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.     

Mechanism of p53 stabilization by ATM after DNA damage

p53 suppresses tumor development by responding to unauthorized cell proliferation, growth factor or nutrient deprivation, and DNA damage. Distinct pathways have been identified that cause p53 activation, including ARF-dependent response to oncogene activation, ribosomal protein-mediated response to abnormal rRNA synthesis, and ATM-dependent response to DNA damage. Elucidating the mechanisms of these signaling events are critical for understanding tumor suppression by p53 and development of novel cancer therapeutics. More than a decade of research has established the ATM kinase as a key molecule that activates p53 after DNA damage. Our recent study revealed that ATM phosphorylation of MDM2 is likely to be the key step in causing p53 stabilization. Upon activation by ionizing irradiation, ATM phosphorylates MDM2 on multiple sites near its RING domain. These modifications inhibit the ability of MDM2 to poly-ubiquitinate p53, thus leading to its stabilization. MDM2 phosphorylation does not inactivate its E3 ligase activity per se, since MDM2 self-ubiquitination and MDMX ubiquitination functions are retained. The selective inhibition of p53 poly-ubiquitination is accomplished through disrupting MDM2 oligomerization that may provide a scaffold for processive elongation of poly ubiquitin chains. These findings suggest a novel model of p53 activation and a general mechanism of E3 ligase regulation by phosphorylation.     

Requirement for HDM2 activity in the rapid degradation of p53 in neuroblastoma

The wild type p53 tumor suppressor protein is rapidly degraded in normal cells by MDM2, the ubiquitin ligase that serves as the key regulator of p53 function by modulating protein stability. Cellular exposure to genotoxic stress triggers the stabilization of p53 by multiple pathways that converge upon interference with MDM2 function. In this study, we first investigated the ability of HDM2 (MDM2 human homologue) to degrade endogenous p53 in neuroblastoma (NB). Although the p53 protein in NB has been reported to be constitutively stabilized, we find that HDM2 in NB is functional and facilitates the rapid turnover of p53 in nonstressed cells via the proteasome pathway. Second, we examined the relationship between p53 and HDM2 in the adriamycin-mediated stabilization of p53 in NB. We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2. In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway.     

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.     

Oligomerization is required for p53 to be efficiently ubiquitinated by MDM2.

Wild-type p53 is degraded in part through the ubiquitin proteolysis pathway. Recent studies indicate that MDM2 can bind p53 and promote its rapid degradation although the molecular basis for this degradation has not been clarified. This report demonstrates that MDM2 can promote the ubiquitination of wild-type p53 and cancer-derived p53 mutants in transiently transfected cells. Deletion mutants that disrupted the oligomerization domain of p53 displayed low binding affinity for MDM2 and were poor substrates for ubiquitination. However, efficient MDM2 binding and ubiquitination were restored when an oligomerization-deficient p53 mutant was fused to the dimerization domain from another protein. These results indicate that oligomerization is required for p53 to efficiently bind and be ubiquitinated by MDM2. p53 ubiquitination was inhibited in cells exposed to UV radiation, and this inhibition coincided with a decrease in MDM2 protein levels and p53.MDM2 complex formation. In contrast, p53 dimerization was unaffected following UV treatment. These results suggest that UV radiation may stabilize p53 by blocking the ubiquitination and degradation of p53 mediated by MDM2.     

RYBP stabilizes p53 by modulating MDM2

The mouse double minute 2 (MDM2)–p53 interaction regulates the activity of p53 and is a potential target for human cancer therapy. Here, we report that RYBP (RING1‐ and YY1‐binding protein), a member of the polycomb group (PcG), interacts with MDM2 and decreases MDM2‐mediated p53 ubiquitination, leading to stabilization of p53 and an increase in p53 activity. RYBP induces cell‐cycle arrest and is involved in the p53 response to DNA damage. Expression of RYBP is decreased in human cancer tissues compared with adjacent normal tissues. These results show that RYBP is a new regulator of the MDM2–p53 loop and that it has tumour suppressor activity.     

Positive regulation of p53 stability and activity by the deubiquitinating enzyme Otubain 1

The ubiquitin (Ub)-proteasome system plays a pivotal role in the regulation of p53 protein stability and activity. p53 is ubiquitinated and destabilized by MDM2 and several other Ub E3s, whereas it is deubiquitinated and stabilized by Ub-specific protease (USP)7 and USP10. Here we show that the ovarian tumour domain-containing Ub aldehyde-binding protein 1 (Otub1) is a novel p53 regulator. Otub1 directly suppresses MDM2-mediated p53 ubiquitination in cells and in vitro. Overexpression of Otub1 drastically stabilizes and activates p53, leading to apoptosis and marked inhibition of cell proliferation in a p53-dependent manner. These effects are independent of its catalytic activity but require residue Asp88. Mutation of Asp88 to Ala (Otub1(D88A)) abolishes activity of Otub1 to suppress p53 ubiquitination. Further, wild-type Otub1 and its catalytic mutant (Otub1(C91S)), but not Otub1(D88A), bind to the MDM2 cognate E2, UbcH5, and suppress its Ub-conjugating activity in vitro. Overexpression of Otub1(D88A) or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage. Together, these results reveal a novel function for Otub1 in regulating p53 stability and activity.     

SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination

The p53 tumour suppressor has a key role in the control of cell growth and differentiation, and in the maintenance of genome integrity. p53 is kept labile under normal conditions, but in response to stresses, such as DNA damage, it accumulates in the nucleus for induction of cell-cycle arrest, DNA repair or apoptosis. Mdm2 is an ubiquitin ligase that promotes p53 ubiquitination and degradation. Mdm2 is also self-ubiquitinated and degraded. Here, we identified a novel cascade for the increase in p53 level in response to DNA damage. A new SUMO-specific protease, SUSP4, removed SUMO-1 from Mdm2 and this desumoylation led to promotion of Mdm2 self-ubiquitination, resulting in p53 stabilization. Moreover, SUSP4 competed with p53 for binding to Mdm2, also resulting in p53 stabilization. Overexpression of SUSP4 inhibited cell growth, whereas knockdown of susp4 by RNA interference (RNAi) promoted of cell growth. UV damage induced SUSP4 expression, leading to an increase in p53 levels in parallel with a decrease in Mdm2 levels. These findings establish a new mechanism for the elevation of cellular p53 levels in response to UV damage.     

Stabilization of the MDM2 oncoprotein by mutant p53

MDM2 is a short-lived protein that regulates p53 degradation. We report here that transient coexpression of MDM2 and several p53 hotspot mutants resulted in stabilization and increased expression of MDM2. Ectopic expression of the mutant p53(175H) allele by recombinant adenovirus infection or stable transfection also stabilized endogenous MDM2 in p53-null cells. A panel of human tumor cell lines expressing different endogenous mutant p53 alleles also contained stabilized nuclear MDM2 at elevated levels when compared with p53-null cells. MDM2 was present in complexes with mutant p53 in tumor cells, and stabilization of MDM2 required direct binding to mutant p53. These results reveal a novel property of mutant p53 and a unique feature of tumors with p53 missense mutations. Accumulation of stable MDM2 may contribute to tumorigenesis through its p53-independent transforming functions.     

MDM2 can promote the ubiquitination, nuclear export, and degradation of p53 in the absence of direct binding

MDM2 can bind the N terminus of p53 and promote its ubiquitination and export from the nucleus to the cytoplasm, where p53 can then be degraded by cytoplasmic proteasomes. Several studies have reported that an intact MDM2 binding domain is necessary for p53 to be targeted for ubiquitination, nuclear export, and degradation by MDM2. In the current study, we examined whether the MDM2 binding domain of p53 could be provided in trans through oligomerization between two p53 molecules. p53 proteins mutated in their MDM2 binding domains were unable to bind MDM2 directly and were resistant to MDM2-mediated ubiquitination, nuclear export, and degradation when expressed with MDM2 alone. However, these same p53 mutants formed a complex with MDM2 and were efficiently ubiquitinated, exported from the nucleus, and degraded when co-expressed with MDM2 and wild-type p53. Moreover, this effect required MDM2 binding by wild-type p53 as well as oligomerization between wild-type p53 and the MDM2 binding-deficient p53 mutants. Taken together, these results support a model whereby MDM2 binding-deficient forms of p53 can bind MDM2 indirectly through oligomerization with wild-type p53 and are subsequently targeted for ubiquitination, nuclear export, and degradation. These findings may have important implications regarding the DNA damage response of p53.