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.     

Novel isatin-derived molecules activate p53 via interference with Mdm2 to promote apoptosis

The p53 protein is a key tumor suppressor in mammals. In response to various forms of genotoxic stress p53 stimulates expression of genes whose products induce cell cycle arrest and/or apoptosis. An E3-ubiquitin ligase, Mdm2 (mouse-double-minute 2) and its human ortholog Hdm2, physically interact with the amino-terminus of p53 to mediate its ubiquitin-mediated degradation via the proteasome. Thus, pharmacological inhibition of the p53-Mdm2 interaction leads to overall stabilization of p53 and stimulation of its anti-tumorigenic activity. In this study we characterize the biological effects of a novel class of non-genotoxic isatin Schiff and Mannich base derivatives (ISMBDs) that stabilize p53 on the protein level. The likely mechanism behind their positive effect on p53 is mediated via the competitive interaction with Mdm2. Importantly, unlike Nutlin, these compounds selectively promoted p53-mediated cell death. These novel pharmacological activators of p53 can serve as valuable molecular tools for probing p53-positive tumors and set up the stage for development of new anti-cancer drugs.     

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.     

Absence of point mutations at codon 17 of the mdm2 gene (serine 17) in human primary tumors

Mdm2 is a phosphoprotein that interacts with protein p53, inhibiting its activity. A serine located in position 17 of Mdm2, has been implicated in its phosphorylation process. We hypothesize that point mutations at serine 17 could block its phosphorylation and thereby increase the p53-Mdm2 interaction. This mechanism could increase the p53 degradation and cause a loss of the protective effect of p53 against tumorigenesis. This hypothesis was based on recent studies in vitro, demonstrating that when serine 17 is mutated, the DNA-dependent protein kinase, activated by genomic damage, is unable to phosphorylate it. Thus, we investigated whether structural point mutations at exon 3 of the Mdm2 gene, affecting codon 17, were present in 162 human primary tumors, 70 breast carcinomas, 14 bladder tumors, 18 colon adenocarcinomas and 60 testicular tumors. Direct sequencing of a fragment (204 bp) of exon 3 of the Mdm2 gene that contains the codon 17 showed no mutations at this position, independently of the presence or absence of p53 gene mutations in the same tumors. These results do not support the hypothesis that mutations in the Mdm2 gene at this level are involved in the tumorigenic process of human cancers.     

Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase

The Murine double-minute clone 2 (Mdm2) onco-protein is the principal regulator of the tumour suppressor, p53. Mdm2 acts as an E3-type ubiquitin ligase that mediates the ubiquitylation and turnover of p53 under normal, unstressed circumstances. In response to cellular stress, such as DNA damage, the Mdm2–p53 interaction is disrupted. Part of the mechanism of uncoupling p53 from Mdm2-mediated degradation involves hypo-phosphorylation of a cluster of phosphorylated serine residues in the central acidic domain of Mdm2. Here, we show that two of the residues within this domain that are phosphorylated in vivo, Ser-260 and Ser-269, are phosphorylated by CK2 in vitro. Treatment of cells with the CK2 inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), leads to the induction of p53 and downstream targets of p53 including Mdm2 itself and p21. These data are consistent with the idea that CK2-mediated phosphorylation of Mdm2 may regulate Mdm2-mediated p53 turnover.     

Cytoplasmically "sequestered" wild type p53 protein is resistant to Mdm2-mediated degradation.

The Mdm2 oncoprotein mediates p53 degradation at cytoplasmic proteasomes and is the principal regulator for maintaining low, often undetectable levels of p53 in unstressed cells. However, a subset of human tumors including neuroblastoma constitutively harbor high levels of wild type p53 protein localized to the cytoplasm. Here we show that the abnormal p53 accumulation in such cells is due to a profound resistance to Mdm2-mediated degradation. Overexpression of Mdm2 in neuroblastoma (NB)(1) cell lines failed to decrease the high steady state levels of endogenous p53. Moreover, exogenous p53, when introduced into these cells, was also resistant to Mdm2-directed degradation. This resistance is not due to a lack of Mdm2 expression in NB cells or a lack of p53-Mdm2 interaction, nor is it due to a deficiency in the ubiquitination state of p53 or proteasome dysfunction. Instead, Mdm2-resistant p53 from NB cells is associated with covalent modification of p53 and masking of the modification-sensitive PAb 421 epitope. This system provides evidence for an important level of regulation of Mdm2-directed p53 destruction in vivo that is linked to p53 modification.