 (18) (Fig. 2E). The level of polyubiquitinated p53 was somewhat increased by overexpression of MDM2, whereas monoubiquitinated p53 was present at high levels, suggesting that MDM2 preferentially catalyzes monoubiquitination of p53 under these conditions (19). Furthermore, we examined the differences in the ubiquitination pattern of substrates due to overexpression of E3 in lysates prepared at different time points (Fig. S3). Although c-Myc was highly ubiquitinated 24 h posttransfection in cells expressing FBXW7, other ubiquitinated substrates accumulated at high levels 48 h posttransfection. Therefore, in subsequent experiments, we used lysates prepared from cells harvested 48 h posttransfection.
Fig. 2.
Detection of ubiquitination activity using TR-TUBE. Ubiquitination assays using the TR-TUBE method are shown. Cells (1.3 × 106) were cotransfected with 3.5 μg of plasmid encoding FLAG-TR-TUBE in combination with 3.5 μg of plasmid encoding emp, WT F-box protein [Skp2 (A), FBXW7 (W7; B), and FBXW1 (W1; C and D)], its dominant-negative mutant (ΔF), or MDM2 (E). Transfected cells were treated with or without MG132 for 4 h before harvesting. Cell lysates obtained 48 h posttransfection were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates were analyzed by immunoblotting. Vertical bars and arrows denote the positions of ubiquitinated substrates and unmodified substrates, respectively.

Development of a Method to Identify Substrates for a Ubiquitin Ligase.

Recently, a large number of ubiquitination sites and ubiquitinated proteins were identified using the anti-diGly antibody (8, 9, 20). In those studies, this antibody was used for direct immunoprecipitation of trypsinized lysates prepared from cells treated with proteasome inhibitor. Therefore, to assess the efficiency of identification of ubiquitinated proteins using TR-TUBE, we compared three methods for enriching ubiquitinated peptides for liquid chromatography (LC)-tandem MS (MS/MS) analysis (Fig. 3A). These analyses were performed using starting material from ∼1 × 107 293T cells. The first and second methods used direct peptide immunoprecipitation, utilizing anti-diGly antibody, of trypsinized cell lysates from MG132-treated cells [diGly (MG132)] or TR-TUBE–overexpressing cells [diGly (FLAG-TR-TUBE)]. In the direct peptide immunoprecipitation, cells were lysed in the presence of 9 M urea and the denatured proteins were diluted with Hepes buffer before trypsin digestion. By contrast, the third method used anti-FLAG antibody enrichment of TR-TUBE–associated proteins before peptide immunoprecipitation [FLAG and diGly (FLAG-TR-TUBE)]. Although the numbers of unique peptides containing the K-ε-GG motif (diGly peptides) and ubiquitinated proteins did not differ significantly between these three methods, the ratios of diGly peptides to total identified peptides and ubiquitinated proteins were drastically different (Fig. 3B). In the dual-enrichment method, more than 95% of identified peptides included the K-ε-GG motif. In addition, overexpression of TR-TUBE markedly decreased the abundance of identified peptides derived from ubiquitin, because TR-TUBE protected polyubiquitin chains on substrates from trypsin digestion (Fig. S4). These results show that initial enrichment of ubiquitinated proteins using TR-TUBE reduces the background without loss of diGly peptides.
Fig. 3.
Development of a method for identifying the substrates of an ubiquitin ligase. (A) Schematic indicating the steps in the substrate identification processes. In the diGly (MG132) method, 293T cells were transfected with HA-empty (empty), ΔF, or WT F-box protein, and the transfectants were treated with MG132 for 4 h before harvesting. In the diGly (TR-TUBE) and FLAG and diGly (TR-TUBE) methods, cells were transfected with FLAG-TR-TUBE in combination with HA-empty, ΔF, or WT F-box protein. In the diGly (MG132) and diGly (TR-TUBE) methods, cells were lysed 48 h posttransfection in urea-based solution and diluted WCLs were digested with trypsin. In the FLAG and diGly (TR-TUBE) method, cells were lysed and immunoprecipitated with anti-FLAG antibody and the eluted proteins from immunoprecipitates were digested with trypsin. The tryptic peptides were further enriched in another immunoaffinity step for peptides containing the K-ε-GG motif (peptide IP with anti-diGly antibody), followed by MS analysis. (B) Comparison of the numbers of diGly peptide (Upper) and ubiquitinated protein (Lower) numbers identified by the three methods described in A. Five individual experiments were performed for each method. (C) Comparison of the efficiency of identification of Skp2 substrates by the three methods described in A. Total peptide spectrum match numbers (# PSMs) of the indicated proteins (p27, p21, CDT1, and CKS1B) obtained from LC-MS/MS analysis using cells expressing HA-empty (blue bars), dominant-negative mutant (Skp2ΔF, green bars), or WT Skp2 (red bars) with or without FLAG-TR-TUBE are shown. Three individual experiments were performed. (D) Detection of ubiquitinated p27 and CDT1 in MG132-treated cells and TR-TUBE–expressing cells. Vertical bars indicate ubiquitinated substrates, and arrows indicate the positions of unmodified substrates. (E) Testing the ubiquitination of CKS1B by Skp2. The arrow indicates the position of 6× Myc-CKS1B.
To evaluate the performance of the TR-TUBE system in identification of ubiquitin ligase substrates, we next compared the number of peptide spectrum matches (PSMs) as a semiquantitative index of three known Skp2 substrates (p27, p21/CDKN1A, and CDT1), in mock-transfected cells, Skp2-expressing cells, and Skp2 ΔF-expressing cells (Fig. 3C). In the first method, using MG132-treated cells, p27 and p21 were barely detectable, whereas CDT1 was reproducibly observed. However, levels of the diGly peptides of these proteins were not always elevated in Skp2-expressing cells. By contrast, in the methods using cells expressing TR-TUBE, especially in the dual-enrichment method, the levels of diGly peptides derived from the substrates were elevated in Skp2-expressing cells. Although p27 itself was stabilized by MG132 treatment, ubiquitinated p27 was barely detectable in MG132-treated cell lysates (Fig. 3D). By contrast, high-molecular-weight smears of both p27 and CDT1 were clearly detected in cells coexpressing TR-TUBE and WT Skp2. Notably, low levels of ubiquitinated CDT1 were detected in MG132-treated cells expressing either WT or mutant Skp2, but not in untreated cells, consistent with the results of the MS analyses. Thus, although there was little difference between the three methods with regard to the efficiency of identification of ubiquitinated proteins, enrichment of TR-TUBE–associated proteins before diGly peptide immunoprecipitation is an effective method for identifying substrates of an overexpressed ubiquitin ligase.
In addition, we found that the levels of two ubiquitinated peptides derived from CKS1B were markedly elevated in Skp2-expressing cells. CKS1B is an essential cofactor of SCFSkp2 that is necessary for the ubiquitination of p27 (21), and CKS1B itself is ubiquitinated by the APC/CCdh1 ubiquitin ligase (22). Hence, we investigated whether Skp2 ubiquitinates CKS1B by coexpressing myc-tagged CKS1B and TR-TUBE in cells, followed by immunoprecipitation of TR-TUBE and immunoblot analysis (Fig. 3E). As predicted, the expression of Skp2 stimulated the ubiquitination of CKS1B.
The identified ubiquitination sites of Skp2 substrates are listed in Table S1, all of which were included in the PhosphoSite Plus and neXtProt databases.

Identification of Substrates for Uncharacterized Ubiquitin Ligases.

Our next goal was to develop a method for the systematic identification of ubiquitin ligase–substrate pairs. For this purpose, we examined the F-box proteins constituting the SCF complex in 293T cells and identified 12 F-box proteins from a FLAG-Cul1 immunoprecipitate (Table S2). We attempted to find substrates for FBXO21, which is ubiquitously expressed and has not yet been well characterized.
To screen for substrates of FBXO21, we performed LC-MS/MS analysis of peptides prepared by the dual-enrichment method from cells coexpressing FLAG-TR-TUBE and HA-empty, HA-FBXO21ΔF, or HA-FBXO21. In three independent analyses, we selected substrate candidates whose PSM numbers and protein scores increased in FBXO21-expressing cells but decreased in FBXO21ΔF-expressing cells (Table S3). Of these candidates, we picked threonyl-tRNA synthetase (TARS) and EP300 interacting inhibitor of differentiation 1 (EID1) because of their reproducibility (Fig. 4A). We also confirmed that the interaction of FBXO21 with TARS or EID1 was stabilized by Skp1 coexpression (23) and treatment with the Nedd8 E1 enzyme inhibitor MLN4924, which stabilizes Cullin-RING ligase substrates (20) (Fig. S5A). We cloned several F-box proteins and investigated whether they bound EID1 or TARS in the presence of MLN4924 (Fig. S5B). Although TARS was detectable at low levels in a few F-box protein immunoprecipitates, the levels of TARS and EID1 were most prominent in FBXO21 immunoprecipitate.
Fig. 4.
Identification of substrates for FBXO21. (A) Screening of FBXO21 substrates by the dual-enrichment method using TR-TUBE and anti-diGly antibody. In three sets of independent MS analyses, we selected proteins whose PSM numbers (# PSMs) increased in cells expressing FBXO21 and decreased in cells expressing FBXO21 mutant. The total PSM numbers of identified substrates (TARS and EID1) obtained from LC-MS/MS analysis using cells expressing HA-empty (blue bars), ΔF (green bars), or WT FBXO21 (red bars) with FLAG-TR-TUBE are shown. (B and C) Ubiquitination assay of EID1. Forty-four hours after FLAG-TR-TUBE in combination with emp, WT FBXO21 (F21), its mutant (ΔF), or siRNA transfection, cells were treated with or without MG132 for 4 h. Anti-FLAG immunoprecipitates were analyzed by immunoblotting. Arrows show the positions of EID1. cont, control. (D) Ubiquitination assay of EID1 by using TR-TUBE and various F-box proteins. Cells were transfected with plasmids encoding HA-TR-TUBE and each FLAG-tagged F-box protein. Anti-HA immunoprecipitates were analyzed by immunoblotting. The arrow shows the position of EID1. (E) RNAi-mediated knockdown of FBXO21. Forty-eight hours after siRNA (si) transfection, FBXO21 and EID1 protein levels in WCLs were assessed by immunoblotting. (F) Quantitative RT-PCR analysis. Total RNA was prepared from 293T cells 48 h after cells were transfected with control or FBXO21-specific siRNA. The data shown are representative of three independent experiments. (G) Forty-eight hours after siRNA transfection, cells were incubated with 2 μg/mL cycloheximide (CHX). In parallel, cells were treated with 1 μM MLN4924 for 1 h before addition of cycloheximide. Cells were harvested at the indicated times after cycloheximide treatment, and WCLs were analyzed by immunoblotting. (H) Stabilization of EID1 by depletion of FBXO21. Twenty-four hours or 48 h after transfection of the indicated siRNAs, protein levels in WCL were analyzed by immunoblotting. (I) Ubiquitination of EID1 by MDM2 and F21. The arrow shows the position of EID1.
EID1, first cloned as an RB1-binding protein, has the ability to inhibit p300 (24) and can also interact with the orphan nuclear receptor SHP (25). To determine whether the interaction of EID1 with FBXO21 is mediated by RB1 or SHP, we sought to identify the regions on EID1 that are necessary for FBXO21 interaction (Fig. S5C). Deletion of the C-terminal RB1-binding site of EID1 did not affect the binding to FBXO21, but deletion of an additional nine residues abolished interaction, suggesting that their interaction is independent on RB1 and SHP. Next, we used immunoblot analysis to evaluate the ability of FBXO21 to ubiquitinate EID1. Ubiquitinated EID1 was clearly detected in both FBXO21-expressing and MG132-treated cells (Fig. 4B), and knockdown of FBXO21 by siRNA suppressed accumulation of ubiquitinated EID1 in MG132-treated cells (Fig. 4C). Further, ubiquitination of EID1 was clearly increased by overexpression of FBXO21, but not other the F-box proteins tested (Fig. 4D). As shown in Fig. 4E, knockdown of FBXO21 by siRNA stabilized EID1 protein levels but did not affect EID1 mRNA levels (Fig. 4F). Consistent with the steady-state levels of EID1, FBXO21 knockdown, as well as treatment of cells with MLN4924, led to stabilization of EID1 in a cycloheximide chase experiment, extending the t1/2 from <30 min to >3 h (Fig. 4G). These results indicate that EID1 is degraded by the proteasome following ubiquitination by SCFFBXO21.
MDM2, rather than Cullin-RING ubiquitin ligase, is the ubiquitin ligase for EID1 (26, 27). However, siRNA-mediated knockdown of MDM2 or RB1 did not stabilize EID1 protein in 293T cells (Fig. 4H). RB1 was stabilized by knockdown of MDM2 at 24 h, consistent with the fact that MDM2 promotes degradation of RB1 (28, 29). In addition, interaction with EID1 causes a conformational rearrangement of RB1 (29, 30), and their interaction may cause RB1 stabilization because the RB1 level was also increased by knockdown of FBXO21 (Fig. 4H). Furthermore, we examined the ubiquitination activity of MDM2 for EID1 (Fig. 4I). However, ubiquitinated EID1 was barely detectable in MDM2-expressing cells, suggesting that the stability of EID1 is regulated by SCFFBXO21 under normal conditions.
TARS is an aminoacyl-tRNA synthetase that is as abundant in cells as ribosomal proteins (31). Although TARS is a highly abundant and stable protein due to its fundamental roles in protein synthesis, 13 residues in TARS are ubiquitinated by Cullin-RING ubiquitin ligases (9), and we found that four sites were ubiquitinated by SCFFBXO21 (Table S1). Indeed, ubiquitination of TARS was stimulated by overexpressed FBXO21 (Fig. S6 A and B). However, the levels of endogenous TARS protein remained constant regardless of whether FBXO21 was knocked down (Fig. S6 CE). Furthermore, we showed that FBXO21 interacted with the TARS editing domain, which includes all of the identified ubiquitination sites within the TARS protein (Fig. S6F and Table S1). In Escherichia coli, severe oxidative stress reduced overall translational fidelity by impairing the editing activity of TARS (24). Actually, ubiquitination of TARS by SCFFBXO21 was slightly elevated in H2O2-treated cells (Fig. S6G), but the stress did not affect its stability (Fig. S6H). These results suggest that TARS is regulated by SCFFBXO21 ubiquitination under stress conditions but that its degradation is not detectable because the protein is so abundant. Editing-defective aminoacyl-tRNA synthetase causes protein misfolding and neurodegeneration (32), and stresses other than oxidative stress may damage its editing activity. Further studies are needed to determine how FBXO21 detects damage in the TARS editing domain.


In this study, we developed the TR-TUBE system, which is useful for detecting ubiquitin ligase activity and identifying substrates of specific E3 ubiquitin ligases. In this system, overexpressed E3 ubiquitinates its endogenous substrates by using ubiquitination-related factors present in cells, and TR-TUBE prevents degradation and deubiquitination of these substrates, allowing detection of the specific activity of an E3 and isolation of its substrates. Although TR-TUBE immunoprecipitates contain excess ubiquitin, TR-TUBE can protect ubiquitin chains on substrates from trypsin digestion (Fig. S4) and reduce the proportion of peptides derived from ubiquitin, which hinder identification of substrates in LC-MS/MS analysis. In trypsinized TR-TUBE immunoprecipitates, substrates of coexpressed Skp2 could be detected by LC-MS/MS analysis; however, we also detected other proteins that are abundant in complex with these substrates, as well as Skp2 itself, but no diGly peptides other than those diGly peptides derived from ubiquitin and Skp2. Therefore, the second enrichment with anti-diGly antibody is remarkably effective for identification of substrates of a particular ubiquitin ligase.
All of the ubiquitination sites of substrates identified in this study were included among the known ubiquitination sites contained in databases (Table S1). However, some sites identified in CDT1 and TARS differed from the sites reported to be ubiquitinated by the Cullin-RING ubiquitin ligase (9). In addition to three reported ubiquitination sites (K132, K153, and K165) in CDT1, which our system failed to detect, K24 of CDT1 was ubiquitinated by SCFSkp2. CDT1 is ubiquitinated by both SCFSkp2 (14) and Cul4-Ddb1 (33), suggesting that the reported sites are ubiquitinated by Cul4-Ddb1. Notably, although the ubiquitination sites of p27 have been analyzed by site-directed mutagenesis (34), these sites have not previously been assigned by proteomic analyses. The diGly peptides derived from p27 were barely detectable in peptide immunoprecipitates of trypsinized cell lysates from cells treated with proteasome inhibitor, but they were effectively detected in lysates from TR-TUBE–expressing cells, suggesting that deubiquitination of p27, rather than instability of the protein itself, hampers detection of the diGly peptides.
Recently, the ubiquitin ligase substrate trapping method was developed for the isolation of ubiquitinated substrates in yeast. In that method, ligase–substrate affinity is increased by fusing the ligase to a tandem ubiquitin-associated domain (35). Although this method should also be useful for identification of the substrates of ubiquitin ligases, it requires parameters such as linker length and configuration to be optimized for each F-box protein. By contrast, the TR-TUBE strategy is very simple, requiring only coexpression of TR-TUBE and the E3. Furthermore, the TR-TUBE method is not restricted to SCF-type ubiquitin ligases, and is also potentially useful for other E3 families. By using a combination of two enrichment strategies, TR-TUBE and the anti–K-ε-GG antibody, we succeeded in identifying ubiquitinated substrates from small amounts of cell lysate. Thus, the TR-TUBE system represents a practical means for obtaining important insights into the functions of ubiquitin ligases.

Materials and Methods

For immunoaffinity purification for ubiquitinated protein identification, WCL prepared from a 10-cm cell culture dish harvested 48 h posttransfection (∼1 × 107 cells) was incubated for 1 h with anti-FLAG monoclonal antibody (anti-DDDDK)–conjugated agarose beads (MBL International). Bead-bound proteins were eluted FLAG peptide (Sigma). Proteins were reduced in 5 mM Tris [2-carboxy-ethyl] phosphine hydrochloride for 30 min at 50 °C, and then alkylated with 10 mM methylmethanethiosulfonate, and alkylated proteins were digested overnight at 37 °C with 1 μg of trypsin (Promega). After tryptic digestion, ubiquitinated peptides were enriched by using the PTMScan ubiquitin remnant motif (K-ε-GG) kit (Cell Signaling). The eluted peptides were desalted using GL-Tip SDB and GL-Tip GC (GL Sciences) prior to LC-MS analysis. Detailed methods are provided in SI Materials and Methods.


This work was supported by a Grant-in-Aid [Japan Society for the Promotion of Science (JSPS) KAKENHI Grant 24580152] for Scientific Research on a Priority Area (to Y.Y.), a Grant-in-Aid (JSPS KAKENHI Grant 24112008) for Scientific Research on Innovative Areas (to Y.S.), Grants-in-Aid (JSPS KAKENHI Grants 2611377 and 13J07852) for JSPS Fellows (to H.T. and H.Y., respectively), and a Grant-in-Aid (JSPS KAKENHI Grant 21000012) for Specially Promoted Research (to K.T.).

Supporting Information

Supporting Information (PDF)
Supporting Information


C Grabbe, K Husnjak, I Dikic, The spatial and temporal organization of ubiquitin networks. Nat Rev Mol Cell Biol 12, 295–307 (2011).
D Komander, M Rape, The ubiquitin code. Annu Rev Biochem 81, 203–229 (2012).
RJ Deshaies, CA Joazeiro, RING domain E3 ubiquitin ligases. Annu Rev Biochem 78, 399–434 (2009).
J Jin, et al., Systematic analysis and nomenclature of mammalian F-box proteins. Genes Dev 18, 2573–2580 (2004).
L Busino, et al., SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins. Science 316, 900–904 (2007).
MA Davis, et al., The SCF-Fbw7 ubiquitin ligase degrades MED13 and MED13L and regulates CDK8 module association with Mediator. Genes Dev 27, 151–156 (2013).
MK Tan, HJ Lim, JW Harper, SCF(FBXO22) regulates histone H3 lysine 9 and 36 methylation levels by targeting histone demethylase KDM4A for ubiquitin-mediated proteasomal degradation. Mol Cell Biol 31, 3687–3699 (2011).
G Xu, JS Paige, SR Jaffrey, Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28, 868–873 (2010).
W Kim, et al., Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44, 325–340 (2011).
SA Sarraf, et al., Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496, 372–376 (2013).
R Hjerpe, et al., Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10, 1250–1258 (2009).
T Sasaki, M Funakoshi, JA Endicott, H Kobayashi, Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain. Biochem Biophys Res Commun 336, 530–535 (2005).
AC Carrano, E Eytan, A Hershko, M Pagano, SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1, 193–199 (1999).
X Li, Q Zhao, R Liao, P Sun, X Wu, The SCF(Skp2) ubiquitin ligase complex interacts with the human replication licensing factor Cdt1 and regulates Cdt1 degradation. J Biol Chem 278, 30854–30858 (2003).
M Yada, et al., Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7. EMBO J 23, 2116–2125 (2004).
P Tan, et al., Recruitment of a ROC1-CUL1 ubiquitin ligase by Skp1 and HOS to catalyze the ubiquitination of I kappa B alpha. Mol Cell 3, 527–533 (1999).
NV Dorrello, et al., S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 314, 467–471 (2006).
AK Hock, KH Vousden, The role of ubiquitin modification in the regulation of p53. Biochim Biophys Acta 1843, 137–149 (2014).
M Li, et al., Mono- versus polyubiquitination: Differential control of p53 fate by Mdm2. Science 302, 1972–1975 (2003).
MJ Emanuele, et al., Global identification of modular cullin-RING ligase substrates. Cell 147, 459–474 (2011).
D Ganoth, et al., The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nat Cell Biol 3, 321–324 (2001).
T Bashir, NV Dorrello, V Amador, D Guardavaccaro, M Pagano, Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature 428, 190–193 (2004).
Y Yoshida, A Murakami, K Tanaka, Skp1 stabilizes the conformation of F-box proteins. Biochem Biophys Res Commun 410, 24–28 (2011).
J Ling, D Söll, Severe oxidative stress induces protein mistranslation through impairment of an aminoacyl-tRNA synthetase editing site. Proc Natl Acad Sci USA 107, 4028–4033 (2010).
A Båvner, L Johansson, G Toresson, JA Gustafsson, E Treuter, A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP. EMBO Rep 3, 478–484 (2002).
S Miyake, et al., Cells degrade a novel inhibitor of differentiation with E1A-like properties upon exiting the cell cycle. Mol Cell Biol 20, 8889–8902 (2000).
B Ye, et al., Pcid2 inactivates developmental genes in human and mouse embryonic stem cells to sustain their pluripotency by modulation of EID1 stability. Stem Cells 32, 623–635 (2014).
C Uchida, et al., Enhanced Mdm2 activity inhibits pRB function via ubiquitin-dependent degradation. EMBO J 24, 160–169 (2005).
P Sdek, et al., MDM2 promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma protein. Mol Cell 20, 699–708 (2005).
M Hassler, et al., Crystal structure of the retinoblastoma protein N domain provides insight into tumor suppression, ligand interaction, and holoprotein architecture. Mol Cell 28, 371–385 (2007).
NA Kulak, G Pichler, I Paron, N Nagaraj, M Mann, Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11, 319–324 (2014).
JW Lee, et al., Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443, 50–55 (2006).
J Jin, EE Arias, J Chen, JW Harper, JC Walter, A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell 23, 709–721 (2006).
M Shirane, et al., Down-regulation of p27(Kip1) by two mechanisms, ubiquitin-mediated degradation and proteolytic processing. J Biol Chem 274, 13886–13893 (1999).
KG Mark, M Simonetta, A Maiolica, CA Seller, DP Toczyski, Ubiquitin ligase trapping identifies an SCF(Saf1) pathway targeting unprocessed vacuolar/lysosomal proteins. Mol Cell 53, 148–161 (2014).

Information & Authors


Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 112 | No. 15
April 14, 2015
PubMed: 25827227


Submission history

Published online: March 31, 2015
Published in issue: April 14, 2015


  1. ubiquitin-binding protein
  2. ubiquitin ligase
  3. ubiquitination


This work was supported by a Grant-in-Aid [Japan Society for the Promotion of Science (JSPS) KAKENHI Grant 24580152] for Scientific Research on a Priority Area (to Y.Y.), a Grant-in-Aid (JSPS KAKENHI Grant 24112008) for Scientific Research on Innovative Areas (to Y.S.), Grants-in-Aid (JSPS KAKENHI Grants 2611377 and 13J07852) for JSPS Fellows (to H.T. and H.Y., respectively), and a Grant-in-Aid (JSPS KAKENHI Grant 21000012) for Specially Promoted Research (to K.T.).


This article is a PNAS Direct Submission.



Yukiko Yoshida1 [email protected]
Protein Metabolism Project,
Yasushi Saeki
Laboratory of Protein Metabolism, and
Arisa Murakami
Protein Metabolism Project,
Laboratory of Protein Metabolism, and
Junko Kawawaki
Protein Metabolism Project,
Hikaru Tsuchiya
Laboratory of Protein Metabolism, and
Hidehito Yoshihara
Laboratory of Protein Metabolism, and
Mayumi Shindo
Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan
Keiji Tanaka1 [email protected]
Laboratory of Protein Metabolism, and


To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: Y.Y., Y.S., and K.T. designed research; Y.Y., Y.S., A.M., J.K., H.T., and H.Y. performed research; Y.Y., Y.S., H.T., H.Y., and M.S. analyzed data; and Y.Y., Y.S., and K.T. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

Metrics & Citations


Note: The article usage is presented with a three- to four-day delay and will update daily once available. Due to ths delay, usage data will not appear immediately following publication. Citation information is sourced from Crossref Cited-by service.

Citation statements



If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.

Cited by


    View Options

    View options

    PDF format

    Download this article as a PDF file


    Get Access

    Login options

    Check if you have access through your login credentials or your institution to get full access on this article.

    Personal login Institutional Login

    Recommend to a librarian

    Recommend PNAS to a Librarian

    Purchase options

    Purchase this article to access the full text.

    Single Article Purchase

    A comprehensive method for detecting ubiquitinated substrates using TR-TUBE
    Proceedings of the National Academy of Sciences
    • Vol. 112
    • No. 15
    • pp. 4507-E1967







    Share article link

    Share on social media