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.
Highlights
We report for the first time that REG␥ 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
As expected, when acetylated FLAGREG␥ was incubated with recombinant His-SIRT1 and NADϩ, REG␥ 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 REG␥, we cannot exclude their regulation in other residues in REG␥
By transiently expressing a FLAGtagged REG␥ and an HA-tagged REG␥ construct or corresponding mutant constructs in HEK293 cells followed by IP and Western blot analysis (Fig. 5C), we found that REG␥-WT interacted better with each other, whereas REG␥-K195R monomeric interactions were compromised, suggesting a role of acetylation at this position in REG␥ protein-protein interactions
Summary
We report for the first time that REG␥ 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. Biological analysis of cells containing REG␥-WT or REG␥-K195R mutant indicates an impact of acetylation on REG␥-mediated regulation of cell proliferation and cell cycle progression REG␥ 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 [3,4,5]. We illustrate that acetylation of REG␥ at the lysine 195 residue by CBP is important for the degradation of REG␥ substrates, such as p21 and HCV core proteins. Functional analysis in cells containing REG␥-WT or REG␥K195R mutation has validated the crucial role of acetylation in REG␥-mediated regulation of cell proliferation and cell cycle progression
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