Predicted Functions of MdmX in Fine-Tuning the Response of p53 to DNA Damage

Tumor suppressor protein p53 is regulated by two structurally homologous proteins, Mdm2 and MdmX. In contrast to Mdm2, MdmX lacks ubiquitin ligase activity. Although the essential interactions of MdmX are known, it is not clear how they function to regulate p53. The regulation of tumor suppressor p53 by Mdm2 and MdmX in response to DNA damage was investigated by mathematical modeling of a simplified network. The simplified network model was derived from a detailed molecular interaction map (MIM) that exhibited four coherent DNA damage response pathways. The results suggest that MdmX may amplify or stabilize DNA damage-induced p53 responses via non-enzymatic interactions. Transient effects of MdmX are mediated by reservoirs of p53∶MdmX and Mdm2∶MdmX heterodimers, with MdmX buffering the concentrations of p53 and/or Mdm2. A survey of kinetic parameter space disclosed regions of switch-like behavior stemming from such reservoir-based transients. During an early response to DNA damage, MdmX positively or negatively regulated p53 activity, depending on the level of Mdm2; this led to amplification of p53 activity and switch-like response. During a late response to DNA damage, MdmX could dampen oscillations of p53 activity. A possible role of MdmX may be to dampen such oscillations that otherwise could produce erratic cell behavior. Our study suggests how MdmX may participate in the response of p53 to DNA damage either by increasing dependency of p53 on Mdm2 or by dampening oscillations of p53 activity and presents a model for experimental investigation.     

MdmX protein is essential for Mdm2 protein-mediated p53 polyubiquitination

Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.     

MdmX binding to ARF affects Mdm2 protein stability and p53 transactivation

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity.     

MdmX promotes bipolar mitosis to suppress transformation and tumorigenesis in p53-deficient cells and mice

Mdm2 and MdmX are structurally related p53-binding proteins that function as critical negative regulators of p53 activity in embryonic and adult tissue. The overexpression of Mdm2 or MdmX inhibits p53 tumor suppressor functions in vitro, and the amplification of Mdm2 or MdmX is observed in human cancers retaining wild-type p53. We now demonstrate a surprising role for MdmX in suppressing tumorigenesis that is distinct from its oncogenic ability to inhibit p53. The deletion of MdmX induces multipolar mitotic spindle formation and the loss of chromosomes from hyperploid p53-null cells. This reduction in chromosome number, not observed in p53-null cells with Mdm2 deleted, correlates with increased cell proliferation and the spontaneous transformation of MdmX/p53-null mouse embryonic fibroblasts in vitro and with an increased rate of spontaneous tumorigenesis in MdmX/p53-null mice in vivo. These results indicate that MdmX has a p53-independent role in suppressing oncogenic cell transformation, proliferation, and tumorigenesis by promoting centrosome clustering and bipolar mitosis.     

MdmX protects p53 from Mdm2-mediated degradation

The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.     

NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2

As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2–p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53‐mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.     

S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response

p53 mediates DNA damage‐induced cell‐cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR‐S6K1 through p38α MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2‐mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR‐S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53‐dependent cell death. These findings thus establish mTOR‐S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1–Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging‐controlling Mdm2–p53 and mTOR‐S6K pathways.     

The promyelocytic leukemia protein protects p53 from Mdm2-mediated inhibition and degradation

The p53 protein is kept labile under normal conditions. This regulation is governed largely by its major negative regulator, Mdm2. In response to stress however, p53 accumulates and becomes activated. For this to occur, the inhibitory effects of Mdm2 have to be neutralized. Here we investigated the role of the promyelocytic leukemia protein (PML) in the activation of p53 in response to stress. We found that PML is critical for the accumulation of p53 in response to DNA damage under physiological conditions. PML protects p53 from Mdm2-mediated ubiquitination and degradation, and from inhibition of apoptosis. PML neutralizes the inhibitory effects of Mdm2 by prolonging the stress-induced phosphorylation of p53 on serine 20, a site of the checkpoint kinase 2 (Chk2). PML recruits Chk2 and p53 into the PML nuclear bodies and enhances p53/Chk2 interaction. Our results provide a novel mechanistic explanation for the cooperation between PML and p53 in response to DNA damage.