Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation

Abstract

Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation.A lteration/deficiency in activation 3 (ADA3) protein is a conserved component of key chromatin-modifying complexes that contain either GCN5 or PCAF histone acetyl transferases (HATs), such as SAGA (Spt/Ada/Gcn5) in yeast, ATAC (ADA2a-containing complex), STAGA (SPT3/TAFII31/GCN5 acetyltransferase), and TFTC (TATA binding protein free-TAF containing complex) in metazoans (1-7). Within these complexes, ADA3 associates with GCN5 and ADA2 to form the HAT module. ADA3 has also been shown to associate with p300, the most well-defined HAT of mammalian systems (8, 9). ADA3 is essential for the HAT activity of p300-and GCN5-containing HAT complexes toward histones (10-14) as well as of nonhistone proteins, such as p53 and ␤-catenin (15, 16).Although strongly implicated in the regulation of HAT activity of ADA3-containing complexes, additional functions for ADA3 have been reported. For example, we identified ADA3 as a novel human papillomavirus E6 oncoprotein-binding protein (17), and additional studies revealed that ADA3 binds to nuclear hormone receptors, such as estrogen receptor and retinoid acid receptor, and enhances their transcriptional activation function (8,(18)(19)(20)(21). Recent studies have identified an essential role of ADA3 in normal cell cycle progression and maintenance of genomic stability (5, 13, 22, 23).Whether ADA3's role in these processes is merely a passive structural one or is actively regulated is unknown. Posttranslational modification represents one potential mechanism to regulate ADA3 function, and in fact yeast ADA3 was found to be modified by acetylation (24). Consistent with this idea, we observed that human ADA3 is also acetylated in vitro by its interacting HAT p300 (13). H...

Epidermal Growth Factor Receptor activation promotes ADA3 acetylation through the AKT-p300 pathway

“… 6,7 We have also shown that ADA3 itself is acetylated by its interacting KATs. 7 In the present study, we demonstrate that ADA3 acetylation is regulated by growth factor receptor activation through a novel signaling pathway that involves AKT and p300 phosphorylation.…”

“… 3,27 To begin to explore the mechanistic links between ADA3 and cell proliferation, we recently demonstrated that ADA3 is post-translationally modified by acetylation, and that acetylation is critical for the role of ADA3 to promote cell cycle progression. 7 In this study, we reveal new mechanistic links that control ADA3 acetylation during cell cycle progression downstream of prototype receptor tyrosine kinases of the EGFR family.…”

“…We have recently shown that p300 acetylates ADA3 and that ADA3 acetylation is required for its role in promoting cell proliferation. 7 To explore the upstream mechanisms that might control ADA3 acetylation during cell proliferation, we used a TERT immortalized human mammary epithelial cell line 76N-TERT, which is completely dependent on EGFR-mediated signaling for proliferation. 13 Cells were deprived of EGF and serum-derived growth factors for 72 hours, and then stimulated with EGF for 15 or 30 min followed by western blotting, to assess the levels of phosphorylation of relevant downstream effectors.…”

“…We have recently identified K109, −122, −124, −194 and −222 on ADA3 as sites of in vivo acetylation by p300. 7 HEK-293T cells were transfected with wild-type ADA3 or an ADA3 mutant, in which all 5 of these lysine residues were mutated to arginine, referred to as 5KR mutant. These cells were then treated with cycloheximide to block new protein synthesis, and the levels of ADA3 proteins in cell lysates harvested at various time points were assessed by western blotting to determine the relative half-lives of the wild-type and mutant ADA3.…”

The Response of Rhodotorula mucilaginosa to Patulin Based on Lysine Crotonylation

“…Histone post-translational modifications (PTMs) play an important role in diverse processes, such as organismal development, cell differentiation, cell death, and also contribute to cancer development due to epigenetic deregulation ( Berdasco and Esteller, 2010 ; Tan et al, 2011 ). There are many types of histone PTMs including methylation, acetylation, phosphorylation, and histone ubiquitination ( Zhao et al, 2010 ; Tan et al, 2011 ; Zhang et al, 2011 ; Olsen and Mann, 2013 ; Weinert et al, 2013 ; Liu and Gao, 2016 ; Mohibi et al, 2016 ; Nishiyama et al, 2016 ; Penard-Lacronique and Bernard, 2016 ). In recent years, histone lysine crotonylation (Kcr), a new PTM has been discovered.…”