In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Summary

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.

Abstract

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.

Introduction

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 manner^1,2. One of the key players in this network is the retinoblastoma (Rb) protein, the first cloned tumor suppressor^1,3. 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 growth^4,5. 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 apoptosis^3,6,7.

Post-translational modifications play a pivotal role in the regulation of RB function^8,9. 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 expression^4,10. 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 repair^{14,15,16,17,18}. 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.

Protocol

1. Detection of Endogenous Rb SUMOylation at the Early G1 Phase

1. Cell culture and cell cycle synchronization.
   1. Maintain HEK293 cells in growth medium containing Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% Pen-Strep and 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂ in an incubator.
   2. Synchronize the HEK293 cells at the G0 phase.
      1. Count the HEK293 cells using a hemocytometer and seed ~1.5 x 10⁷ cells in a 15 cm dish with 25ml growth medium for 24 h before treatment. After reaching a confluence of 70% - 80%, aspirate the medium and wash the cells twice with 5 mL prewarmed phosphate-buffered saline (PBS).
      2. Add 25 mL DMEM containing 1% Penicillin-Streptomycin and incubate the cells at 37 °C for 72 h. Wash the cells off the plate using 3 mL ice-cold PBS. Transfer the cells into a new 5 mL tube.
      3. Subject a small portion of cells to cell cycle analysis by flow cytometry (step 1.2); collecting the remaining majority of the cells by centrifugation at 200 x g for 5 min at room temperature, and then store them in an ultra-lower temperature freezer until analysis of Rb SUMOylation.
   3. To obtain G1-phase cells, at the end of the G0 synchronization process, remove the DMEM and add back fresh growth medium, thus allowing the HEK293 cells to re-enter the cell cycle. Then, collect the cells at different G1 phases (early G1: 30 min; G1: 2h) for cell cycle analysis and further SUMO assay.
   4. Synchronize the HEK293 cells at the S phase using the double-thymidine block method.
      1. Grow HEK293 cells to a confluency of 50% and wash cells with prewarmed PBS. Then add growth medium supplemented with 2.5 mM thymidine for 18 h (first block).
      2. Remove the thymidine-containing medium, and then wash the cells twice with prewarmed PBS. Add fresh growth medium for 14 h to release the cells.
      3. Discard the medium with a pipette and add growth medium supplemented with 2.5 mM thymidine for another 18 h (second block) before conducting an analysis of the cell cycle and Rb SUMOylation.
   5. For the G2/M synchronization, plate ~1.5 × 10⁷ HEK293 cells to a 15 cm dish with 25 mL growth medium for 24 h. Then add nocodazole to the medium until a final concentration of 400 ng/mL is obtained. Finally, incubate the cells for 16 h before conducting the analysis of the cell cycle and Rb SUMOylation.
2. Cell cycle analysis by flow cytometry.
   1. Re-suspend the synchronized HEK293 in PBS, and then fix the cell suspension using ice-cold 70% ethanol for 2 h at 4 °C. Note that to minimize cell clustering, add the cell suspension dropwise to the ice-cold 100% ethanol to obtain a final concentration of 70% ethanol while gently vortexing.
   2. Centrifuge the cells for 5 min at 500 x g, then carefully discard the supernatant and wash the cells twice with PBS. Repeat the centrifuge step.
   3. Add 500 µL PBS containing 50 µg/mL nucleic acid stain propidium iodide, 0.1% Triton X-100 and 1 µg/mL RNase A to the cells and mix well. Incubate the cells for 15 min at 37 °C.
   4. Store the samples at 4 °C until analysis by flow cytometry.
3. Immunoprecipitation of endogenous Rb protein.
   1. Prepare the HEK293 cell lysates.
      1. Lyse the synchronized HEK293 cells by gently re-suspending them in 1 mL of ice-cold radio-immunoprecipitation assay (RIPA) lysis buffer (Table 1) containing freshly added 20 mM N-Ethylmaleimide, an isopeptidase inhibitor that could block SUMO proteases and stabilize SUMO conjugates.
      2. Further homogenize the cells on ice by Dounce homogenization, sonication or simply passing through 10 times through a 21 G needle attached on a 2 mL syringe. Then, incubate the cells on ice for 5 min.
         NOTE: The anionic detergent sodium dodecyl sulfate (SDS) in the RIPA buffer is crucial for the later determination of the Rb-SUMO, as it could eliminate the unspecific SUMO signal that is derived from the non-covalent interaction between the Rb protein and other non-Rb SUMO-species.
   2. Centrifuge the cell lysates at 18,000 x g for 30 min at 4 °C. Transfer the supernatants to new 1.5 mL micro-centrifuge tubes.
      NOTE: The protocol can be paused here. The proteins can be stored at -80 °C for at least 6 months.
   3. To prepare the input control of each sample for the later Western blot, save a small portion of the above-described supernatant to a new tube and store at -80 °C.
   4. To the above supernatants, add 1 µg of non-specific mouse immunoglobulin G (IgG) of the same species and isotype as the monoclonal Rb antibody, and 20 µL of 50% protein A/G-sepharose slurry. Then, incubate for 1 h at 4 °C with gentle rotation.
   5. Centrifuge the samples at 3,000 x g for 3 min at 4 °C. Carefully collect the supernatants without disturbing the beads, and transfer them to new 1.5 mL micro-centrifuge tubes. To each of the samples, add 5 µL Rb primary antibody and 40 µL of 50% protein A/G-sepharose slurry. Then, incubate overnight at 4 °C with gentle rotation.
   6. Collect the beads by centrifugation at 3,000 x g for 3 min at 4 °C, and carefully remove the supernatant by pipetting. Note that in order to avoid bead loss, do not aspirate the beads dry.
   7. Wash the beads four times with 1 mL of the RIPA buffer. Each time, mix the tubes well by rotation them at 4 °C for 15 min. Collect the beads by low speed centrifugation at 3,000 x g for 3 min at 4 °C and then discard the supernatants.
   8. After removing supernatants from the final wash, re-suspend the beads in 30 µL 1x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Table 1) and mix well.
      1. Incubate the tubes at 100 °C in a heat block for 10 min. Centrifuge the samples at 12,000 x g for 1 min to pellet the beads. Carefully collect the supernatants without disturbing the beads, and transfer them to 1.5 mL micro-centrifuge tubes.
   9. Analyze the samples by 4% - 20% gradient SDS-PAGE gel and Western blot or store them at -20 °C for later use.

2. Analysis of 6XHis-tagged Exogenous Rb SUMOylation in Human Cells

1. Cell culture and transfection.
   1. Seed ~6 × 10⁶ HEK293 cells in a 10 cm dish and incubate for 24 h in growth medium under normal cell culture conditions to acquire 75%-85% confluent cells. Before transfection, remove the growth medium and add 6 mL of prewarmed reduced serum medium (see Table of Materials) and then place the dishes back into the incubator.
   2. For the Rb constitutive SUMOylation assay, add 15 µg each of the 6XHis-tagged Rb-Ubc9-WT, Rb-Ubc9-C93S, Ubc9-WT and Ubc9-C93S plasmids in 500 µL reduced serum medium in 1.5 mL micro-centrifuge tubes, respectively.
      1. To analyze the SUMO-conjugation of wild type Rb and its SUMOylation-defective mutant, mix 10 µg of either 6XHis-tagged Rb-WT or Rb-K720R plasmid together with GFP-SUMO1 (10 µg) in 500 µL reduced serum medium in 1.5 mL micro-centrifuge tubes¹³.
   3. Meanwhile, in a separate tube, mix 50 µL transfection reagent (see Table of Materials) in 500 µL reduced serum medium, and incubate at room temperature for 5 min.
   4. Fully combine the two separate tubes described above, and incubate at room temperature for 15 min. Add the transfection reagent/plasmid complexes to the cells and continue to culture for 8 h at 5% CO₂ and 37 °C.
   5. Replace reduced serum medium with growth medium, and incubate the cells under normal culture conditions for 48 h after transfection.
2. Nickel affinity pull down of the 6XHis-tagged Rb protein.
   1. Lyse the transfected HEK293 cells and extract the total proteins as described Section 1.3.1.
   2. Centrifuge the cell lysates at 18,000 x g for 30 min at 4 °C. Carefully collect the supernatants and transfer them to clean tubes.
      NOTE: The protein can be frozen at this point for more than 6 months at -80 °C.
   3. To prepare the input control of each sample for the later Western blot, save a small portion of the supernatants described above to a new tube and store at -80 °C.
   4. Wash 25 µL of 50% nickel nitrilotriacetic acid (Ni-NTA) agarose beads with RIPA buffer twice in a 1.5 mL micro-centrifuge tube for each sample. Collect the beads by centrifugation at 3,000 x g for 3 min at 4 °C.
   5. Add 1 M imidazole to each sample to obtain a final concentration of 10 mM. Then, add each sample to the tube containing the prepared Ni-NTA agarose beads.
   6. Incubate the beads and the lysates for 2 h at 4 °C with gentle rotation. Spin the beads at 3,000 x g for 3 min at 4 °C, and carefully remove the supernatants by pipetting. To avoid bead loss, do not aspirate the beads dry.
   7. Wash the beads with 1 mL of wash buffer containing 20 mM imidazole (Table 1). Each time, mix the tubes well by rotation at 4 °C for 15 min. Collect the beads by spinning at 3,000 x g for 3 min at 4 °C, and discard the supernatants.
   8. After the final wash, add 30 µL of elution buffer containing 250 mM imidazole (Table 1) to each sample and flick to mix. Then, incubate for 20 min at 4 °C to elute the proteins.
   9. Mix each sample with 6× SDS-PAGE loading buffer (Table 1). Then, incubate the tubes at 100 °C in a heat block for 10 min.
   10. Centrifuge at 12,000 x g for 1 min to pellet the beads. Carefully collect the supernatants without disturbing the beads, and transfer the samples to 1.5 ml micro-centrifuge tubes. The samples can be analyzed by Western blot or stored at -20 °C for later use.

3. Western Blot

1. Load the immunoprecipitation or pull down samples obtained from the previous steps onto 4-20% gradient SDS-PAGE gels. Conduct electrophoresis at 120 V for 90 min to separate the proteins.
2. Transfer the proteins from the gel to a polyvinylidene difluoride (PVDF) membrane by electroblotting at 300 mA for 90 min at 4 °C using the tank transfer method. Block the PVDF membrane in block buffer containing 5% nonfat milk (Table 1) for 1 h at room temperature.
3. Incubate the membrane with primary antibody in antibody dilution buffer containing 3% bovine serum albumin (BSA) (Table 1) overnight at 4 °C. For primary antibodies, use a working concentration of 0.5 µg/mL (anti-SUMO1 antibody), or a dilution of 1:2000 (anti-Rb and anti-Tubulin antibodies), or 1:5,000 (anti-GFP and anti-His antibody).
4. Wash the membrane 3x for 10 min each time using 1x tris-buffered saline with Tween (TBST) buffer (Table 1). Incubate the membrane with species specific Horseradish Peroxidase (HRP)-conjugated secondary antibodies diluted 1:5,000 in block buffer containing 5% nonfat milk (Table 1). Wash the membrane 3x for 10 min each time using 1x TBST buffer.
5. Incubate the membrane with enhanced chemiluminescence (ECL) working solution. Cover the membrane with plastic wrap and expose it to X-ray film depending on the strength of signal.

Results

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 pro...

Discussion

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 t...

Materials

Name	Company	Catalog Number	Comments
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