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

Phosphatases in the cellular response to DNA damage

In the last fifteen years, rapid progress has been made in delineating the cellular response to DNA damage. The DNA damage response network is composed of a large number of proteins with different functions that detect and signal the presence of DNA damage in order to coordinate DNA repair with a variety of cellular processes, notably cell cycle progression. This signal, which radiates from the chromatin template, is driven primarily by phosphorylation events, mainly on serine and threonine residues. While we have accumulated detailed information about kinases and their substrates our understanding of the role of phosphatases in the DNA damage response is still preliminary. Identifying the phosphatases and their regulation will be instrumental to obtain a complete picture of the dynamics of the DNA damage response. Here we give an overview of the DNA damage response in mammalian cells and then review the data on the role of different phosphatases and discuss their biological relevance

Rad54B serves as a scaffold in the DNA damage response that limits checkpoint strength

The strength of the DNA damage checkpoint critically influences cell fate, yet the mechanisms behind the fine tuning of checkpoint strength during the DNA damage response (DDR) are poorly understood. Here we show that ​Rad54B—a SNF2 helicase-like DNA-repair protein—limits the strength of both the G1/S and G2/M checkpoints. We find that ​Rad54B functions as a scaffold for ​p53 degradation via its direct interaction with the ​MDM2–​MDMX ubiquitin–ligase complex. During the early phases of the DDR, ​Rad54B is upregulated, thereby maintaining low checkpoint strength and facilitating cell cycle progression. Once the ​p53-mediated checkpoint is established, ​Rad54B is downregulated, and high checkpoint strength is maintained. Constitutive upregulation of ​Rad54B activity, which is frequently observed in tumours, promotes genomic instability because of checkpoint override. Thus, the scaffolding function of ​Rad54B dynamically regulates the maintenance of genome integrity by limiting checkpoint strength.UTokyo Research掲載「がん発生の基盤となる仕組みを探る」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/discovery-of-a-basic-mechanism-of-cancer-development/UTokyo Research "Discovery of a basic mechanism of cancer development" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/discovery-of-a-basic-mechanism-of-cancer-development

It\u27s getting complicated-A fresh look at p53-MDM2-ARF triangle in tumorigenesis and cancer therapy

Anti-tumorigenic mechanisms mediated by the tumor suppressor p53, upon oncogenic stresses, are our bodies\u27 greatest weapons to battle against cancer onset and development. Consequently, factors that possess significant p53-regulating activities have been subjects of serious interest from the cancer research community. Among them, MDM2 and ARF are considered the most influential p53 regulators due to their abilities to inhibit and activate p53 functions, respectively. MDM2 inhibits p53 by promoting ubiquitination and proteasome-mediated degradation of p53, while ARF activates p53 by physically interacting with MDM2 to block its access to p53. This conventional understanding of p53-MDM2-ARF functional triangle have guided the direction of p53 research, as well as the development of p53-based therapeutic strategies for the last 30 years. Our increasing knowledge of this triangle during this time, especially through identification of p53-independent functions of MDM2 and ARF, have uncovered many under-appreciated molecular mechanisms connecting these three proteins. Through recognizing both antagonizing and synergizing relationships among them, our consideration for harnessing these relationships to develop effective cancer therapies needs an update accordingly. In this review, we will re-visit the conventional wisdom regarding p53-MDM2-ARF tumor-regulating mechanisms, highlight impactful studies contributing to the modern look of their relationships, and summarize ongoing efforts to target this pathway for effective cancer treatments. A refreshed appreciation of p53-MDM2-ARF network can bring innovative approaches to develop new generations of genetically-informed and clinically-effective cancer therapies

Simulating Molecular Mechanisms Of the Mdm2-Mediated Regulatory Interactions: a Conformational Selection Model Of the Mdm2 Lid Dynamics

Diversity and complexity of MDM2 mechanisms govern its principal function as the cellular antagonist of the p53 tumor suppressor. Structural and biophysical studies have demonstrated that MDM2 binding could be regulated by the dynamics of a pseudo-substrate lid motif. However, these experiments and subsequent computational studies have produced conflicting mechanistic models of MDM2 function and dynamics. We propose a unifying conformational selection model that can reconcile experimental findings and reveal a fundamental role of the lid as a dynamic regulator of MDM2-mediated binding. In this work, structure, dynamics and energetics of apo-MDM2 are studied as a function of posttranslational modifications and length of the lid. We found that the dynamic equilibrium between closed and semi-closed lid forms may be a fundamental characteristic of MDM2 regulatory interactions, which can be modulated by phosphorylation, phosphomimetic mutation as well as by the lid size. Our results revealed that these factors may regulate p53-MDM2 binding by fine-tuning the thermodynamic equilibrium between preexisting conformational states of apo-MDM2. In agreement with NMR studies, the effect of phosphorylation on MDM2 interactions was more pronounced with the truncated lid variant that favored the thermodynamically dominant closed form. The phosphomimetic mutation S17D may alter the lid dynamics by shifting the thermodynamic equilibrium towards the ensemble of semi-closed conformations. The dominant semi-closed lid form and weakened dependence on the phosphorylation seen in simulations with the complete lid can provide a rationale for binding of small p53-based mimetics and inhibitors without a direct competition with the lid dynamics. The results suggested that a conformational selection model of preexisting MDM2 states may provide a robust theoretical framework for understanding MDM2 dynamics. Probing biological functions and mechanisms of MDM2 regulation would require further integration of computational and experimental studies and may help to guide drug design of novel anti-cancer therapeutics

The Role of MDM2 Phosphorylation in P53 Responses to DNA Damage and Tumor Suppression: A Dissertation

The p53 tumor suppressor protein is upregulated in response to DNA damage and other stress signals. The upregulation of p53 involves freeing it from negative regulation imposed by Mdm2 and MdmX (Mdm4). Accumulating evidence indicates that phosphorylation of Mdm proteins by different stress-activated kinases such as ATM or c-Abl significantly impacts p53 functions. We have previously shown that ATM phosphorylation of Mdm2 Ser394 is required for robust p53 stabilization and activation following DNA damage. This dissertation describes in vivo examination of the mechanism by which Mdm2 Ser394 phosphorylation impacts p53 activities and its contribution to suppression of oncogene and DNA damage-induced tumors. We determine that phosphorylation of Mdm2 Ser394 regulates p53 activity by modulating Mdm2 stability and paradoxically delays Myc-driven lymphomagenesis while increasing lymphomagenesis in sub-lethally irradiated mice. c-Abl phosphorylates the residue neighboring Mdm2 Ser394, Mdm2 Tyr393. This dissertation describes the generation of a novel Mdm2Y393F mutant mouse to determine if c-Abl phosphorylation of Mdm2 regulates p53-mediated DNA damage responses or tumor suppression in vivo. Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes. The studies presented herein further our understanding of the mechanisms by which DNA damage-associated kinases stabilize and activate p53, and influence p53-dependent responses and tumor suppression. A better understanding of the in vivo effects of Mdm2 phosphorylation may facilitate the development of novel therapeutics capable of stimulating p53 anti-tumor activity or alleviating p53- dependent toxicities in non-malignant tissues

Expression levels of MDM2 and MDMX proteins and their associated effects on P53 in human liposarcomas

Background: Inactivation of wild type P53 by its main cellular inhibitors, MDM2 and MDMX, is a well-recognised feature of tumour formation in liposarcomas (LS). MDM2 over-expression has been detected in approximately 80% of liposarcomas, but only limited information is available about MDMX expression levels. On commencing this work, we were not aware of any study that had described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and / or MDMX single and dual affinity antagonist compounds have emerged as alternative approaches for potential targeted therapeutic strategies in LS. Methods: After appropriate optimisation and confirmation of experimental techniques, a case series of 64 pathologically characterised liposarcomas of various sub-types was analysed by immunohistochemistry, to simultaneously assess the expression levels of P53, MDM2 and MDMX. P53 mutation status was investigated in cases that over-expressed P53. Results: 83% of cases over-expressed MDM2 and 69% co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly, in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels, suggesting an interaction between MDM2 and MDMX that resulted in reduced efficiency of MDM2 when degrading P53. No increased incidence of P53 mutations was detected in cases that over-expressed P53 compared to the general population of LSs. Conclusions: The results of the study indicated that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53 in human liposarcomas. This suggests that careful characterisation of all these markers will be necessary when considering in vivo evaluation of novel MDM blocking compounds as a therapeutic strategy to restore wild type P53 functions