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 (Delta80-100), 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.

FIGURE 1.

MDM2 regulates ATF3 levels in the DNA damage response. A and B, A549 cells were treated with 0.4 μg/ml DOX, 2 nm AD, or 1.5 μm CPT and lysed at the indicated times for immunoblotting (A). Densitometry was used to quantitate ATF3 levels (B). C, A549 cells were treated as in A. Total RNA was prepared and subjected to real-time RT-PCR to quantify ATF3 mRNA levels. D–F, mdm2-wild-type (p53^−/−/mdm2^+/+) or -deficient (p53^−/−/mdm2^−/−) MEF cells were treated with 0.4 μg/ml DOX for the indicated times and then subjected to immunoblotting (D) or real-time RT-PCR (F). ATF3 levels were quantitated by densitometry, and the results are shown in E. G and H, indicated cells were treated with 0.4 μg/ml DOX for 8 h. After removal of DOX, 100 μg/ml cycloheximide (CHX) was added into culture medium. Cells were harvested at the indicated times for immunoblotting. ATF3 levels were quantitated by densitometry, and the results are shown in H. I, p53^−/−/mdm2^+/+ or p53^−/−/mdm2^−/− MEF cells were treated with 0.4 μg/ml DOX for 24 and 48 h and stained with propidium iodide for flow cytometry analysis as described previously (3). Percentages of subG₀/G₁ cells were used to calculate the folds of increase in apoptosis rates after DOX treatments.

FIGURE 2.

MDM2 regulates the basal level of ATF3. A and B, p53^−/−/mdm2^+/+and p53^−/−/mdm2^−/− MEF cells were harvested and subjected to immunoblotting (A) or real-time RT-PCR (B). C, LNCaP cells were infected with lentiviruses expressing MDM2-specific shRNA (shM2-1 or shM2-2) for 3 days and then lysed for immunoblotting. D, LNCaP cells expressing shM2-1 or shLuc were lysed for real-time RT-PCR assays. E, p53-deficient (p53^−/−) HCT116 cells were infected with lentiviruses expressing shM2 or shLuc and subjected to immunoblotting as in C.

FIGURE 3.

MDM2 promotes ATF3 degradation. A, H1299 cells were transfected with ATF3, GFP, and increasing amounts of MDM2 as indicated. Cells were lysed, and GFP expression levels were quantitated using a fluorescence spectrophotometer to normalize transfection efficiencies. Normalized cell lysates were then subjected to immunoblotting. B, H1299 cells were transfected as indicated and subjected to immunoblotting as in A. C and D, H1299 cells were transfected and treated with 100 μg/ml cycloheximide for different time. ATF3 levels were quantitated by densitometry, and the results are shown in D.

FIGURE 4.

MDM2 binds directly to ATF3. A, H1299 cells were transfected as indicated and lysed for IP assays using the ATF3 antibody (α-ATF3) or IgG. Precipitated proteins were subjected to immunoblotting (IB) to detect MDM2 and ATF3. B, LNCaP cell lysates were incubated with the ATF3 antibody or IgG at 4 °C overnight. Immunoprecipitated proteins were then subjected to immunoblotting. C, 100 ng of purified ATF3 protein was incubated with 200 ng of purified GST-MDM2 protein or BSA at 4 °C overnight. Protein complexes were pulled down by glutathione-agarose and subjected to immunoblotting.

FIGURE 5.

MDM2 is a bona fide E3 ubiquitin ligase for ATF3. A, purified ATF3 protein was incubated with E1, E2, MDM2, and/or ubiquitin as indicated and then subjected to immunoblotting using the ATF3 antibody. B, purified ATF3 or Δ102–139 protein was subjected to in vitro ubiquitination assay as in A. C, FLAG-tagged ATF3 or Δ102–139 protein was expressed with MDM2 in H1299 cells as indicated. Cell lysates were immunoprecipitated with the anti-FLAG antibody. D, PC3 cells were transfected with ATF3, MDM2, and/or FLAG-Ub as indicated. Cell lysates were immunoprecipitated with the ATF3 antibody and subjected to immunoblotting (IB) for ubiquitinated proteins using the FLAG antibody. E, H1299 cells were transfected with FLAG-ATF3, MDM2, and/or HA-Ub as indicated, followed by IP assays using the FLAG antibody. Ubiquitinated proteins were detected with an anti-HA antibody.

FIGURE 6.

ATF3 via its basic region binds to the RING finger of MDM2. A, GST-MDM2 fusion proteins were immobilized on glutathione-agarose and incubated with in vitro translated ATF3 protein. After extensive washes, bound proteins were eluted, subjected to SDS-PAGE, and detected by immunoblotting. The lower panel shows Coomassie Blue staining of the fusion proteins. B, GST-ATF3 fusion proteins were immobilized and incubated with in vitro translated MDM2 as in A. The bound MDM2 protein was detected by immunoblotting. The lower panel shows Ponceau S staining of the blot.

FIGURE 7.

MDM2-mediated ATF3 degradation requires the binding of MDM2 to ATF3. A, H1299 cells were transfected with the indicated plasmids and subjected to IP using the ATF3 antibody. B, ATF3 or Δ80–100 was expressed with or without MDM2 and subjected to immunoblotting as in Fig. 3A. C, schematic representation of negative-feedback loops for tight controls of p53 activity in cellular stress responses is shown.