MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.The tumor suppressor gene p53 encodes a transcription factor that is activated in response to various forms of stress, leading to the induction of a number of genes whose products mediate either cell cycle arrest or apoptosis (1). Under most physiological conditions, p53 activity is tightly controlled, primarily through the ability of MDM2 to target p53 for degradation, which ensures cell survival. Current model of p53 activation suggests that diverse stress signals converge on a single regulatory node, namely the p53-MDM2 module, and interfere with the ability of MDM2 to target p53 for degradation (2). Analogous to MDM2, MDMX ablation is also associated with p53-dependent embryonic death in mice, placing MDMX in the category of essential p53 negative regulators (3). In contrast to MDM2, however, MDMX lacks ubiquitin E3 ligase activity and is unable to target p53 for ubiquitin-proteasome-dependent proteolysis (4). Moreover, MDMX was reported to inhibit MDM2-mediated p53 degradation (4 -6), contradicting the role of MDMX as defined by the genetic study. To resolve these conflicting results and gain better understanding of why neither gene product can compensate for the loss of the other, we generated MDMX-deficient cells using small interference RNA (siRNA) 1 and carried out biochemical analysis of MDM2 in these cells. In conjunction with the use of MEFs derived from either single or double knock-out mice, our loss-of-function approach allowed us to obtain compelling evidence at the molecular level to highlight mutual dependence of MDM2 and MDMX in their functional inhibition of p53 and provide support for the findings obtained in genetic studies. /MDM2Ϫ/Ϫ MEFs (Dr. Carl Maki, Harvard School of Public Health), were maintained in minimal essential medium supplemented with 10% fetal bovin...

In response to DNA damage or replication fork stress, the Fanconi anemia (FA) pathway is activated, leading to monoubiquitination of FancD2 and FancI and their co-localization in foci. Here we show that, in the chicken DT40 cell system, multiple alanine-substitution mutations in 6 conserved and clustered S/TQ motifs of FancI largely abrogate monoubiquitination as well as focus formation of both FancI and FancD2, resulting in loss of DNA repair function. Conversely, FancI carrying phospho-mimic mutations on the same 6 residues induces constitutive monoubiquitination and focus formation of FancI and FancD2, and protects against cell killing and chromosome breakage by DNA interstrand crosslinking agents. We propose that the multiple phosphorylation of FancI serves as a molecular switch in activation of the FA pathway. Mutational analysis of putative phosphorylation sites in human FANCI indicates that this switch is evolutionarily conserved.

Although genetic studies have demonstrated that MDMX is essential to maintain p53 activity at low levels in non-stressed cells, it is unknown whether MDMX regulates p53 activation by DNA damage. We show here that DNA damage-induced p53 induction is associated with rapid down-regulation of the MDMX protein. Significantly, interference with MDMX down-regulation results in the suppression of p53 activation by genotoxic stress. We also demonstrate that DNA damage-induced MDMX reduction is mediated by MDM2, which targets MDMX for proteasomal degradation by a distinct mechanism that permits preferential MDMX degradation and therefore ensures optimal p53 activation.