Multiple Roles for Acetylation in the Interaction of p300 HAT with ATF-2 †

ArticleinBiochemistry 46(28):8207-16 · August 2007with10 Reads
DOI: 10.1021/bi7000054 · Source: PubMed
The transcriptional coactivator paralogues p300 and CBP contain acetyltransferase domains (HAT) and catalyze the lysine acetylation of histones and other proteins as an important aspect of their functions. Prior studies revealed that the basic leucine zipper domain (b-ZIP) of transcription factor ATF-2 (also called CRE-BP1) can interact with the CBP HAT domain. In this study, we have examined the ATF-2 b-ZIP interaction with the p300 HAT domain and shown that p300 HAT autoacetylation can enhance the binding affinity. Pull-down assays revealed that hyperacetylated p300 HAT is more efficiently retained by immobilized ATF-2 b-ZIP than hypoacetylated p300 HAT. Loop deleted p300 HAT lacking autoacetylation was retained about as well as hyperacetylated p300 HAT, suggesting that the loop and ATF-2 compete for p300 HAT binding. While ATF-2 b-ZIP is a weak inhibitor of hypoacetylated p300 HAT acetylation of a histone H4 peptide, hyperacetylated p300 HAT is much more potently inhibited by ATF-2 b-ZIP. Moreover, we showed that ATF-2 b-ZIP could serve as an acetyltransferase substrate for p300 HAT. Using mass spectrometry, two p300 HAT lysine acetylation sites were mapped in ATF-2 b-ZIP. Immunoprecipitation-Western blot analysis with anti-acetyl-lysine antibody revealed that ATF-2 can undergo reversible acetylation in vivo. Mutational analysis of the two ATF-2 b-ZIP acetylation sites revealed their potential contributions to ATF-2-mediated transcriptional activation. Taken together, these studies suggest multiple roles for protein acetylation in the regulation of transcription by p300/CBP and ATF-2.
    • "SOD1 contains 11 lysine (K) residues, which are K4, K10, K24, K31, K37, K71, K76, K92, K123, K129 and K137. As lysine lysine (K)-arginine (R) replacement is widely used to generate acetylationdeficient mutants181920, each of the lysine was individually mutated to a nonacetylatable arginine, and the impact on SOD1 acetylation was examined. Among the 11 mutants, only the K71R mutation largely abolished SOD1 acetylation (Figure 1B) and the treatment of deacetylase inhibitors failed to increase the detectable signaling of acetylation (Figure 1C), indicating the acetylation of SOD1 occurred at K71. "
    [Show abstract] [Hide abstract] ABSTRACT: Cancer cells are characterized by a high dependency on antioxidant enzymes to cope with the elevated rates of reactive oxygen species (ROS). Impairing antioxidant capacity in cancer cells disturbs the ROS homeostasis and exposes cancer cells to massive oxidative stress. In this study, we have discovered that superoxide dismutase 1 (SOD1), a major player in maintaining the cellular redox status, was acetylated at lysine 71. This acetylation, which was primarily deacetylated by Sirtuin 1 (SIRT1), suppressed the enzymatic activity of SOD1 via disrupting its association with copper chaperone for SOD1 (CCS). More importantly, genotoxic agents, such as camptothecin (CPT), induced SOD1 acetylation by disrupting its binding with SIRT1. CPT-induced SOD1 acetylation was stimulated by its provoked ROS, suggesting a positive feedback loop, in which ROS per se impairs the antioxidative defence of cancer cells and reinforces oxidative stress stimulated by anticancer agents. The intrinsic abundance of SOD1 acetylation varied among cancer cells, and high level of SOD1 acetylation was correlated with elevated sensitivity to CPT. Together, our findings gained mechanistic insights into how cytotoxic agents fine tune the intracellular ROS homeostasis to strengthen their anticancer effects, and suggested SOD1 acetylation as a candidate biomarker for predicting response to CPT-based chemotherapy.
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    • "To further understand the role of p300-mediated acetylation in Skp2 stability, various Skp2 constructs were transfected cells, and their expression levels were monitored during cell-cycle progression. A K/R replacement was used to generate acetylation-deficient mutants while both K/L (Rea et al., 2000; Schwer et al., 2002) and K/Q replacements (Karanam et al., 2007; Kemper et al., 2009) were used to mimic lysine-acetylation. WT-Skp2 levels are decreased in early-to mid-G1 phase where APC/Cdh1 is highly active. "
    [Show abstract] [Hide abstract] ABSTRACT: Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.
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    • "ATF2 is also phosphorylated by ataxia-telangiectasia mutated (ATM) kinase (Fig. 1), which mediates its transcription-independent role in the DNA damage response (Bhoumik et al., 2005). In addition to being phosphorylated, ATF2 is also acetylated on Lys357 and Lys374 by p300/CREB-binding protein (CBP, also known as CREBBP), which contributes to its transcriptional activity (Karanam et al., 2007). Binding of ATF2 suppresses the acetyltransferase activity of the transcriptional coactivator p300/CBP. "
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    Article · Jun 2012
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