MDM2 Mediates Ubiquitination and Degradation of Activating Transcription Factor 3

Abstract

Activating transcription factor 3 (ATF3) is a common stress sensor, and its rapid induction by cellular stresses (e.g. DNA damage) is crucial for cells to mount appropriate responses (e.g. activating the tumor suppressor p53) and maintain homeostasis. Although emerging evidence suggests that dysregulation of ATF3 contributes to occurrences of human diseases including cancer, the mechanism(s) by which ATF3 expression is regulated is largely unknown. Here, we demonstrate that mouse double minute 2 (MDM2) is a bona fide E3 ubiquitin ligase for ATF3 and regulates ATF3 expression by promoting its degradation. MDM2 via its C-terminal RING finger can bind to the Basic region of ATF3 and mediate the addition of ubiquitin moieties to the ATF3 leucine zipper domain. As a consequence, ATF3, but not a mutant deficient in MDM2 binding (⌬80 -100), is degraded by MDM2-mediated proteolysis. Consistent with these results, ablation of MDM2 in cells not only increases basal ATF3 levels, but results in stabilization of ATF3 in late stages of DNA damage responses. Because ATF3 was recently identified as a p53 activator, these results suggest that MDM2 could inactivate p53 through an additional feedback mechanism involving ATF3. Therefore, we provide the first evidence demonstrating that ATF3 is regulated by a posttranslational mechanism. ATF32 is a member of the ATF/CREB family of transcription factors, and its expression is rapidly induced by a large variety of cellular stresses including DNA damage, wounds, and cellular injury (1). ATF3 can bind to DNA (via the ATF/CREB consensus sequence, 5Ј-TGACGTCA-3Ј) (1) and other proteins (e.g. Smad3, p53, and E6) (2-4), resulting in alterations in gene expression and cellular functions. Although consequences of stress-induced ATF3 expression are not well understood, recent evidence links ATF3 to several important pathways, including TGF␤ signaling (2), the Toll-like receptor 4 pathway (5), the eIF2 kinase-mediated endoplasmic reticulum stress response (6), as well as the p53 activation pathway (3), suggesting that dysregulation of ATF3 could contribute to occurrences of many human diseases including cancer. Indeed, although we previously showed that ATF3 deficiency promotes oncogenic transformation (3, 7), recent unbiased cDNA array studies have revealed that ATF3 expression is down-regulated in common human cancers (for a review of these data, see Ref. (8).Although induction of ATF3 expression is a common characteristic of stress responses (1), the mechanisms by which ATF3 expression is regulated during these processes remain largely unknown. It has been shown that ATF3 expression can be regulated by transcription factors such as ATF2, Smad3, and NF-B (2, 9, 10) and controlled by signaling mediated by p38 or JNK/SAPK (9, 11). Moreover, an atypical p53-binding site was identified in the ATF3 promoter (12). However, whether p53 regulates ATF3 expression still remains to be firmly validated, even though a marginal effect of p53 on ATF3 expression was reported in specific cells and in response...

The Stress Response Mediator ATF3 Represses Androgen Signaling by Binding the Androgen Receptor

“…This mechanism is reminiscent of mechanisms utilized by many other AR repressors (60) and is in line with our previous findings that ATF3 can regulate cellular functions independent of its transcriptional activity (59,64). Whereas both the basic region and the ZIP domain of ATF3 can mediate proteinprotein interactions (25,41,59,64), we found that ATF3 directly bound AR through the ZIP domain. c-Jun, a bZIP protein capable of promoting prostate cancer cell growth (10), was previously shown to interact with AR (49), suggesting that the characteristic leucine residues in the ZIP domain might be responsible for AR binding.…”

“…To test whether ATF3 affected AR nuclear translocation, we coexpressed ATF3 (fused to mCherry) and AR (fused to green fluorescent protein [GFP]) in PC3 cells and examined cells under a fluorescence microscope. As expected (41,64), ATF3 was predominantly localized in the nucleus, and this subcellular localization pattern was not altered in the presence of androgen (Fig. 5A).…”

The Stress-responsive Gene ATF3 Mediates Dichotomous UV Responses by Regulating the Tip60 and p53 Proteins

“…Indeed, we previously showed that the binding of ATF3 to Tip60 can promote the removal of ubiquitin chains by the deubiquitinase USP7, thereby preventing Tip60 from proteasomal degradation (17). Because ATF3 can interact with MDM2 (38) and the latter E3 ubiquitin ligase was suggested to be involved in UV-induced Tip60 expression (16), there is also a possibility that ATF3 stabilized Tip60 by regulating MDM2 in the UV response. However, we did not find evidence that MDM2 could mediate the degradation of Tip60 (data not shown).…”

The Many Faces of MDM2 Binding Partners

“…Importantly, deregulation of ATF3 is documented to contribute to tumorigenesis, presumably through lack of appropriate DNA damage responses. 98 Protein phosphatase 2A (PP2A) is a serine-threonine phosphatase involved in the DNA damage response. PP2A is a heterotrimer comprised of a catalytic C subunit, a structural A subunit, and several regulatory B subunits.…”