Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.
The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.
The accurate control of cell cycle progression in eukaryotic cells is based on a tight regulatory network, which ensures that particular events take place in an ordered manner1,2. One of the key players in this network is the retinoblastoma (Rb) protein, the first cloned tumor suppressor1,3. The Rb protein is thought to be a negative regulator of cell cycle progression, especially for the G0/G1 to S phase transition, and tumor growth4,5. Failure of Rb function either directly leads to the most common intraocular malignancy in children, retinoblastoma, or contributes to the development of many other types of cancer5. Moreover, Rb is involved in many cellular pathways including cell differentiation, chromatin remodeling, and mitochondria-mediated apoptosis3,6,7.
Post-translational modifications play a pivotal role in the regulation of RB function8,9. Phosphorylation is one such modification, and it usually leads to Rb inactivation. In quiescent G0 cells, Rb is active with a low phosphorylation level. As cells progress through G0/G1 phase, Rb is sequentially hyper-phosphorylated by a series of cyclin-dependent protein kinases (CDKs) and cyclins, such as cyclin E/CDK2 and cyclin D/CDK4/6, which inactivate Rb and eliminate its ability to repress cell-cycle related gene expression4,10. Rb could also be modified by small ubiquitin-related modifier (SUMO)11,12,13.
SUMO is a ubiquitin-like protein that is covalently attached to a variety of target proteins. It is crucial for diverse cellular processes, including cell cycle regulation, transcription, protein cellular localization and degradation, transport, and DNA repair14,15,16,17,18. The SUMO conjugation pathway consists of the dimeric SUMO E1 activating enzyme SAE1/UBA2, the single E2 conjugating enzyme Ubc9, multiple E3 ligases, and SUMO-specific proteases. Generally, nascent SUMO proteins must be proteolytically processed to generate the mature form. The mature SUMO is activated by the E1 heterodimer and then transferred to the E2 enzyme Ubc9. Finally, the C-terminal glycine of SUMO is covalently conjugated to the target lysine of a substrate, and this process is usually facilitated by E3 ligases. The SUMO protein can be removed from the modified substrate by specific proteases. A previous study by the authors of this paper revealed that SUMOylation of Rb increases its binding to CDK2, leading to hyper-phosphorylation at the early G1 phase, a process which is necessary for cell cycle progression13. We also demonstrated that the loss of Rb SUMOylation causes a decreased cell proliferation. Moreover, it was recently demonstrated that the SUMOylation of Rb protects the Rb protein from proteasomal turnover, thus increasing the level of Rb protein in cells19. Therefore, SUMOylation plays an important role in Rb function in various cellular processes. To further study the functional consequence and physiological relevance of Rb SUMOylation, it is important to develop an effective method to analyze the SUMO status of Rb in human cells or patient tissues.
SUMOylation is a reversible, highly dynamic process. Thus, it is usually difficult to detect the SUMO-modified proteins under completely endogenous conditions. This paper presents a method to detect endogenous Rb SUMOylation. Furthermore, it shows how to detect exogenous Rb SUMOylation of both wild-type Rb and its SUMO-deficient mutation11. In particular, Jacobs et al. described a method to increase the SUMO modification of a given substrate specifically by Ubc9 fusion-directed SUMOylation (UFDS)20. Based on this method, this protocol describes how to analyze the forced SUMOylation of Rb and its functional consequences. Given that hundreds of SUMO substrates have been described previously and more putative SUMO substrates have been identified from many proteomic-based assays, this protocol can be applied to analyze the SUMO-modification of these proteins in human cells.
1. Detection of Endogenous Rb SUMOylation at the Early G1 Phase
2. Analysis of 6XHis-tagged Exogenous Rb SUMOylation in Human Cells
3. Western Blot
To detect endogenous Rb SUMOylation during cell cycle progression, this study first synchronized HEK293 cells at five different stages of the cell cycle (G0, early G1, G1, S, and G2/M) as described in the protocol section of this paper. The quality of synchronization was confirmed by using the nucleic acid stain with propidium iodide followed by flow cytometry analysis (Figure 1). Next, the cells were collected and lysed by denaturing RIPA buffer.The SUMO proteases inhibitor, N-Ethylmaleimide, was added to a final concentration of 20 mM to preserve the native SUMO signal during the experiments. After immunoprecipitation of the endogenous Rb species under denaturing conditions to block the non-covalent unspecific interaction, and the following western blot using an anti-SUMO1 antibody, the presence of the SUMO signal was specifically detected at the early G1 phase (Figure 2). Although the global SUMOylation was enhanced at the S/G2/M phase, at the time point of Rb SUMOylation, it had not changed (Figure 2, input panel). This finding suggests that the SUMOylation of Rb is not simply the consequence of altered global SUMO conjugation activity. Thus, these results show that the protein immunoprecipitation and Western blot analysis described in this paper allow for the detection of SUMOylation of the endogenous Rb protein.
To detect the forced SUMOylation of the Rb protein, this study generated a constitutive SUMOylated Rb construct by fusing Ubc9, the sole SUMO E2 ligase, to its C-terminal allowing for efficient and selective SUMOylation of Rb (Figure 3A). The loss-of-function mutation of Ubc9, C93S, is also fused to the C-terminal of Rb to serve as a control (Figure 3A). To further strengthen the specificity of the SUMO-Rb signal, non-fused Ubc9 alone was constructed as well. All four of the His-tagged plasmids were transfected into the HEK293 cells for 48 h before they were lysed in RIPA buffer supplemented with 20 mM N-Ethylmaleimide. All the proteins were then subjected to a pull down assay, as described in the protocol section of this paper. The eluted Rb proteins were analyzed by Western blot. The Ubc9 fusion-directed SUMOylation of Rb was detected using an anti-SUMO1 antibody (Figure 3B, SUMO1 panel), whereas the Ubc9 defective mutation, C93S, failed to produce this signal. Note that the highly efficient SUMO-modification caused by Ubc9-fusion leads to a higher molecular weight band that could even be directly detected by using the Rb antibody, and it corresponds to the SUMO signal (Figure 3B, Rb panel). Moreover, Ubc9 alone did not cause any SUMO conjugation, further confirming the Rb-specific SUMOylation (Figure 3B).
Because SUMO1 is conjugated to lysine 720 of the Rb protein11, to further analyze the Rb SUMOylation, this study generated a SUMO-deficient mutation by replacing this lysine residue with an arginine (K720R). To facilitate the detection of SUMO-conjugation of the two transiently expressed Rb proteins, a GFP-SUMO1 construct was added to enhance the intracellular SUMO signal. His-tagged wild type or mutant Rb was co-transfected into HEK293 cells together with GFP-SUMO1, followed by pull down assay and analysis of Rb-SUMO1 conjugation capability. As observed, the K720R mutant totally abolished the SUMO-modification of Rb, further strengthening the proposed method's ability to detect the Rb SUMOylation (Figure 4).
Figure 1: Validation of the cell cycle synchronization assay by flow cytometry. HEK293 cells were cultured and synchronized at the G0, early G1, G1, S, and G2/M phases of the cell cycle as described in this protocol. Cell cycle distributions were determined by fluorescence-activated cell sorting (FACS). The represented data shows an example of the quantitative analysis of the FACS assay (n = 1). Please click here to view a larger version of this figure.
Figure 2: Rb is SUMOylated at early G1 phase. HEK293 cells were synchronized at different phases of the cell cycle as described in this protocol. The cells were collected and lysed in RIPA buffer supplemented with 20 mM N-Ethylmaleimide. The input controls were loaded on a 4% – 20% SDS-PAGE gradient gel, transferred, and blotted with anti-SUMO1 and anti-Tubulin, as indicated. The remaining cell lysates were then subjected to immunoprecipitation using anti-Rb antibody at a dilution of 1:200. The resulting eluents were separated by 4% – 20% SDS-PAGE gradient gel and immunoblotted using anti-SUMO1 antibody and anti-Rb antibody. This experiment was repeated twice with the same result. This figure has been modified with permission13. Please click here to view a larger version of this figure.
Figure 3. Detection of the SUMO-modification of Rb-Ubc9 fusion protein. (A) Diagram of the Ubc9 fusion-directed SUMOylation (UFDS) constructs of Rb. (B) Constitutive SUMOylation of Rb caused by UFDS. HEK293 cells transiently transfected with His-tagged UFDS constructs were lysed in RIPA buffer with 20 mM N-Ethylmaleimide. The total lysate of each sample was incubated with Ni-NTA agarose beads to pull down the His-tagged Rb-Ubc9 fusion proteins or Ubc9 (used as the negative control), respectively. The purified proteins were separated by 4% – 20% SDS-PAGE gradient gel and immunoblotted with anti-SUMO1 and anti-His antibodies. His-Rb: 6XHis tagged Rb-Ubc9 fusion proteins; His-Ubc9: 6XHis tagged Ubc9 only. Rb-Ubc9: unmodified Rb-Ubc9; Rb-Ubc9-S: SUMOylated Rb-Ubc9; Hyper-pRb-Ubc9: hyper-phosphorylated Rb-Ubc9. The results shown in the figure are representative of three independent trials. This figure has been modified with permission13. Please click here to view a larger version of this figure.
Figure 4. The SUMO-deficient K720R mutation reduces the SUMOylation of Rb. HEK293 cells were transiently co-transfected with the wild type or mutant Rb-His constructs together with GFP-SUMO1. The cells were collected and lysed in RIPA buffer supplemented with 20 mM N-Ethylmaleimide. A small portion of the lysates was directly analyzed by Western blot as the input control. The rest of the lysates were incubated with Ni-NTA agarose beads to pull down the His-tagged Rb proteins as described in this protocol, and they were immunoblotted with anti-SUMO1 and anti-His antibodies. Note that the lysine-to-arginine mutation at 720 of Rb totally abolishes the SUMO modification of this protein. The experiment was conducted once with a similar result to that previously reported11. This figure has been modified with permission13. Please click here to view a larger version of this figure.
Solution | Components | Comments |
RIPA Lysis Buffer | 50 mM Tris, pH = 8.0; 150 mM NaCl; 1% NP-40; 1% sodium deoxycholate; 0.1% SDS | Add protease inhibitor cocktail and N-ethylmaleimide immediately before use. |
Wash Buffer | 50 mM NaH2PO4 pH = 8.0; 300 mM NaCl; 20 mM imidazole | Used for Pull down assay only |
Elution Buffer | 50 mM NaH2PO4 pH = 8.0; 300 mM NaCl; 250 mM imidazole | Used for Pull down assay only |
6x Loading Buffer | 0.5 M Tris, pH = 6.8; 30% glycerol; 10% SDS; 5% β-mercaptoethanol; 0.1% bromphenol blue | Store at -20 °C |
10x Running Buffer | 0.25M Tris, pH 8.6; 1.9M Glycine; 1% SDS | To make 1x Running Buffer: mix 100 ml 10x Running Buffer with 900 mL ddH2O |
10x Transfer Buffer | 0.25 M Tris, pH 8.3, 1.9 M Glycine | To make 1x Transfer Buffer: mix 100 mL 10x Transfer Buffer with 200 ml Methanol and 700 mL ddH2O |
10x TBS Buffer | In 1 L of ddH2O: 24.08 g Tris pH 7.4; 80 g NaCl | To make 1x TBST Buffer: mix 100 mL 10x TBS Buffer with 900 mL ddH2O and 1 mL of Tween-20 |
Blocking Buffer | 1x TBST with 5% nonfat dry milk | Prepare the buffer immediately before use |
Antibody Dilution Buffer | 1x TBST with 3% BSA and 0.02% sodium azide | This Buffer could be stored at 4 °C for at least 6 month |
Table 1: Solutions and buffers.
This paper describes a protocol to detect and analyze the endogenous SUMOylation of Rb in human cells. As this method is specifically focused on the endogenous Rb protein without any alternation of global SUMO-related signal, it is an important tool for investigating Rb-SUMO modification under completely natural physiologic circumstances.
To achieve this aim, it is important to keep in mind that: 1) although SUMO comprises four isoforms (SUMO1-4, each encoded by different genes)in comparison to ubiquitination, all the SUMO species are much less abundant; 2) for most SUMO target proteins, only a small portion of a given protein is SUMOylated at steady state, which is the outcome of the constant competition between the enzymes involved in SUMO conjugation and deconjugation14,21; and 3) SUMO targets can undergo rapid cycles of SUMOylation and deSUMOylation. For example, recent data obtained by the authors of this current paper demonstrate that Rb-SUMO1 exists only for a very short window of time in the early G1 phase, which is consistent with the conception that SUMOylation is a reversible, highly dynamic process13. All these facts make it challenging to directly detect the endogenous SUMOylation of a given protein. Thus, to successfully detect this, it is vital to identify the exact conditions under which this modification occurs. For instance, in this proposed protocol, the timing of the cell cycle synchronization is crucial for the detection of Rb SUMOylation. An optimized experimental procedure is also important for the successful detection of the low-level SUMO-modified species of a given protein. For example, proper sonication or passing-through needles could prevent the formation of sticky and viscous components due to genomic DNA, thus proper sonication can facilitate total protein extraction. Moreover, a good monoclonal antibody with high affinity to the target protein could be helpful for improving the quantity and specificity of immunoprecipitation. In summary, the proposed method is important for further exploring the physiological conditions under which endogenous Rb is SUMOylated, which is the premise of the following overexpression-based functional assay.
To amplify the SUMO signal of a given protein, it is common to co-overexpress this protein as well as other SUMO-related proteins (usually Ubc9 and SUMO protein) in cells. Although a simple Western blot using an antibody against the substrate is the easiest way to find the higher molecular weight, SUMOylated form of this protein, we failed to detect the SUMO-Rb signal with this method. Thus, this paper only focused on a method to detect the SUMO-modification of the precipitated exogenous Rb protein. Moreover, this method described how to artificially enhance or eliminate the SUMO-conjugation of Rb. As the sole E2 ligase, Ubc9 has been found to directly transfer SUMO to specific targets22. Thus, by fusing to the C terminal of Rb, Ubc9 significantly promotes the SUMO-modification of Rb. Using this method, the authors of this paper successfully demonstrated that SUMOylation of Rb sufficiently promoted its own phosphorylation by increasing its binding to CDK213. Moreover, to study the functional consequences of the loss of Rb SUMOylation, the authors generated a SUMO-deficient Rb construct, as previously reported11. By using the method proposed in this current paper, it was shown that SUMO-Rb is necessary for normal cell cycle progression and cell proliferation13.
In addition to SUMO1, SUMO2 and SUMO3 also play important roles in protein SUMOylation14,17. SUMO2 and SUMO3 are often referred to as SUMO2/3 because they are closely related and share 97% identity. SUMO1 shares a 50% similarity with SUMO2/3. It is widely accepted that SUMO1 is responsible for normal cell physiological functions and maintenance, whereas SUMO-2/3 is predominantly involved in cell stress responses15,17,23,24. Given that Rb could also be SUMOylated by SUMO2/3 with unknown function12, the method proposed in this study is still suitable for the detection and analysis of this modification of Rb. In addition to Rb, the methods described here can be widely applied to the detection and functional analysis of the SUMOylation of a variety of target proteins.
The authors have nothing to disclose.
This study was supported by grants from the Science and Technology Commission of Shanghai (Grant No. 14411961800) and National Natural Science Foundation of China (Grant No. 81300805).
Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific | 11995065 | |
Opti-MEM | Thermo Fisher Scientific | 31985070 | |
Fetal Bovine Serum (FBS) | Thermo Fisher Scientific | 26140079 | |
Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | |
Phosphate-buffered Saline (PBS) | Thermo Fisher Scientific | 10010023 | |
Trypsin-EDTA | Thermo Fisher Scientific | 25200056 | |
Thymidine | Sigma | T9250 | |
Nocodazole | Sigma | M1404 | |
propidium iodide | Thermo Fisher Scientific | P3566 | |
Triton X-100 | AMRESCO | 694 | |
RNase A | Thermo Fisher Scientific | EN0531 | |
N-Ethylmaleimide | Sigma | E3876 | |
Sodium Dodecyl Sulfate (SDS) | AMRESCO | M107 | |
Nonidet P-40 Substitute (NP-40) | AMRESCO | M158 | |
protease inhibitor | Roche | 5892970001 | |
Mouse Immunoglobulin G (IgG) | Santa Cruz Biotechnology | sc-2025 | |
Rb antibody | Cell Signaling Technology | #9309 | |
Protein A/G-Sepharose Beads | Santa Cruz Biotechnology | sc-2003 | |
Lipofectamine-2000 | Thermo Fisher Scientific | 11668019 | |
Nickel Nitrilotriacetic Acid (Ni-NTA) Agarose Beads | Qiagen | 30230 | |
Imidazole | Sigma | I0250 | |
4%-20% Gradient SDS-PAGE Gel | BIO-RAD | 4561096 | |
Polyvinylidene Difluoride (PVDF) Membrane | Millipore | IPVH00010 | |
Tween-20 | AMRESCO | M147 | |
Tubulin antibody | Abmart | M30109 | |
SUMO1 antibody | Thermo Fisher Scientific | 33-2044 | |
GFP antibody | Abmart | M20004 | |
Horseradish Peroxidase (HRP) secondary antibody | Jackson ImmunoResearch Laboratories | 715-035-150 | |
enhanced chemiluminescence (ECL) Kit | Thermo Fisher Scientific | 32106 |