ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage

The p53 tumor suppressor is activated after DNA damage to maintain genomic stability and prevent transformation. Rapid activation of p53 by ionizing radiation is dependent on signaling by the ATM kinase. MDM2 and MDMX are important p53 regulators and logical targets for stress signals. We found that DNA damage induces ATM-dependent phosphorylation and degradation of MDMX. Phosphorylated MDMX is selectively bound and degraded by MDM2 preceding p53 accumulation and activation. Reduction of MDMX level by RNAi enhances p53 response to DNA damage. Loss of ATM prevents MDMX degradation and p53 stabilization after DNA damage. Phosphorylation of MDMX on S342, S367, and S403 were detected by mass spectrometric analysis, with the first two sites confirmed by phosphopeptide-specific antibodies. Mutation of MDMX on S342, S367, and S403 each confers partial resistance to MDM2-mediated ubiquitination and degradation. Phosphorylation of S342 and S367 in vivo require the Chk2 kinase. Chk2 also stimulates MDMX ubiquitination and degradation by MDM2. Therefore, the E3 ligase activity of MDM2 is redirected to MDMX after DNA damage and contributes to p53 activation.     

The MDM2-p53 interaction

Activation of the p53 protein protects the organism against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. MDM2, a p53-specific E3 ubiquitin ligase, is the principal cellular antagonist of p53, acting to limit the p53 growth-suppressive function in unstressed cells. In unstressed cells, MDM2 constantly monoubiquitinates p53 and thus is the critical step in mediating its degradation by nuclear and cytoplasmic proteasomes. The interaction between p53 and MDM2 is conformation-based and is tightly regulated on multiple levels. Disruption of the p53-MDM2 complex by multiple routes is the pivotal event for p53 activation, leading to p53 induction and its biological response. Because the p53-MDM2 interaction is structurally and biologically well understood, the design of small lipophilic molecules that disrupt or prevent it has become an important target for cancer therapy.     

DNA damage-induced MDMX degradation is mediated by MDM2

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.     

MDM2 promotes ubiquitination and degradation of MDMX

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Mitogenic signals activate p53 by induction of ARF expression, which inhibits p53 ubiquitination by MDM2. Recent studies showed that the MDM2 homolog MDMX is also an important regulator of p53. We present evidence that MDM2 promotes MDMX ubiquitination and degradation by the proteasomes. This effect is stimulated by ARF and correlates with the ability of ARF to bind MDM2. Promotion of MDM2-mediated MDMX ubiquitination requires the N-terminal domain of ARF, which normally inhibits MDM2 ubiquitination of p53. An intact RING domain of MDM2 is also required, both to interact with MDMX and to provide E3 ligase function. Increase of MDM2 and ARF levels by DNA damage, recombinant ARF adenovirus infection, or inducible MDM2 expression leads to proteasome-mediated down-regulation of MDMX levels. Therefore, MDMX and MDM2 are coordinately regulated by stress signals. The ARF tumor suppressor differentially regulates the ability of MDM2 to promote p53 and MDMX ubiquitination and activates p53 by targeting both members of the MDM2 family.     

The tumour suppressor RASSF1A promotes MDM2 self-ubiquitination by disrupting the MDM2-DAXX-HAUSP complex

The tumour suppressor p53, which accumulates in response to DNA damage and induces cell-cycle arrest and apoptosis, has a key function in the maintenance of genome integrity. Under normal conditions, the antiproliferative effects of p53 are inhibited by MDM2, a ubiquitin ligase that promotes p53 ubiquitination and degradation. MDM2 is also self-ubiquitinated and degraded. Here, we show that the tumour suppressor RASSF1A regulates G(1)-S cell-cycle progression in a p53-dependent manner by promoting MDM2 self-ubiquitination and preventing p53 degradation. Importantly, RASSF1A associates with MDM2 and death-domain-associated protein (DAXX) in the nucleus, thereby disrupting the interactions between MDM2, DAXX, and the deubiquitinase, HAUSP, and enhancing the self-ubiquitin ligase activity of MDM2. Moreover, RASSF1A partially contributes to p53-dependent checkpoint activation at early time points in response to DNA damage. These findings reveal a new and important function for RASSF1A in regulating the p53-MDM2 pathway.     

MDM2 and promyelocytic leukemia antagonize each other through their direct interaction with p53

p53 can be regulated through post-translational modifications and through interactions with positive and negative regulatory factors. MDM2 binding inhibits p53 and promotes its degradation by the proteasome, whereas promyelocytic leukemia (PML) activates p53 by recruiting it to multiprotein complexes termed PML-nuclear bodies. We reported previously an in vivo and in vitro interaction between PML and MDM2 that is independent of p53. In the current study, we investigated whether interaction between MDM2 and PML can indirectly affect p53 activity. Increasing amounts of MDM2 inhibited p53 activation by PML but could not inhibit PML-mediated activation of a p53 fusion protein that lacked the MDM2-binding domain. Conversely, increasing amounts of PML could overcome p53 inhibition by MDM2 but could not overcome MDM2-mediated inhibition of a p53 fusion protein that lacked the PML-binding domain. These results demonstrate that MDM2 and PML can antagonize each other through their direct interaction with p53 and suggest the combined effects of MDM2 and PML on p53 function are determined by the relative level of each protein. Furthermore, these results imply that interactions between MDM2 and PML by themselves have little or no effect on p53 activity.     

DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms

The MDM2 homolog MDMX is an important regulator of p53 activity during embryonic development. MDMX inactivation in mice results in embryonic lethality in a p53-dependent fashion. The expression level of MDMX is not induced by DNA damage, and its role in stress response is unclear. We show here that ectopically expressed MDMX is mainly localized in the cytoplasm. DNA damage promotes nuclear translocation of MDMX in cells with or without p53. Coexpression of MDM2 or p53 is sufficient to induce MDMX nuclear translocation, suggesting that activation of p53 and induction of MDM2 expression can contribute to this process. Stable transfection of MDMX into U2OS cells does not alter p53 level but results in reduced p53 DNA-binding activity and reduced MDM2 expression. The ability of ARF (alternate reading frame of INK4a) to activate p53 is also significantly inhibited by expression of MDMX. These results suggest that MDMX function may be regulated by DNA damage. Furthermore, MDMX may complement MDM2 in regulating p53 during embryonic development due to its ability to inhibit p53 in the presence of ARF.     

ATM activates p53 by regulating MDM2 oligomerization and E3 processivity

Rapid activation of p53 by ionizing irradiation is a classic DNA damage response mediated by the ATM kinase. However, the major signalling target and mechanism that lead to p53 stabilization are unknown. We show in this report that ATM induces p53 accumulation by phosphorylating the ubiquitin E3 ligase MDM2. Multiple ATM target sites near the MDM2 RING domain function in a redundant manner to provide robust DNA damage signalling. In the absence of DNA damage, the MDM2 RING domain forms oligomers that mediate p53 poly ubiquitination and proteasomal degradation. Phosphorylation by ATM inhibits RING domain oligomerization, specifically suppressing p53 poly ubiquitination. Blocking MDM2 phosphorylation by alanine substitution of all six phosphorylation sites results in constitutive degradation of p53 after DNA damage. These observations show that ATM controls p53 stability by regulating MDM2 RING domain oligomerization and E3 ligase processivity. Promoting or disrupting E3 oligomerization may be a general mechanism by which signalling kinases regulate ubiquitination reactions, and a potential target for therapeutic intervention.     

Mutual dependence of MDM2 and MDMX in their functional inactivation of p53

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.