Regulation of MDM2 E3 Ligase Activity by Phosphorylation after DNA Damage

Abstract

MDM2 is a major regulator of p53 by acting as a ubiquitin E3 ligase. The central acidic domain and C-terminal RING domain of MDM2 are both indispensable for ubiquitination of p53. Our previous study suggested that ATM phosphorylation of MDM2 near the C terminus inhibits RING domain oligomerization, resulting in p53 stabilization after DNA damage. We present here evidence that these modifications allosterically regulate the functions of both acidic domain and RING domain of MDM2. Using chemical cross-linking, we show that the MDM2 RING domain forms oligomers including dimer and higher-order complexes in vivo. RING domain dimerization efficiency is negatively regulated by upstream sequence. ATM-mediated phosphorylation of the upstream sequence further inhibits RING dimerization. Forced oligomerization of MDM2 partially overcomes the inhibitory effect of phosphorylation and stimulates p53 ubiquitination. Furthermore, the ability of MDM2 acidic domain to bind p53 core domain and induce p53 misfolding are also suppressed by the same C-terminal ATM sites after DNA damage. These results suggest that the acidic domain and RING domain of MDM2 are both allosterically coupled to the intervening ATM sites, which enables the same modification to regulate multiple MDM2 functions critical for p53 ubiquitination.The p53 pathway is critical for maintenance of genomic stability and preventing development of cancer. The most notable feature of p53 is its stabilization after exposure to a wide range of stress signals such as hyperproliferation, nucleotide depletion, and DNA damage. These responses may be essential for its function as a tumor suppressor (14). In normal cells, p53 is present at very low levels due to rapid degradation through the ubiquitin-dependent proteasome pathway. p53 turnover is regulated by MDM2, which binds p53 and functions as an ubiquitin E3 ligase to promote p53 degradation by the proteasome (15,16,21). Additional E3 ligases such as Pirh2 and Cop1 have also been implicated as regulators of p53 turn over (12,24). However, their physiological significance in p53 regulation and stress response still remain to be established. Current evidence suggests that MDM2 is a major and indispensable regulator of p53 level (18,32).MDM2 and p53 interact through their N-terminal domains in a high-affinity binding and through their central domains in a weak binding (48). Upon complex formation, the MDM2 C-terminal RING domain recruits ubiquitin-conjugating enzyme E2 that performs the transfer of activated ubiquitin to p53 lysine residues. To date, the understanding of molecular mechanisms that lead to p53 stabilization by different pathways remain incomplete. An important group of MDM2 regulators are proteins that bind to the central acidic domain, such as ARF, L5, L11, and L23 (37, 50). These basic proteins are important for mediating mitogenic stress and ribosomal stress signals to p53. They have been shown to inhibit MDM2-mediated p53 ubiquitination, but little is known about the mechanisms. They may act by neutralizing ...

Limiting the power of p53 through the ubiquitin proteasome pathway

“…Mdm4 loss leads to a p53-dependent embryo-lethal phenotype (Parant et al 2001;Migliorini et al 2002). While Mdm2 forms homodimers, it forms heterodimers with Mdm4 more efficiently (Cheng et al 2011). At physiological levels, Mdm2 homodimers predominantly monoubiquitinate p53 and promote its nuclear export.…”

Casein Kinase 1α Regulates an MDMX Intramolecular Interaction To Stimulate p53 Binding

“…In the case of MDM2, we showed that phosphorylation of ATM sites near the RING domain inhibits RING homodimerization (8). Furthermore, phosphorylation of the ATM sites inhibits the binding between the MDM2 acidic domain and p53 core domain, suggesting that the MDM2 acidic domain conformation is regulated by the C-terminal ATM sites (8). There is also evidence that the p53 binding pocket of MDM2 allosterically communicates with the acidic domain, such that interaction of the MDM2 N-terminal pocket with the p53 N terminus stimulates a secondsite interaction between the MDM2 acidic domain and the p53 core (50).…”

“…9d shows many similarities between MDMX and MDM2 regarding their interdomain relationships. In the case of MDM2, we showed that phosphorylation of ATM sites near the RING domain inhibits RING homodimerization (8). Furthermore, phosphorylation of the ATM sites inhibits the binding between the MDM2 acidic domain and p53 core domain, suggesting that the MDM2 acidic domain conformation is regulated by the C-terminal ATM sites (8).…”

“…MDM2 is phosphorylated by ATM, ATR, and C-Abl near the C terminus (32,46,47). MDM2 phosphorylation after DNA damage correlates with reduced dimerization and oligomerization of the RING domain, suggesting that higher-order complex formation is needed for efficient polyubiquitination of p53 (7,8). ATM phosphorylation of MDM2 at S395 also stimulates RNA binding by the RING domain, which has been shown to inhibit E3 ligase activity (11, 28).…”

“…ATM phosphorylation of MDM2 at S395 also stimulates RNA binding by the RING domain, which has been shown to inhibit E3 ligase activity (11,28). Furthermore, MDM2 phosphorylation by ATM regulates the interaction between the MDM2 acidic domain and p53 DNA binding domain (8). This interaction is important for inducing p53 misfolding and promoting p53 ubiquitination.…”