Site-specific acetylation of the proteasome activator REGγ directs its heptameric structure and functions

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.

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

FIGURE 1.

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.

FIGURE 2.

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.

FIGURE 3.

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.

FIGURE 4.

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.

FIGURE 5.

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.

FIGURE 6.

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.

FIGURE 7.

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.