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The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2013 Apr 23;288(23):16567–16578. doi: 10.1074/jbc.M112.437129

Site-specific Acetylation of the Proteasome Activator REGγ Directs Its Heptameric Structure and Functions*

Jiang Liu ‡,§,¶,1, Ying Wang ‡,§,¶,1, Lei Li ‡, Li Zhou ‡, Haibin Wei ‡, Qingxia Zhou ‡, Jian Liu , Weicang Wang ‡, Lei Ji ‡, Peipei Shan ‡, Yan Wang ‡, Yuanyuan Yang ‡, Sung Yun Jung , Pei Zhang ‖, Chuangui Wang ‡, Weiwen Long , Bianhong Zhang ‡,2, Xiaotao Li ‡,¶,3
PMCID: PMC3675592  PMID: 23612972

Background: REGγ mediates degradation of numerous proteins. How REGγ activity is regulated remains unclear.

Results: REGγ acetylation at lysine 195 promotes its activity by enhancing monomeric interactions and heptameric formation of the REGγ molecules.

Conclusion: Site-specific acetylation of REGγ is important for its structural architecture and enzymatic function.

Significance: Our discovery provides basis for additional venue to intervene the REGγ proteasome function.

Keywords: Cell Biology, Gene Regulation, Proteasome, Protein Degradation, Protein Self-assembly, Protein-Protein Interactions, Acetylation, K195, REGγ, Heptamerization

Abstract

The proteasome activator REGγ has been reported to promote degradation of steroid receptor coactivator-3 and cyclin-dependent kinase inhibitors p21, p16, and p19 in a ubiquitin- and ATP-independent manner. A recent comparative analysis of REGγ expression in mouse and human tissues reveals a unique pattern of REGγ in specific cell types, suggesting undisclosed functions and biological importance of this molecule. Despite the emerging progress made in REGγ-related studies, how REGγ function is regulated remains to be explored. In this study, we report for the first time that REGγ can be acetylated mostly on its lysine 195 (Lys-195) residue by CREB binding protein (CBP), which can be reversed by sirtuin 1 (SIRT1) in mammalian cells. Site-directed mutagenesis abrogated acetylation at Lys-195 and significantly attenuated the capability of REGγ to degrade its target substrates, p21 and hepatitis C virus core protein. Mechanistically, acetylation at Lys-195 is important for the interactions between REGγ monomers and ultimately influences REGγ heptamerization. Biological analysis of cells containing REGγ-WT or REGγ-K195R mutant indicates an impact of acetylation on REGγ-mediated regulation of cell proliferation and cell cycle progression. These findings reveal a previously unknown mechanism in the regulation of REGγ assembly and activity, suggesting a potential venue for the intervention of the ubiquitin-independent REGγ proteasome activity.

Introduction

The proteasome activator REGγ (also known as PA28γ, PSME3, Ki antigen) belongs to the REG or 11 S family of proteasome activators that have been shown to bind and activate the 20 S proteasome (1, 2). REGγ has been reported to promote degradation of some important regulatory proteins such as steroid receptor coactivator-3 and cyclin-dependent kinase inhibitors p21, p16, and p19 in a ubiquitin- and ATP-independent manner (3–5). Moreover, REGγ facilitates the turnover of tumor suppressor p53 by promoting MDM2-mediated p53 ubiquitination (6) and regulating p53 cellular distribution (7). Furthermore, REGγ is overexpressed in some cancers (8, 9) and is linked to multiple cancer-related pathways (10). A unique expression pattern of REGγ in cell specific manner has been documented, suggesting undisclosed functions and biological importance of this molecule (11). Despite recent progress made in this field, how REGγ is regulated in mammalian cells is largely unknown.

Post-translational modification is an important process in regulating protein structures and functions. Acetylation occurs as a co-translational and post-translational modification of histones and non-histone proteins such as p53 and tubulins (12). In fact, proteomic studies have identified thousands of acetylated mammalian proteins (13, 14), of which chromatin proteins and enzymes are highly represented. Acetylation commonly occurs at a lysine residue and can affect protein nuclear localization, stability, transcriptional activity, DNA binding, and interactions with other proteins and cofactors (12, 15), indicating that acetylation has a considerable impact on protein functions. Several studies suggest that acetylation can alter protein structures or protein-protein interactions (16–18). For example, acetylation of KLF5 transcription factor enhances its interaction with Smad4 to promote transcription of target genes (16). Thompson et al. (17) demonstrate that acetylation of the putative inhibitory loop of p300 may open the locked gate and activate its acetyltransferase activity.

Protein acetylation is a reversible process that is governed by the opposing actions of histone acetyltransferases and histone deacetylases. CBP4 and p300 (E1A binding protein p300) possess strong histone acetyltransferase activity and act on both histone and non-histone proteins (19, 20). Histone deacetylases are classified into four classes and two families: classical (classes I, II, and IV) and Sir2 (silent information regulator 2)-related protein (sirtuin) families (class III) (21). Among the seven members of mammalian sirtuins (SIRT1–7), SIRT1 is the most studied and strongly implicated in cellular regulation through its deacetylase activity (22).

In this study, we illustrate that acetylation of REGγ at the lysine 195 residue by CBP is important for the degradation of REGγ substrates, such as p21 and HCV core proteins. However, SIRT1, a deacetylation enzyme, can interact with REGγ and remove acetylation group at Lys-195, attenuating REGγ activity. Further study reveals that blocking acetylation at Lys-195 significantly reduces interactions between REGγ monomers and ultimately influences the formation of heptamer. Finally, functional analysis in cells containing REGγ-WT or REGγ-K195R mutation has validated the crucial role of acetylation in REGγ-mediated regulation of cell proliferation and cell cycle progression.

EXPERIMENTAL PROCEDURES

Cell Culture and Reagents

HEK293/293T, H1299, HeLa, and A549 cells were purchased from ATCC and maintained in DMEM (Invitrogen), 10% FBS (Invitrogen), and penicillin/streptomycin (Invitrogen). The HEK293 REGγ inducible cell lines were generated by the Flp-InTM T-RExTM system (Invitrogen). REGγ integration in REGγ−/− mouse embryonic fibroblast (MEF) stable cells were generated by lentivirus infection for 2 days and then selected by puromycin (Invitrogen, 3 μg/ml). The antibodies used in this study included anti-REGγ (Invitrogen), anti-FLAG, anti-β-actin (Sigma), anti-CBP, anti-p21 (BD Biosciences), anti-HA, anti-AcK (Cell Signaling Technology and Abcam), anti-SIRT1 (Millipore), and anti-FLAG M2 Affinity Gel (Sigma). The CBP siRNA SMARTpool was purchased from Dharmacon, Inc. Other purchased reagents were proteasome inhibitor MG132 (Sigma), Cycloheximide (Sigma), trichostatin A (Sigma), niacinamide (Sigma), resveratrol (Sigma), BCA protein assay kits (Thermo Scientific), and CellTiter 96® AQueous non-radioactive cell proliferation assay (MTS) reagents (Promega). All of the experiments shown in the study were repeated at least three times.

Plasmid Constructs and Site-directed Mutagenesis

The mammalian expression vector pCDNA5/FRT/TO (Invitrogen) was modified to express REGγ or FLAG-tagged REGγ at the N terminus. HA-tagged REGγ and HCV core-173 constructs were generated in the pSG5 vector. pCDH-CMV-EF1-REGγ was constructed by inserting a digested PCR fragment into the lentivirus expression vector pCDH-CMV-EF1-Puro. GST-tagged REGγ was generated in pGEX-4T-1 vector. pPAL7- REGγ was constructed into pPAL7 vector. His-SIRT1 was generated in pET28a vector. pCDNA3.1-p21 was generated into the pCDNA3.1 vector. pCDNA FLAG-CBP was kindly provided by Dr. Qin Feng (Department of Molecular and Cellular Biology, Baylor College of Medicine), pCDNA3 FLAG-SIRT1, pCDNA3 SIRT1, and pCDNA3 SIRT1 H363Y were provided by Dr. Qiang Tong (Departments of Pediatrics, Medicine, Molecular Physiology & Biophysics, Baylor College of Medicine). Lysine-to-arginine mutations in REGγ or its FLAG/HA-tagged versions were generated by site-directed mutagenesis at residues Lys-6, Lys-14, and Lys-195. All of the constructs were verified by DNA sequencing.

Mass Spectrometry

The HEK293 FLAG-REGγ inducible cells were treated with doxycycline 1 μg/ml for 48 h to induce highly expressed FLAG-REGγ. The cells were lysed with lysis buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40). FLAG-REGγ was immunoprecipitated from precleared cell lysates by incubation with anti-FLAG M2 Affinity Gel overnight at 4 °C. The immunoprecipitates were washed three times with NETN buffer (20 mm Tris-HCl (pH 8.0), 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40). The washed beads were boiled with SDS-PAGE loading buffer. FLAG-REGγ were resolved by SDS-PAGE and stained with Coomassie Blue. FLAG-REGγ protein bands were cut, destained, and digested with trypsin or V8 protease overnight in the NH4HCO3 buffer. Mass spectrometry was performed as described previously (5). Recombinant REGγ protein was purified by Profinity eXactTM Protein Purification System (Bio-Rad). Recombinant REGγ was resolved by SDS-PAGE and stained with Coomassie Blue. After in-gel digestion and peptide extraction, the peptides were analyzed on an Orbitrap Elite spectrometer connected to an EASY-nLC 1000 UPLC system using a nanoelectrospray ion source (Thermo Scientific).

Immunoprecipitation and Detection of REGγ Acetylation in Cells

To detect REGγ acetylation in cells, HEK293/A549/HeLa cells were harvested and lysed in the lysis buffer described above. The lysates were centrifuged, and the supernatants were incubated with 1 μg of anti-REGγ antibody overnight at 4 °C with 1 μg of rabbit IgG (Santa Cruz Biotechnology) as a control. Each sample was incubated with protein A/G Plus-agarose beads (Santa Cruz Biotechnology) for 3 h. Immunoprecipitated REGγ was resolved on a 10% SDS-PAGE gel and analyzed by Western blotting with anti-AcK antibody. The nitrocellulose membrane was stripped by the RestoreTM Western blot Stripping Buffer (Thermo) and then probed with anti-REGγ antibody. For reciprocal immunoprecipitation, HEK293 cell lysate was incubated with anti-AcK antibody overnight at 4 °C and then incubated with protein A/G plus-agarose beads for 3 h. The immunoprecipitates were resolved on a 10% SDS-PAGE gel and analyzed by Western blotting with anti-REGγ antibody. FLAG-tagged REGγ was immunoprecipitated from 293 REGγ-inducible cells with anti-FLAG M2 affinity gel. FLAG-REGγ was eluted with 200 μg/ml FLAG peptide for 1 h. FLAG-REGγ was incubated with anti-AcK antibody overnight at 4 °C in 80 μl of buffer (50 mm Tris, 137 mm NaCl, 1 mm EDTA, 10 mm NaF, 0.1 mm Na3VO4, 1 mm DTT, 10% glycerol, 0.5% Nonidet P-40, pH 7.8), together with 50 mm sodium butyrate, 6.6 μm trichostatin A (TSA), 10 mm nicotinamide (NAM), and protease inhibitors. The mixture was incubated with protein A/G-agarose beads for 3 h. The supernatant unbound REGγ and the immunoprecipitated acetylated REGγ was resolved on a SDS-PAGE gel and analyzed by Western blotting with anti-REGγ antibody. For deacetylase inhibition, the HEK293 FLAG-REGγ inducible cells were treated with 1 μg/ml doxycycline (for 48 h), 6.6 μm TSA, and 10 mm NAM for 6 h prior to cell harvest. Cells were lysed in FLAG lysis buffer (50 mm Tris, 137 mm NaCl, 1 mm EDTA, 10 mm NaF, 0.1 mm Na3VO4, 1% Nonidet P-40, 1 mm DTT, 10% glycerol, pH 7.8) containing fresh protease inhibitors (Roche Applied Science), 6.6 μm TSA, and 10 mm NAM. Cell extracts were immunoprecipitated with anti-FLAG M2 affinity gel. REGγ acetylation was analyzed by Western blotting with a Pan-anti-AcK antibody.

REGγ Acetylation and Deacetylation Assays in Vitro

FLAG-tagged CBP was expressed in HEK293T cells and immunoprecipitated by anti-FLAG M2 affinity gel. FLAG-CBP was eluted with 200 μg/ml FLAG peptide (Sigma) for 1 h. GST-tagged REGγ was expressed and purified as described previously (5). 5 μg of GST-REGγ was incubated with 3 μg of FLAG-CBP in 30 μl of histone acetyltransferase buffer (250 mm Tris-HCl, pH 8.0, 500 μm EDTA, 5 mm DTT, 50% glycerol, 50 mm sodium butyrate, 6.6 μm TSA, 10 mm NAM, and protease inhibitors) with or without addition of 5 mm acetyl-CoA. After 3 h at 30 °C, samples were resolved on SDS-PAGE and analyzed by Western blot. In vitro deacetylation assays were performed as follows. FLAG-REGγ was immunoprecipitated from HEK293 FLAG-REGγ inducible cells transfected with CBP, and the enriched REGγ proteins were eluted with FLAG peptide. Acetylated FLAG-REGγ was incubated with recombinant His-SIRT1 in 50 μl of deacetylation buffer (50 mm Tris-HCl, pH 8.0, 50 mm NaCl, 4 mm MgCl2, 0.5 mm DTT, 0.1 mm PMSF, 5% glycerol, 0.02% Nonidet P-40, and protease inhibitors) in the presence or absence of 50 μm NAD+ for 2 h at 30 °C. The reactions were subjected to SDS-PAGE and analyzed by Western blot.

Transfections and REGγ Activity Detection

Plasmid CBP (1 μg) was transiently transfected into HEK293 cells for 32 h using FuGENE HD DNA transfection reagent (Roche Applied Science). REGγ acetylation levels were detected as described above. Additional experiments were performed in the HEK293 FLAG-REGγ-inducible cells by expressing CBP in the presence or absence of SIRT1, followed by determination of REGγ acetylation. In HeLa cells, SIRT1-WT and the deacetylase mutant, SIRT1-H363Y, were ectopically expressed for 32 h, and protein expressions were examined by Western blotting using anti-SIRT1, anti-REGγ, and anti-p21. For RNA interference, HEK293 cells were transfected with 60 nm ON-TARGET plus siRNA specific for human CBP using LipofectamineTM 2000 transfection reagent (Invitrogen). After 72 h of transfection, cells were harvested and lysed for Western blot analysis of CBP, REGγ, and p21 protein levels. The p21 and/or HCV core protein levels were measured by Western blotting to estimate REGγ activity. In the cells, the p21 or HCV core construct was either co-transfected with a control vector or REGγ-WT, REGγ-K6R, REGγ-K14R, REGγ K195R, or REGγ K195Q. HEK293 REGγ-inducible cells were incubated with 100 mg/ml cycloheximide with or without additional of doxycycline (1 μg/ml, for 48 h) at indicated time for p21 protein half-life analysis.

Co-immunoprecipitation and Protein-Protein Interaction Analysis

HEK293 cells were cultured in the presence of 20 μm MG132 for 8 h before harvest. REGγ was immunoprecipitated from precleared cell lysates with 1 μg of anti-REGγ overnight at 4 °C, with 1 μg of rabbit IgG in the control group. Each sample was incubated with protein A/G Plus-agarose beads for 3 h. The immunoprecipitates were washed three times with immunoprecipitation (IP) washing buffer (50 mm Tris, 137 mm NaCl, 1 mm EDTA, 10 mm NaF, 0.1 mm Na3VO4, 1 mm DTT, 10% glycerol, pH 7.8), and the co-immunoprecipitated proteins were analyzed by Western blotting with a SIRT1 antibody. The nitrocellulose membrane was stripped by the stripping buffer and then probed with anti-REGγ as references. For the reciprocal co-immunoprecipitation, HEK293 cell lysates were incubated with a SIRT1 antibody followed by detection of REGγ in the immunoprecipitates with Western blot analysis. For interactions between REGγ monomers, HEK293 cells were transfected with HA-tagged and/or FLAG-tagged REGγ-WT or K195R mutant for 48 h, along with MG132 (20 μm for 8 h) before harvest. The cell lysates were incubated with anti-FLAG M2 affinity gel overnight at 4 °C. The co-immunoprecipitated HA-REGγ were examined by Western blot with an anti-HA antibody.

Analysis of REGγ Heptamer by Native PAGE and FPLC

The HEK293 FLAG-REGγ-WT or K195R inducible cells were cultured in the presence of doxycycline (1 μg/ml, for 48 h), 6.6 μm TSA, and 10 mm NAM (for 6 h) prior to harvest. Following cell lysis, the supernatants were prepared in NativePAGETM sample buffer (Invitrogen) and resolved by NativePAGETM Novex® Bis-Tris gel system (Invitrogen). Transferred PVDF membranes were stained with Ponceau S to display the NativeMarkTM unstained protein standard (Invitrogen). After decolorization, the PVDF membrane was probed with a FLAG antibody to detect the REGγ heptamer complex. HEK293 cells were transiently transfected with 1 μg of FLAG-REGγ-WT, K195R, K195Q, or K188F (a heptamerization-defective mutant) into a six-well plate for 32 h, REGγ heptamer complexes were detected by native PAGE as described above. To investigate endogenous REGγ heptamerization, HEK293 FLAG-REGγ-inducible cells cultured in the presence or absence of TSA, NAM, or resveratrol were fractionated through a Superose 6 column. The elution profiles of REGγ were monitored by Western blotting. The FPLC was performed as described (5).

MTS Assay and Cell Cycle Analysis

MTS assay was performed by seeding HEK293 FLAG-REGγ-inducible cells in a 96-well plates at 3 × 103 cells per well and were cultured for 5 days. Doxycycline (1 μg/ml) was added to cells at day 1. Cells were incubated with MTS solution at 37 °C for 2 h, and the absorbances (490 nm) were measured and analyzed. Cell cycle analysis was carried out by estimating DNA contents with flow cytometry. HEK293 FLAG-REGγ-inducible cells were fixed in ice-cold 70% ethanol, incubated overnight at −20 °C, and stained with propidium iodide/Triton X-100-containing RNase A solution for 15 min at 37 °C. Cell cycle analysis was performed by BD CantoII Cell Analyzers. The data were analyzed using Flowjo software.

RESULTS

REGγ Is Acetylated in Mammalian Cells

Recent studies reveal a variety of physiological functions of REGγ in growth, cell proliferation, and cancer progression (9–11, 23, 24). How REGγ function is regulated remains to be elucidated. Our previous proteomic analysis (5) indicates REGγ as a protein with different post-translational modification. To determine whether REGγ could be an acetylated protein, we carried out IP of REGγ in lysates from different mammalian cells followed by Western blot analysis using a general anti-acetyl-lysine (anti-AcK) antibody. In HEK293, endogenous REGγ is clearly recognized in the IP enriched sample by the anti-AcK antibody (Fig. 1A). Similarly, in human cancer cell lines, including A549 and HeLa, REGγ protein acetylation can also be detected (Fig. 1, B and C). Alternatively, we performed a reciprocal IP analysis with the anti-AcK antibody followed by Western blotting using anti-REGγ. The result showed a single band at the molecular weight identical to the size of REGγ, indicative of an acetylated REGγ (Fig. 1D). To further test our observation, we treated cells with histone deacetylase inhibitors TSA and NAM (25, 26) to enhance cellular levels of acetylated proteins. In the doxycycline-induced FLAG-REGγ over-expressing HEK293 cells, we found a notable increase in the acetylation of FLAG-REGγ with TSA/NAM treatment (Fig. 1E). Taken together, these results suggest that REGγ can be acetylated in mammalian cells.

FIGURE 1.

FIGURE 1.

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REGγ is acetylated in mammalian cells. Endogenous REGγ in HEK293 (A), A549 (B), or HeLa cells (C) were immunoprecipitated with anti-REGγ antibody. Acetylation of REGγ was detected by immunoblotting with an anti-AcK antibody. D, a reciprocal immunoprecipitation was performed with the anti-AcK antibody using lysates from HEK293 cells, and immunoprecipitated REGγ was examined by immunoblotting with anti-REGγ antibody. E, HEK293 cells inducibly expressing FLAG-REGγ were cultured in the presence of doxycycline (1 μg/ml) for 48 h, with 6.6 μm TSA and 10 mm NAM for 6 h before harvesting the cells. FLAG-REGγ was immunoprecipitated by FLAG M2 affinity gel. REGγ acetylation was examined as in A–C. Asterisk refers to nonspecific bands.

Lys-195 Is a Major Acetylation Site in REGγ

To identify the potential acetylated residues, doxycycline-induced FLAG-REGγ in HEK293 cells was immunoprecipitated with FLAG beads, resolved by SDS-PAGE, and analyzed by MS/MS (LC-MS/MS). MS/MS spectrum showed acetylation at three lysine residues at positions 6 (Lys-6), 14 (Lys-14), and 195 (Lys-195), respectively (Fig. 2A and data not shown). This was endorsed in part by a different mass spectrometry study with identification of acetylation at Lys-195 (13). The mass score (score difference average) for REGγ acetylation at 195 is above the mean average of nearly 2000 acetylated proteins identified (13), reflecting relative abundance of this site specific acetylation. As a control, the recombinant REGγ protein purified from Escherichia coli was resolved by SDS-PAGE and cut out for LC-MS/MS analysis. To our surprise, bacterially generated recombinant REGγ was also acetylated at Lys-195 (Fig. 2B). Sequence analysis of REGγ from multiple species reveals that the Lys-195 site and surrounding residues are highly conserved across the animal kingdom (Fig. 2A, lower panel), with Lys-6 and Lys-14 slightly less conserved among vertebrates (Fig. 2A, upper panel). To define the ratio of acetylated REGγ in total molecules, we purified FLAG-tagged REGγ from 293 REGγ-inducible cells followed by the reciprocal immunoprecipitation with anti-AcK antibody. We found that about half of the REGγ molecules are acetylated (Fig. 2C, lanes 4 and 5) compared with the unbound REGγ protein (Fig. 2C, lanes 2 and 3). To analyze acetylation, normally Lys→Gln (KQ) substitution is used to mimic lysine acetylation, whereas Lys→Arg (KR) substitution is to eliminate acetylation target site without neutralization of the positive charges (27–29). Therefore, we generated doxycycline-inducible REGγ-expressing HEK293 cells, including acetylation-defective mutations of K6R, K14R, or K195R in FLAG-REGγ. In these stable cell lines, the K195R mutation significantly reduced REGγ acetylation levels, whereas K6R and K14R mutations had little impact on the overall acetylation in REGγ (Fig. 2D). A similar result was found in HEK293 cells transiently expressing FLAG-REGγ-K195R mutant, which was poorly recognized by the anti-AcK antibody compared with FLAG-REGγ-WT (Fig. 2E). These results emphasize that Lys-195 is a major acetylation site in REGγ, reflecting a potentially important role of this lysine residue in modulating REGγ activity.

FIGURE 2.

FIGURE 2.

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Lys-195 is a major acetylation site in REGγ. A, sequence analysis of REGγ from multiple species indicates that Lys-195 is a highly conserved acetylation site, with a slightly less conservation at Lys-6 and Lys-14 among vertebrates, consistent with the results of mass spectrometry. B, LC-MS/MS spectrum of IAK(ac)YPHVEDYR identified from recombinant REGγ protein expressed in E. coli. The search result was opened by Viewer in MaxQuant package. The precursor ion m/z showed a mass shift of 42.01 Da, b3, b4, b7–10, and y3–7, y9, y10 fragment ions were found in MS/MS spectrum. The acetylated peptide hits were filtered by 1% false discovery rate at protein, peptide, and site level. C, FLAG-tagged REGγ was purified from 293 REGγ-inducible cells by FLAG peptide. Acetylated REGγ was immunoprecipitated by anti-AcK antibody. The unbound REGγ and bound acetylated REGγ were examined by immunoblotting with anti-FLAG antibody. D, HEK293 cells inducibly expressing FLAG-REGγ were treated with 1 μg/ml doxycycline for 48 h. FLAG-REGγ WT and mutants were immunoprecipitated with anti-FLAG M2 affinity gel, and acetylation status was examined by immunoblotting with anti-AcK antibody. E, in HEK293 cells, transiently expressed FLAG-REGγ-WT and FLAG-REGγ K195R were immunoprecipitated with anti-FLAG M2 affinity gel, and acetylations were determined by immunoblotting with anti-AcK antibody.

Blocking Acetylation at Lys-195 Attenuates REGγ Activity

To understand the biological consequence of REGγ acetylation, expressions of p21 and a truncated HCV core-173 protein, well known substrates of the REGγ proteasome (3, 4, 30), are utilized to evaluate REGγ activity. We analyzed the ability of REGγ acetylation-defective mutants K6R, K14R, and K195R to promote degradation of p21 and HCV core-173 in HEK293 cells as well as the human lung cancer cell line H1299. The latter cell line lacks p53 expression and therefore avoids the impact of REGγ on p21 degradation through down-regulation of p53 (6). Although REGγ mutations at Lys-6 or Lys-14 had minor effect to attenuate REGγ-mediated degradation of p21 protein (data not shown), REGγ mutation at Lys-195 significantly reduced the capacity of REGγ to degrade HCV core-173 and p21 proteins in H1299 cells (Fig. 3, A, lane 3, and B, lane 3). As controls, REGγ-WT retained the activity to promote degradation of HCV core-173 and p21 proteins (Fig. 3, A, lane 2, and B, lane 2). In the REGγ inducible HEK293 cells, we also obtained results similar to what we observed in H1299 cells (data not shown), indicating that acetylation of REGγ occurs in different cell types. Next, we generated an acetylation-mimetic REGγ K195Q mutant, which is capable of accelerating p21 degradation in comparison with REGγ K195R in H1299 cells, but to a less extent compared with REGγ-WT (Fig. 3B). To understand the action of endogenous REGγ acetylation mutants in cells without wild type REGγ, we generated REGγ derivatives in lentivirus, including REGγ-WT, K195R, K195Q, along with a vector control, and stably integrated these constructs in REGγ−/− MEF cells. Despite that stable integration of REGγ-WT in REGγ−/− MEF cells does not function efficiently in its degradation of endogenous p21 probably due to compensation mechanisms, the stable cells expressing REGγ-WT and REGγ-K195Q in the parental REGγ−/− MEFs had comparable effects on p21 degradation. However, REGγ-K195R showed a remarkable inhibition of p21 protein degradation (Fig. 3C). Even though the Lys→Gln substitution does not always faithfully mimic the acetylation status of the lysine residue (29, 31), our data indicate that acetylation at Lys-195 is crucial for maintaining REGγ activity in its degradation of target proteins.

FIGURE 3.

FIGURE 3.

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Blocking acetylation at Lys-195 attenuates REGγ activity. A, H1299 cells were co-transfected with HA-HCV core-173 and REGγ plasmids for 32 h. The HA-HCV core protein levels were determined by immunoblotting. B, REGγ-WT, K195R and K195Q were co-transfected with p21 for 32 h in H1299 cells, and p21 levels were determined by immunoblotting. C, endogenous p21 protein levels were examined by immunoblotting in cells stably integrated REGγ derivatives in REGγ−/− MEF stable cells as indicated. Stability of endogenous p21 was examined by cycloheximide (CHX) (100 mg/ml) treatment for the indicated time in HEK293-inducible cells overexpressing REGγ WT (D), REGγ K195R (E), or K195Q (F). An asterisk indicates nonspecific bands.

To explore whether acetylation of REGγ at Lys-195 affects its function in the turnover of substrate protein p21, we used REGγ-inducible HEK293 cells treated with cycloheximide for indicated time periods. As expected, p21 degradation was expedited when overexpressing REGγ WT (Fig. 3D). However, induced overexpression of REGγ K195R mutant had no significant impact on the decay rate of p21 (Fig. 3E). In a parallel experiment, induced overexpression of REGγ K195Q could effectively decrease p21 half-life as REGγ WT did (Fig. 3F). Taken together, these results indicate that acetylation at Lys-195 is critical for retaining REGγ activity.

CBP and SIRT1 Reversely Regulate REGγ Acetylation and Activity

CBP and p300 are transcriptional coactivators with intrinsic histone acetyltransferase activity (32–36) to regulate gene expression. To test whether REGγ could be acetylated by these histone acetyltransferases, CBP was transiently expressed into HEK293 cells. We found significantly increased acetylation level of REGγ with CBP co-transfection (Fig. 4A). In addition, the recombinant GST-tagged REGγ protein could be acetylated by FLAG-tagged CBP in the presence of acetyl-CoA in vitro (Fig. 4B). Next, we investigated whether REGγ is a substrate of the Sir2 families in mammalian cells. Among SIRT1-SIRT7, FLAG-tagged SIRT1 showed strong interaction with GFP-tagged REGγ (data not shown). Furthermore, we found robust interactions between endogenous SIRT1 and REGγ in HEK293 cells by IP analysis using the anti-REGγ antibody (Fig. 4C). Similarly, a reciprocal IP with anti-SIRT1 antibody detected endogenous co-IP of REGγ and SIRT1 in HEK293 cells (Fig. 4D), indicating that REGγ is a potential target of SIRT1. In the inducible FLAG-REGγ-WT expressing HEK293 cells, augmented CBP expression enhanced acetylation in the FLAG-REGγ, whereas co-expressing CBP and SIRT1 blocked the effect by CBP, reflecting a causal relation between SIRT1 and REGγ deacetylation (Fig. 4E, left panel). In contrast, transient overexpressing CBP failed to significantly enhance REGγ acetylation in the FLAG-REGγ-K195R-inducible HEK293 cells and co-expressing SIRT1 and CBP further diminished REGγ acetylation (Fig. 4E, right panel). In addition, we found an obviously increased acetylation level of REGγ after SIRT1 knocking down in 293T cells (Fig. 4F). As expected, when acetylated FLAG-REGγ was incubated with recombinant His-SIRT1 and NAD+, REGγ acetylation level was obviously reduced in vitro (Fig. 4G). These results strongly suggest that CBP and SIRT1 mainly target Lys-195 for acetylation/deacetylation in REGγ, although we cannot exclude their regulation in other residues in REGγ.

FIGURE 4.

FIGURE 4.

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CBP and SIRT1 regulate REGγ acetylation and activity. A, HEK293 cells were transfected with CBP for 32 h, and acetylation of endogenous REGγ was examined by immunoblotting with anti-AcK antibody. B, the recombinant GST-REGγ (5 μg) were incubated with FLAG-CBP (3 μg) in 30 μl of histone acetyltransferase buffer for 3 h at 30 °C. The acetylation level of GST-REGγ was examined by immunoblotting. C, endogenous REGγ protein in HEK293 cells was immunoprecipitated with anti-REGγ antibody and IgG as a control. The coimmunoprecipitated SIRT1 protein levels were determined by immunoblotting with anti-SIRT1 antibody. D, endogenous SIRT1 protein in HEK293 cells was immunoprecipitated with an anti-SIRT1 antibody, and the coimmunoprecipitated REGγ protein levels were determined by immunoblotting with anti-REGγ antibody. E, CBP and SIRT1 were transfected into HEK293 cells inducibly expressing FLAG-REGγ-WT or FLAG-REGγ-K195R as indicated. Acetylation levels of REGγ were examined by immunoblotting with anti-AcK antibody after immunoprecipitation of FLAG-REGγ with anti-FLAG M2 affinity gel. F, endogenous REGγ acetylation levels were examined in SIRT1 knocking down 293T cells by immunoblotting with anti-REGγ antibody. G, acetylated FLAG-REGγ was incubated with recombinant His-SIRT1 in 50 μl of deacetylase buffer for 2 h at 30 °C. REGγ acetylation level was analyzed by immunoblotting. H, HeLa cells were transfected with SIRT1 or SIRT1-H363Y mutant for 32 h. Endogenous p21 and REGγ protein levels were analyzed by immunoblotting with anti-p21 or anti-REGγ antibody. I, HEK293 cells were transiently transfected with siRNA against CBP for 72 h. CBP knockdown efficiency and REGγ p21 protein levels were examined by immunoblotting. CHX, cycloheximide; DOX, doxycycline; Ctrl, control.

Furthermore, we determined whether the influence of SIRT1 on REGγ activity depends on its deacetylase activity. Transient overexpression of SIRT1-WT in HeLa cells significantly increased p21 protein level, whereas exogenously expressed SIRT1-H363Y, a deacetylase-defective mutant (37), failed to inhibit REGγ-dependent degradation of p21 (Fig. 4H). Consistently, we also found that silencing CBP in HEK293 cells enhanced p21 protein levels (Fig. 4I). Taken together, these results suggest that CBP and SIRT1 can regulate REGγ activity through acetylation and deacetylation in REGγ at specific sites.

Acetylation at Lys-195 Is Crucial for the Monomeric Interactions and Overall Structure of REGγ

Based on the discovery that acetylation in REGγ influences its activity, we intended to address how this could be achieved. Previous studies demonstrate that functional REGγ exists as a heptameric ring in cells (8, 38, 39). We tested whether acetylation is involved in regulation of the overall structure of REGγ. In FLAG-REGγ-WT-overexpressing HEK293 cells, REGγ heptamers were easily detected by native PAGE followed by membrane transferring and antibody blotting (Fig. 5A, lane 1). Interestingly, enhancing REGγ acetylation by TSA and NAM treatment greatly increased the amount of REGγ heptamer complexes (Fig. 5A, lane 2). In FLAG-REGγ-K195R-overexpressing HEK293 cells, REGγ heptamer formation was dramatically suppressed even in the presence of TSA and NAM treatment (Fig. 5A, lanes 3 and 4). Furthermore, we transiently expressed FLAG-REGγ-WT and corresponding acetylation mutants in HEK293 cells to examine REGγ heptamerization by native PAGE. The data clearly showed that FLAG-REGγ-WT and the acetylation-mimetic FLAG-REGγ-K195Q mutant formed heptamers in cells (Fig. 5B, lanes 2 and 4). In contrast, the acetylation-defective FLAG-REGγ-K195R mutant and the heptamerization-defective FLAG-REGγ-K188F mutant (38) had poor REGγ heptamer formation (Fig. 5B, lanes 3 and 5). The crystal structure of heptameric REGα (40), which is highly homologous to REGγ, suggests a parallel intermolecular interactions between helix 2 of one monomer with the helix 4 of the neighboring molecule. We then questioned whether acetylation affects association between REGγ monomers. By transiently expressing a FLAG-tagged REGγ and an HA-tagged REGγ construct or corresponding mutant constructs in HEK293 cells followed by IP and Western blot analysis (Fig. 5C), we found that REGγ-WT interacted better with each other, whereas REGγ-K195R monomeric interactions were compromised, suggesting a role of acetylation at this position in REGγ protein-protein interactions. To further test the impact of acetylation on REGγ monomeric interactions, we extended part of the above experiments described in Fig. 5C with additional treatment of either TSA/NAM or SIRT1 co-transfection, alone or in combination. As expected, TSA/NAM treatment enhanced the interactions between FLAG-REGγ and HA-REGγ (Fig. 5D, compare lane 5 with lane 6), whereas SIRT1 attenuated their association in the presence or absence of TSA/NAM (Fig. 5D, compare lane 5 with lane 7; lane 6 with lane 8; and lane 7 with lane 8). Moreover, we performed size exclusion chromatography using lysates from the inducible FLAG-REGγ-WT and FLAG-REGγ-K195R HEK293 cells. Based on the molecular standard, we found that majority of the FLAG-REGγ-WT formed heptamers with a peak at ∼230 kDa (Fig. 5E, upper panel). Upon TSA/NAM treatment and increased REGγ acetylation, a change in fraction pattern occurred with a shift toward the peak fraction, indicating an increase in REGγ heptamerization (Fig. 5E, middle panel). On the contrary, FLAG-REGγ-K195R expressing cells produced a stretched elution pattern ranging from monomers (∼ 30 kDa), various degrees of oligomers, and reduced higher molecular weight fractions (heptamers) (Fig. 5E, lower panel). Similar results were obtained following REGγ deacetylation by resveratrol in the induced FLAG-REGγ-WT overexpressing cells (data not shown). Based on the results that acetylation-defective mutant significantly impaired its heptamerization and monomers association (Fig. 5, A–C), we examined whether REGγ-K195R mutant is unstable, which may be targeted for degradation or association with other protein complexes. Consequently, we found that acetylation-defective mutant REGγ-K195R degrades faster compared with REGγ-WT (Fig. 5F). Collectively, these results demonstrate acetylation at Lys-195 is crucial for REGγ monomeric interactions and assembly of REGγ heptameric complexes.

FIGURE 5.

FIGURE 5.

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Acetylation at Lys-195 is crucial for REGγ heptamerization and interactions between REGγ monomers. A, HEK293 cells inducibly expressing FLAG-REGγ were cultured with 1 μg/ml doxycycline for 48 h, in the presence or absence of TSA and NAM for 6 h before cells were harvested. REGγ heptamerization was determined by native PAGE gel system with anti-FLAG antibody. B, FLAG-REGγ-WT, K195R, K195Q, or K188F was transfected into HEK293 cells for 32 h. Cell lysates were analyzed by native PAGE Gel system to examine REGγ heptamerization. C, FLAG-REGγ and HA-REGγ constructs were transfected into HEK293 cells as indicated. Cell lysates was immunoprecipitated by FLAG M2 affinity gel. Co-immunoprecipitated HA-REGγ was detected by immunoblotting with an anti-HA antibody. D, HEK293 cells were treated with TSA/NAM or TSA/NAM along with transient expression of SIRT1 as indicated, together with co-expression of different tagged REGγ derivatives. Co-immunoprecipitated HA-REGγ was detected by immunoprecipitating FLAG-REGγ and immunoblotting with indicated antibodies. E, cell lysates from the FLAG-REGγ inducible HEK293 cells were subjected to size exclusion chromatography for FPLC analysis. F, stability of FLAG-REGγ WT or K195R mutant was examined by cycloheximide (CHX) treatment for indicated time in HEK293-inducible cells overexpressing FLAG-REGγ WT or K195R.

Acetylation-defective Mutant REGγ Impairs Cell Growth and Cell Cycle Progression

As a broad acting cyclin-dependent kinase inhibitor, p21 plays a central role in cell cycle regulation in many cells types (41, 42). Recent studies indicate that REGγ influences cell cycle through degradation of several cell cycle regulators, including p21, p16, and p19 (3, 4). Thus, we reasoned that blocking REGγ acetylation at Lys-195 may also affect cell proliferation and cell cycle progression. Using the HEK293 cells inducibly expressing FLAG-REGγ-WT and FLAG-REGγ-K195R for the cell proliferation assay, we observed that cells overexpressing FLAG-REGγ-K195R had significantly reduced growth rate at day 2 through day 5 (Fig. 6A). Next, these cells were also subjected to flow cytometry analysis to evaluate the impact of acetylation defect on cell cycle progression. Compared with FLAG-REGγ-WT expressing cells, FLAG-REGγ-K195R expressing cells had an increased population at the G0/G1 phase, and a significantly decreased proportion of S phase cells (Fig. 6B), indicating a reduced cell cycle progression from G0/G1 to S phase transition. Taken together, our results further substantiate an important role for REGγ acetylation in cell growth and cell cycle regulation.

FIGURE 6.

FIGURE 6.

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Acetylation mutation at Lys-195 in REGγ reduces cell proliferation and mitigates cell cycle progression. A, HEK293 cells inducibly expressing FLAG-REGγ were treated with doxycycline after cells were seeded in 96-well plates for 24 h. Absorbance was measured at indicated times. Data were analyzed as means ± S.D. of spectrometric absorbance of three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent the statistic comparisons between growth in HEK293 with FLAG-REGγ-WT and HEK293 with FLAG-REGγ-K195R. B, the HEK293 with FLAG-REGγ-WT and HEK293 with FLAG-REGγ-K195R were treated with doxycycline for 48 h, and DNA contents of the inducible cells in different cell cycles were analyzed by flow cytometry. Each bar indicates the distribution of the cell cycles. Data are reported as means ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 indicate statistic differences between indicated groups.

DISCUSSION

Emerging evidence leads to a renewed attention to the ubiquitin-independent REGγ-proteasome pathway. With the discovery of more and more substrates in this protein degradation pathway (3–6, 30, 43, 44), REGγ is being recognized as an important regulator. Yet, the regulatory input that may alter the biological function of REGγ remains poorly understood. In this study, we demonstrate that site-specific acetylation of REGγ at Lys-195 is crucial for its monomeric interactions and formation of a functional heptameric complex. Modulating acetylation status at this lysine residue has profound impact on the activity of REGγ in substrate protein degradation. Our results provide the first biochemical evidence for a role of acetylation in structural and functional regulation of the REGγ-proteasome complex.

In eukaryotic cells, acetylation is among the most common covalent modifications and ranks among the most important master switches similar to phosphorylation (45). It is now clear that prokaryotes have the capacity to acetylate both the α-amino groups of N-terminal residues and the ϵ-amino groups of lysine side chains, suggesting that acetylation appears to be an ancient reversible modification such as phosphorylation (46). Reversible protein acetylation provides key regulatory switches for cell signaling pathways, which has been shown to affect a diverse array of biochemical properties, including protein activity, protein stability, DNA/protein-protein interactions, and intracellular localization (12, 15). We have defined lysine 195 in REGγ as the mostly affected residue for acetylation/deacetylation mediated by CBP/SIRT1. The REGγ K195R mutant that can no longer be acetylated failed to promote its substrate degradation, whereas the K195Q mutant that mimics a constitutively acetylated state retained the capacity to degrade target proteins, which further correlates with REGγ-dependent regulation in cell growth and cell cycle progression. We also demonstrated reversible acetylation-deacetylation modification at the Lys-195 sites. Our results by no means exclude the possibility of weak acetylation on other sites or regulation by other histone acetyltransferases/histone deacetylases. In fact, Lys-6 and Lys-14 in REGγ can also be acetylated based on mass spectrometry and bioinformatics prediction. We have previously demonstrated that SUMOylation of REGγ can be enhanced in the presence of PIAS1 at Lys-6, Lys-12, and Lys-14, which results in cytoplasmic distribution and stabilization of REGγ (47). It is likely that competition by SUMOylation at Lys-6 and Lys-14 attenuates acetylation at these sites.

Although we failed to generate a Lys-195-specific Ac antibody, the Pan-AcK antibody successfully detected acetylation at Lys-195 in REGγ-WT but not in REGγ-K195R mutant, suggesting high prevalence of this site-specific acetylation in mammalian cells. Based on our comparative analysis of score difference average (which is a standard for estimation of modification signal) for ∼2000 of the acetylated proteins (13), we found the score for acetylation of REGγ at Lys-195 is above the average score, indicating that acetylation of REGγ at Lys-195 is around average levels compared with all proteins examined so far. In addition, our IP Western results with Pan-acetylation antibody suggest that about half of the molecules are acetylated. Because the REGγ molecules form heptamers, it is likely that such an acetylation fraction may be enough for enhancing protein-protein interactions.

Given that REGγ is highly homologous to REGα, the predicted location of Lys-195 in REGγ should be at the very C terminus of the helix 3 based on the structure of REGα (48). Facing the substrate interaction surface, Lys-195 should be easily accessed by enzymes such as SIRT1 to dynamically regulate the disassembly of REGγ. If interactions between REGγ monomers only occur between helixes 2 and 4, we believe Lys-195 acetylation may induce favorable structure to facilitate this interaction. Acetylation in REGγ remarkably enhances monomeric interactions and heptameric formation, which is consistent with previous reports that acetylation can enhance protein-protein interactions (49–51). Whether acetylation of REGγ at Lys-195 is a default or translationally coupled process remains to be investigated. Future crystal structure analysis of REGγ may enable us to understand how this site-specific acetylation facilitates its protein-protein interactions.

Interestingly, the endogenously expressed acetylation-defective REGγ-K195R mutant dramatically impaired the heptameric complex, resulting in an elution pattern ranged from monomers, various degrees of oligomers, and reduced amount of heptamers (Fig. 5E). The results suggest that REGγ indeed has intrinsic properties in self-association (48). It is likely that acetylation may accelerate the oligomerization processes of intracellular REGγ, which may be otherwise targeted for degradation or association with other protein complexes. In support of this idea, we found that acetylated REGγ is more stable, whereas acetylation defective mutant degrades faster (Fig. 5F). Despite that cells stably or transiently expressing REGγ-K195R also have significant amount of endogenous wild type REGγ, we still observe significant impact of K195R mutant on protein-protein interaction and proteolytic functions (Figs. 3 and 5).

To summarize our findings in this study, CBP acetylates REGγ at Lys-195, which promotes its heptamerization and increases REGγ activity in the degradation of targets proteins. In contrast, SIRT1 can bind with REGγ and deacetylates REGγ, which inhibits heptamerization or trigger disassembly of REGγ, leading to inactivation of REGγ capacity (Fig. 7). Our results provide a novel mechanism for the reciprocal regulation of REGγ homeostasis by acetylation. As a potential druggable target, REGγ activity may be modulated by histone acetyltransferase/SIRT inhibitors or activators.

FIGURE 7.

FIGURE 7.

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A working model simplifies the influences of acetylation on REGγ assembly and function. CBP acetylates REGγ at Lys-195, which promotes REGγ to form a heptamer, resulting in augmented REGγ activity. When SIRT1 binds with REGγ, it deacetylates REGγ and inhibits REGγ heptamerization, releasing monomers from REGγ disassembly.

Acknowledgments

We thank Dr. Qin Feng (Department of Molecular and Cellular Biology, Baylor College of Medicine) and Dr. Qiang Tong (Departments of Pediatrics, Medicine, Molecular Physiology & Biophysics, Baylor College of Medicine) for providing plasmids for this study. We appreciate the comments and suggestions from Dr. Ming-Jer Tsai and Dr. Sophia Y. Tsai (Department of Molecular and Cellular Biology, Baylor College of Medicine) for this project.

*

This work was supported in part by an Excellent East China Normal University Ph.D. student award (to J. L.). This work was also supported by National Institutes of Health Grant 1R01CA131914, Norman Hackerman Advanced Research Program Grants 1082318401 and PN004949-0012-2009), and the Pilot/Feasibility Program of the Diabetes & Endocrinology Research Center (P30-DK079638) at Baylor College of Medicine. This work was supported in part by National Natural Science Foundation of China Grants 81261120555, 30870503, 81071657, 31100946, and 31200878; Science and Technology Commission of Shanghai Municipality Grants 11DZ2260300, 10JC1404200, 11ZR1410000, and 12ZR1409300; and National Basic Research Program Grants 2009CB918402 and 2011CB504200.

4
The abbreviations used are:
CBP
CREB binding protein
CREB
cAMP-responsive element-binding protein
HCV
hepatitis C virus
MEF
mouse embryonic fibroblast
TSA
trichostatin A
NAM
nicotinamide
IP
immunoprecipitation.

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