c-Abl phosphorylation of Mdm2 facilitates Mdm2-Mdmx complex formation

Mdm2 and Mdmx are oncoproteins that have essential yet nonredundant roles in development and function as part of a multicomponent ubiquitinating complex that targets p53 for proteasomal degradation. However, in response to DNA damage, Mdm2 and Mdmx are phosphorylated and protect p53 through various mechanisms. It has been predicted that Mdm2-Mdmx complex formation modulates Mdm2 ligase activity, yet the mechanism that promotes formation of Mdm2-Mdmx complexes is unknown. Here, we show that optimal Mdm2-Mdmx complex formation requires c-Abl phosphorylation of Mdm2 both in vitro and in vivo. In addition, Abl phosphorylation of Mdm2 is required for efficient ubiquitination of Mdmx in vitro, and eliminating c-Abl signaling, using c-Abl(-/-) knock-out murine embryonic fibroblasts, led to a decrease in Mdmx ubiquitination. Further, p53 levels are not induced as efficiently in c-Abl(-/-) murine embryonic fibroblasts following DNA damage. Overall, these results define a direct link between genotoxic stress-activated c-Abl kinase signaling and Mdm2-Mdmx complex formation. Our results add an important regulatory mechanism for the activation of p53 in response to DNA damage.     

PTEN protects p53 from Mdm2 and sensitizes cancer cells to chemotherapy.

The PTEN tumor suppressor protein inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signaling that promotes translocation of Mdm2 into the nucleus. When restricted to the cytoplasm, Mdm2 is degraded. The ability of PTEN to inhibit the nuclear entry of Mdm2 increases the cellular content and transactivation of the p53 tumor suppressor protein. Retroviral transduction of PTEN into U87MG (PTEN null) glioblastoma cells increases p53 activity and expression of p53 target genes and induces cell cycle arrest. U87MG/PTEN glioblastoma cells are more sensitive than U87MG/PTEN null cells to death induced by etoposide, a chemotherapeutic agent that induces DNA damage. Previously, tumor suppressor proteins have been supposed to act individually to suppress cancers. Our results establish a direct connection between the activities of two major tumor suppressors and show that they act together to respond to stresses and malignancies. PTEN protects p53 from survival signals, permitting p53 to function as a guardian of the genome. By virtue of its capacity to protect p53, PTEN can sensitize tumor cells to chemotherapy that relies on p53 activity. p53 induces PTEN gene expression, and here it is shown that PTEN protects p53, indicating that a positive feedback loop may amplify the cellular response to stress, damage, and cancer.     

Acetylation is indispensable for p53 activation

The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its activation. We have now identified all major acetylation sites of p53. Although unacetylated p53 retains its ability to induce the p53-Mdm2 feedback loop, loss of acetylation completely abolishes p53-dependent growth arrest and apoptosis. Notably, acetylation of p53 abrogates Mdm2-mediated repression by blocking the recruitment of Mdm2 to p53-responsive promoters, which leads to p53 activation independent of its phosphorylation status. Our study identifies p53 acetylation as an indispensable event that destabilizes the p53-Mdm2 interaction and enables the p53-mediated stress response.     

Caspase-2-mediated cleavage of Mdm2 creates a p53-induced positive feedback loop

Caspase-2 is an evolutionarily conserved caspase, yet its biological function and cleavage targets are poorly understood. Caspase-2 is activated by the p53 target gene product PIDD (also known as LRDD) in a complex called the Caspase-2-PIDDosome. We show that PIDD expression promotes growth arrest and chemotherapy resistance by a mechanism that depends on Caspase-2 and wild-type p53. PIDD-induced Caspase-2 directly cleaves the E3 ubiquitin ligase Mdm2 at Asp 367, leading to loss of the C-terminal RING domain responsible for p53 ubiquitination. As a consequence, N-terminally truncated Mdm2 binds p53 and promotes its stability. Upon DNA damage, p53 induction of the Caspase-2-PIDDosome creates a positive feedback loop that inhibits Mdm2 and reinforces p53 stability and activity, contributing to cell survival and drug resistance. These data establish Mdm2 as a cleavage target of Caspase-2 and provide insight into a mechanism of Mdm2 inhibition that impacts p53 dynamics upon genotoxic stress.     

The p53 and Mdm2 families in cancer.

Cells within an organism are occasionally exposed to either intracellular or environmental stress. Such stress often has genotoxic potential that enhances the probability of cancer. Two gene families, the p53 family (p53, p63 and p73) and the Mdm2 family (Mdm2 and MdmX), serve as major integrators of the signals generated by genotoxic and oncogenic stress. Their co-ordinated modulation ensures an optimal response to stress and decreases the likelihood of cancer. Work over the past year has provided better understanding of the p53-Mdm2 module that lies in the heart of this regulatory network, and of the intricate interplay between the various members of the network.     

The in vivo role of the RP-Mdm2-p53 pathway in signaling oncogenic stress induced by pRb inactivation and Ras overexpression

The Mdm2-p53 tumor suppression pathway plays a vital role in regulating cellular homeostasis by integrating a variety of stressors and eliciting effects on cell growth and proliferation. Recent studies have demonstrated an in vivo signaling pathway mediated by ribosomal protein (RP)-Mdm2 interaction that responds to ribosome biogenesis stress and evokes a protective p53 reaction. It has been shown that mice harboring a Cys-to-Phe mutation in the zinc finger of Mdm2 that specifically disrupts RP L11-Mdm2 binding are prone to accelerated lymphomagenesis in an oncogenic c-Myc driven mouse model of Burkitt's lymphoma. Because most oncogenes when upregulated simultaneously promote both cellular growth and proliferation, it therefore stands to reason that the RP-Mdm2-p53 pathway might also be essential in response to oncogenes other than c-Myc. Using genetically engineered mice, we now show that disruption of the RP-Mdm2-p53 pathway by an Mdm2(C305F) mutation does not accelerate prostatic tumorigenesis induced by inactivation of the pRb family proteins (pRb/p107/p130). In contrast, loss of p19Arf greatly accelerates the progression of prostate cancer induced by inhibition of pRb family proteins. Moreover, using ectopically expressed oncogenic H-Ras we demonstrate that p53 response remains intact in the Mdm2(C305F) mutant MEF cells. Thus, unlike the p19Arf-Mdm2-p53 pathway, which is considered a general oncogenic response pathway, the RP-Mdm2-p53 pathway appears to specifically suppress tumorigenesis induced by oncogenic c-Myc.     

p53-Mdm2 loop controlled by a balance of its feedback strength and effective dampening using ATM and delayed feedback

When the genomic integrity of a cell is challenged, its fate is determined in part by signals conveyed by the p53 tumour suppressor protein. It was observed recently that such signals are not simple gradations of p53 concentration, but rather a counter-intuitive limit-cycle behaviour. Based on a careful mathematical interpretation of the experimental body of knowledge, we propose a model for the p53 signalling network and characterise the p53 stability and oscillatory dynamics. In our model, ATM, a protein that senses DNA damage, activates p53 by phosphorylation. In its active state, p53 has a decreased degradation rate and an enhanced transactivation of Mdm2, a gene whose protein product Mdm2 tags p53 for degradation. Thus the p53-Mdm2 system forms a negative feedback loop. However, the feedback in this loop is delayed, as the pool of Mdm2 molecules being induced by p53 at a given time will mark for degradation the pool of p53 molecules at some later time, after the Mdm2 molecules have been transcribed, exported out of the nucleus, translated and transported back into the nucleus. The analysis of our model demonstrates how this time lag combines with the ATM-controlled feedback strength and effective dampening of the negative feedback loop to produce limit-cycle oscillations. The picture that emerges is that ATM, once activated by DNA damage, makes the p53-Mdm2 oscillator undergo a supercritical Hopf bifurcation. This approach yields an improved understanding of the global dynamics and bifurcation structure of our time-delayed, negative feedback model and allows for predictions of the behaviour of the p53 system under different perturbations.     

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.     

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.     

P53 licensed to kill? Operating the assassin

The p53 protein is a key player in the cellular response to stress. Proper regulation of p53 is imperative for the suppression of tumor development. This regulation is largely governed by its master inhibitor, Mdm2, which both blocks p53 activities and promotes its destabilization. This tight regulation of p53 by Mdm2 must be interrupted under stress conditions in order for p53 to be stabilized in an active form. A combined action of partner proteins and modifying enzymes is essential for the relief of p53 from Mdm2. The recent revelation of p53 association with the PML-nuclear bodies provides one explanation of how this regulatory network is coordinated within the nucleus in response to certain stress conditions. Thus, it is not only the nature of the p53 regulatory complex but also the spatial and temporal context of this association that governs the output inhibitory signals mediated by p53.     

c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination

The p53 protein is subject to Mdm2-mediated degradation by the ubiquitin-proteasome pathway. This degradation requires interaction between p53 and Mdm2 and the subsequent ubiquitination and nuclear export of p53. Exposure of cells to DNA damage results in the stabilization of the p53 protein in the nucleus. However, the underlying mechanism of this effect is poorly defined. Here we demonstrate a key role for c-Abl in the nuclear accumulation of endogenous p53 in cells exposed to DNA damage. This effect of c-Abl is achieved by preventing the ubiquitination and nuclear export of p53 by Mdm2, or by human papillomavirus E6. c-Abl null cells fail to accumulate p53 efficiently following DNA damage. Reconstitution of these cells with physiological levels of c-Abl is sufficient to promote the normal response of p53 to DNA damage via nuclear retention. Our results help to explain how p53 is accumulated in the nucleus in response to DNA damage.     

Mdm2 Plays a Positive Role as an Effector of p53-Dependent Responses

The p53 tumor suppressor is negatively regulated in cells by the Mdm2 protein. Mdm2 has therefore been the focus of intensive research aiming at using it as a target for cancer therapy with the ultimate goal of restoring p53 activity. Several studies have attempted to ablate Mdm2 expression or disrupt its interaction with p53 in cancer cells. While the p53-Mdm2 duo has concentrated a lot of attention, multiple new and diverse functions and targets of Mdm2 have been uncovered. Downregulation of Mdm2 using an siRNA approach has recently provided evidence for a new role of Mdm2 in the p53 response, by modulating the inhibition of the cyclin-dependent kinase 2 (cdk2) by p21. Here, this and other recent findings are discussed that support a new role for Mdm2 in the regulation of p53 response.     

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.