Schwab (ed.), Encyclopedia of Cancer, Springer-Verlag Berlin Heidelberg 2015
The protein human homologue of mouse double minute 2 (MDM2) was first identified as the product of the MDM2 gene amplified in transformed murine cells. It is a p53-binding nuclear protein that antagonizes and downregulates p53 activity. The gene maps to 12q14–q15 and is amplified in certain human tumors, including sarcomas, glioblastomas, and astrocytomas.
Discovery and significance
MDM2 was discovered by Donna George’s laboratory as a gene that is amplified within double-minute chromosomes in 3T3DM cells and encodes a cellular transforming activity that promotes tumorigenicity in xenografts. Its association with p53 was first observed in immunoprecipitation analyses of p53 as a coprecipitating protein migrating with an apparent molecular weight of 95 kDa. Subsequently, it was shown that overexpression of MDM2 leads to increased degradation of p53 via the proteasome, thereby establishing its role as a crucial regulator of p53 levels.
MDM2 is a ubiquitin E3 ligase of the RING (really interesting new gene) finger type. It has several established biological functions and targets in the cell. Its principal function is the regulation (inhibition) of the p53 tumor suppressor protein which it controls through several coordinated activities. These are:
- Mediating mono- and subsequently polyubiquitylation of p53, leading to degradation of p53 by the 26S proteasome
- Exporting p53 to the cytoplasm
- Targeting of p53 to the proteasome
- Inducing a conformational change in p53 that blocks the site-specific DNA-binding function of p53
- Targeting histone deacetylase activity toward p53 leading to deacetylation of C-terminal lysine residues in p53 that are the major targets of ubiquitylation
- Mediating the modification of p53 by NEDD8 and by SUMO-1, SUMO-2, and SUMO-3
In contrast to these inhibitory activities, there are also reports that under conditions of cellular stress, MDM2 can bind to and stimulate translation of p53 mRNA.
The central importance of MDM2 in regulating p53 function in vivo is underpinned by two mouse models. Firstly, Mdm2-null mice die in utero at E5–E6 owing to widespread unregulated p53-dependent apoptosis. Significantly, this lethality can be rescued in a p53-null background, underscoring the critical
level of regulation that MDM2 provides. In contrast to the knockout model, overexpression of Mdm2 in mice inhibits p53 function in vivo and increases susceptibility to various cancers including lymphomas, soft-tissue sarcomas, and osteosarcomas.
Full-length human MDM2 (apparent molecular weight 90 kDa) comprises 491 amino acids and contains several structured and unstructured regions (there are a number of truncated MDM2 isoforms of 85, 76, and 57 kDa). A schematic representation of its structure is given in Fig. 1 which highlights a number of its salient features. These include:
- An N-terminal p53-binding pocket (amino acids 25–110: see below for 3D structure and the development of inhibitors)
- Nuclear import (amino acids 161–163 and 179–185) and export (amino acids 191–202) sequences that permit nucleocytoplasmic shuttling
- An unstructured central acidic domain (amino acids 215–300)
- A zinc finger domain (amino acids 290–335) that mediates interaction with the inhibitory partners, ribosomal proteins L5, L11, and S7
- A C-terminal regulatory region (amino acids 386–429) that is targeted for phosphorylation through DNA damage pathways (see below)
- A RING finger domain (amino acids 436–491) that participates in the transfer of ubiquitin from E2 ligases (e.g., UBCH5) to p53
Three of the structured regions of MDM2 have been resolved: the N-terminal p53-binding pocket (Fig. 2), the zinc finger, and the RING finger domain. The coordinates for these structures are available from the NCBI web site.
MDM4 (also known as MDMX)
The MDM4 protein is structurally related to MDM2. It is a defective ubiquitin ligase and is therefore unable to mediate transfer of ubiquitin to p53. However, it is thought to interact with p53 via its N-terminal p53-binding pocket (which closely resembles that of MDM2) leading to an inhibition of p53-mediated transcription. Importantly, MDM2 is, itself, a relatively weak E3 ligase for p53 but is stimulated by MDM4 through hetero-oligomerization mediated by their respective RING fingers. This hetero-oligomerization also stabilizes MDM2 by blocking its auto-ubiquitylation. The level of MDM4 is not directly controlled by p53-dependent gene expression (as is the case for MDM2: see below).
However, it is regulated by MDM2 via ubiquitylation and degradation. Like MDM2, MDM4 is an essential regulator of p53, and its involvement in controlling p53 activity is strongly supported from experiments with genetically altered mice.
The N-terminal p53-binding pocket
This region of MDM2 (amino acids 25–110) provides a hydrophobic cleft that forms a tight complex with the N-terminus of p53 by accommodating p53 amino acids F19, W23, and L26, respectively (see Fig. 2). Interaction of p53 within this domain of MDM2 leads to ensuing contacts between the two molecules that permit subsequent ubiquitylation of p53 (discussed in detail below).
It was previously thought that the binding of p53 to this domain of MDM2 could sterically block to the interaction of p53 with key transcription factors. However, studies with mouse models that express a mutant Mdm2 protein (C462A) that can still bind through its N-terminus to p53, but cannot mediate ubiquitylation, have challenged this idea. More recent research has found that the C462A Mdm2 mutant can actually stimulate p53-dependent transcription but the mechanism for this has not yet been established.
The N-terminal p53-binding domain of MDM2 is also the target of novel drugs aimed at disrupting the p53/MDM2 interaction (see below).
There are several published structures of the N-terminal p53-binding domain of MDM2 (e.g., see protein database ID: 1YCR).
Nuclear import and export sequences
MDM2 contains two nuclear import sequences and one nuclear export sequence that mediate the ability of MDM2 to shuttle between the nucleus and the cytoplasm. The nuclear export function is essential for the ability of MDM2 to downregulate p53 levels, and its discovery led to a model whereby MDM2 binds to p53 in the nucleus and mediates its export to the cytoplasm where it is degraded.
The acidic domain
The acidic domain is a largely unstructured region in the central part of the protein (amino acids 215–300) and is thought to play a number of key functions. Firstly, several groups identified the acidic domain as mediating a second point of contact with p53 that is required for the ubiquitylation and degradation of p53 (the so-called p53 ubiquitylation signal). In this model, the binding of the N-terminus of p53 to the cleft in the N-terminal domain of MDM2 (the principal p53-binding site) leads to subsequent association of the acidic domain with the p53 core (site-specific DNA-binding domain). This, in turn, permits ubiquitylation of p53 as mediated by the MDM2 RING finger. It was recently shown that two segments of the acidic domain are required to mediate p53 ubiquitylation and destruction: Thus, amino acids 247–258 are required for ubiquitylation of p53, while these amino acids, together with amino acids 270–274, are required to mediate p53 destruction. These new data support previous findings that the acidic domain comprises distinct but possibly overlapping biochemical functions that are required for p53 turnover. However, it has not yet been demonstrated whether or how these regions within the acidic domain contact p53 and mediate their overlapping effects.
The acidic domain is also the interaction site for numerous regulators of MDM2 (see below) including the important ARF (alternative reading frame) tumor suppressor that inhibits MDM2 in response to hyperproliferative signaling (see below). Importantly, it is a focal point for most (if not all) signals that promote the induction of p53.
The zinc finger
The zinc finger domain plays an important role in regulating MDM2 function specifically in response to changes in ribosome integrity and/or availability. Upon reduction in ribosome biogenesis (e.g., through reduced rRNA expression), excess ribosomal proteins L5 and/or L11 bind directly to the MDM2 zinc finger, inhibit MDM2 function, and thereby induce a p53 response. In this manner, MDM2 regulation is linked to cellular growth (via ribosome number) and is sensitive to “ribosomal stress.” The physiological role of the zinc finger was recently underscored through the development and analysis of Mdm2C305F/C305F homozygous mutant mice. The C305F mutation within the zinc finger had previously been shown to prevent ribosomal protein binding. Under normal conditions, Mdm2C305F/C305F mice show no obvious phenotypical or developmental differences from wild-type mice nor do they show any altered susceptibility to the development of spontaneous tumors. Fibroblasts from these mice show a normal DNA damage response to various genotoxic agents, and the mice themselves show levels of p53 target activation and apoptosis in response to whole body irradiation that are indistinguishable from wild-type mice. However, agents that induce ribosomal stress (such as low levels of actinomycin D) fail to induce a robust p53 response in these mice. L5/L11 binding therefore constitutes an MDM2-targeted p53 activation pathway that is essentially independent of the DNA damage and ARF pathways. Notably, L11 is a
transcriptional target of the Myc oncoprotein. Mice expressing Myc under control of the immunoglobulin enhancer (E-mu-Myc mice) develop aggressive B-cell lymphomas. When crossed with Mdm2C305F/C305F mice lymphoma, development and death are accelerated. The zinc finger therefore offers crucial protection against Myc-induced lymphomagenesis in vivo. It does not, however, accelerate prostatic tumorigenesis induced by inactivation of Rb family members and therefore has a selective role in its responsiveness to tumor-associated stimuli.
The structure of the zinc finger has been solved (protein database ID: 2C6B).
The RING finger
The RING finger of MDM2 contains a Cys3-His2-Cys3 consensus which coordinates two ions of zinc leading to proper folding of the domain. It is required to mediate the transfer of ubiquitin to p53 from ubiquitin E2 ligases; to mediate the binding of MDM2 to its homologue, MDM4; and to bind to specific RNA sequences. A cysteine to alanine substitution at residue 464 completely abolishes the ability of MDM2 to ubiquitylate p53 in vitro and in transfected cultured cells. The RING finger is also required to
mediate p53 degradation. Consistent with this idea, a mouse model expressing Mdm2C462A/C464A fails to suppress p53. Additionally, the RING finger is required for the export of p53 to the cytoplasm.
Autoregulatory feedback loop
MDM2 is regulated at several levels. Firstly, MDM2 expression is controlled by p53 as part of a negative feedback loop in which p53 stimulates expression from one of two MDM2 promoters, the “P2” promoter that lies upstream of MDM2 exon 2. The subsequent increased level of MDM2 protein stimulates the ubiquitylation and degradation of its activator, p53, thus maintaining p53 protein levels. In addition to promoting the maintenance of p53 levels homeostatically (there are several other p53 ubiquitin ligases that contribute to the maintenance of its levels), increased MDM2 expression following induction of p53 is thought to be a major player in resetting p53 to preinduction levels. The MDM2 gene has an alternative promoter, “P1,” that is upstream of exon 1. P1 is constitutively active, is independent of p53, and can maintain a low level of MDM2 expression in the absence of p53.
MDM2 protein levels are also regulated by auto-ubiquitylation (i.e., where one molecule of MDM2 ubiquitylates another). There is also evidence thatMDM2 ubiquitylation and turnover can be mediated by additional ubiquitin ligases such as PCAF (p300/CBP-associated factor).
Regulation by protein-protein interactions
MDM2 participates in a wide variety of protein-protein interactions, many of which regulate its function. A list of these regulators is given in Table 1 (interacting proteins for which the binding sites in MDM2 have been mapped are also shown in Fig. 1). Key examples of MDM2-regulating proteins are:
- MDM4: a critical activator of MDM2 (discussed above)
- The ARF (alternative reading frame) tumor suppressor: This protein is encoded, mainly within an alternative reading frame, within the CDKN2A gene which also encodes the p16INK4A tumor suppressor. ARF expression is induced by a variety of hyperproliferative stimuli including activated oncogene products. ARF binds to MDM2 within the central acidic domain leading to inhibition of MDM2 ligase activity and, consequently, p53 ubiquitylation. ARF is also responsible for localizing/anchoring MDM2 into the nucleolus (i.e., into a compartment where it is physically separated from p53).
- Ribosomal proteins L5, L11, L23, and S7: Reduced growth rate or ribosomal stress leads to increased levels of these proteins which then interact with the acidic domain and/or zinc finger of MDM2, with the outcome that they block MDM2-mediated ubiquitylation and degradation of p53.
Regulation by posttranslational modification
In addition to ubiquitylation, MDM2 is subject to other regulatory posttranslational modifications including SUMOylation mediated by UBC9 and acetylation of K466 and K467 within the RING domain by CREB-binding protein (CBP). Importantly, MDM2 is regulated by multisite phosphorylation mediated by several independent but cooperating pathways. The protein kinases within these pathways that phosphorylate MDM2, together with the target sites and the biochemical/biological outcomes of the phosphorylation events, are summarized in Fig. 3. The key features of MDM2 regulation by phosphorylation are as follows:
DNA damage signaling
In response to DNA damage, MDM2 is phosphorylated by the DNA damage-activated protein kinase, ATM, at several C-terminal sites (mainly S395) and by the related kinase, ATR, at S407 (Fig. 3), leading to a transient attenuation of p53 turnover. Mechanistically, these phosphorylation events inhibit RING oligomerization and E3 ligase activity leading to decreased p53 nuclear export and attenuated degradation.
Consistent with such a role, knock-in mice expressing an S394A substitution (S394 is the orthologous human S395 residue in mouse Mdm2), which cannot be phosphorylated, are radioresistant and fail to induce p53. In contrast, mice bearing an S394D substitution (where the presence of a negative charge can mimic constitutive phosphorylation), show a more robust induction of p53 in response to DNA damage, consistent with the idea that S394 (mouse) phosphorylation contributes significantly to p53 induction.
Expression of the WIP1 protein phosphatase (wild-type p53-induced phosphatase, also known as PPM1D) is also induced by p53 in response to DNA damage. WIP1 acts on both p53 itself and on MDM2 where it dephosphorylates Ser395, thereby reversing the effects of ATM/ATR. WIP1 induction is thought to be a major part of the mechanism that attenuates the p53 response and restores homeostasis of the p53/MDM2 feedback loop.
Activation of the DNA damage pathway/pathways also leads to hypophosphorylation of several residues within the acidic domain that are essential for MDM2-mediated degradation of p53. Evidence suggests that under normal homeostatic conditions, these modifications are promoted through the cooperative involvement of protein kinases GSK3-beta, CK1, and CK2. Following DNA damage, stimulation of the DNA-activated protein kinase leads to phosphorylation and activation of protein kinase AKT which, in turn, phosphorylates and inactivates GSK3-beta. These events are thought to underpin the hypophosphorylation of the acidic domain leading to a reduced capacity to degrade p53. It is also possible that DNA damage-induced changes in these modifications may cooperate with those promoted by ATM/ATR.
MDM2 is a target of survival signaling stimuli mediated through the PI3-kinase/AKT pathway. AKT phosphorylates MDM2 at residues flanking its nuclear localization and export sequences (serines 166, 186, 188: see Fig. 2) leading to increased nuclear localization ofMDM2 and, consequently, increased association with and degradation of p53. In this manner, survival signaling, or indeed cancer-associated constitutive activation of AKT signaling, is able to increase the threshold for p53 induction and activation.
Reciprocally, it is significantly easier to induce p53-mediated apoptosis following reduced survival signaling. The PIM family of oncogenic protein kinases, which are activated physiologically through increased gene expression in response to diverse signals, can also phosphorylate MDM2 at serines 166 and 186 (as for AKT, oncogenic activation of PIM may also lead to hyperactivation of MDM2). Additionally, these residues have been shown to be targeted by mTOR-activated S6K1 in response to DNA damage signals. Serines 166 and 186 can therefore act as a convergent signaling node in MDM2 that mediates, and possibly integrates, diverse signals that set a threshold for p53 induction and activity. From the perspective of potential disease susceptibility, there is also new evidence from mouse studies showing that glucocorticoids elevated during chronic restraint lead to increased phosphorylation of MDM2 at these residues via induction of serum- and glucocorticoid-induced protein kinase (SGK1). The increased MDM2 activity suppresses p53 and contributes to increased tumorigenesis in the mice. This may offer a potential explanation for the epidemiological observation that chronic psychological stress is associated with increased cancer susceptibility.
Interaction with other proteins: substrates and regulators
In addition to p53, MDM2 has been shown to interact with a host of other proteins. Many of these proteins are substrates for MDM2-mediated ubiquitylation and/or proteasomal targeting and degradation (see Table 2 and Fig. 1). (Others, as mentioned above and listed in Table 1, are key regulators of MDM2 or can act as cooperating partners.) As a consequence of some of the interactions summarized in Table 2, MDM2 is thought to encompass a number of p53-independent functions in a variety of processes including differentiation, transcriptional regulation, DNA repair, the maintenance of genome stability, and cell cycle control.
MDM2 in cancer
MDM2 is abnormally upregulated in a wide range of human cancers through mechanisms including mainly gene amplification but also increased transcription, mRNA stability, and enhanced translation. Elevated MDM2 levels are thought to contribute to cancer development by suppressing p53 levels, thereby increasing the threshold for inducing p53. However, a proportion of malignancies have both overexpressed MDM2 and mutant p53, suggesting that one or more p53-independent function/functions of MDM2 may contribute to cancer development. Indeed, patients with both MDM2 overexpression and p53 mutation often have a worse prognosis than those with either abnormality alone. This is also reflected in animal models for cancer development: For example, a high proportion of mice expressing E-mu-Myc develop lymphomas in which Mdm2 is overexpressed concomitantly with loss or mutation of p53. Moreover, mice overexpressing Mdm2 in a p53-null background have a higher incidence of certain cancers as compared with p53-null mice. There is also evidence that overexpression of Mdm2 in mouse mammary epithelia of mammary gland (in both p53+/+ and _/_ mice) leads to polyploidy.
In addition to overexpression, mutations within theMDM2 gene have been observed in several types of human cancers. While the significance of these mutations is not fully understood, many occur within the region encoding the zinc finger. These mutant genes give rise to proteins that are impaired in their ability to interact with ribosomal proteins L5 and L11 and cannot, therefore, mediate the induction of p53 in response to ribosomal stress. These data fit with the idea that signaling through the ribosomal protein/MDM2pathway may play a role in p53 activation during the development of at least some types of cancer.
Genetic factors that affect the level of MDM2 expression in different individuals can also affect the susceptibility to cancer development. For example, the small nucleotide polymorphism, SNP309, represents a T to G substitution in the intronic promoter/enhancer region of MDM2, which generates a potent binding site for the transcription factor, Sp1, leading to increased MDM2 transcription. Appropriate cell lines harboring the G allele of SNP309 show increased levels of MDM2 resulting from increased transcription and an elevated threshold for p53 induction and apoptosis in response to chemotherapeutics. Multiple cancer studies have confirmed that individuals harboring the G allele of SNP309 show increased risk, early onset, a relative lack of response to therapy, reduced survival, and poorer outcome as compared with individuals harboring the T allele.
Additionally, there is a gender influence on susceptibility to various cancers arising from the G allele based on the observation that active estrogen signaling promotesMDM2 expression through the increased Sp1 binding at SNP309. Stratification analysis of cohorts of patients with various tumor types supports the idea that the G allele of SNP309 can accelerate tumorigenesis specifically in individuals with active gender-specific hormone pathways such as estrogen signaling.
The principle for developing drugs aimed at inactivating MDM2 is that, by doing so, it should be possible to induce or reactivate p53 in cancers that retain wild-type p53 but have lost the ability (e.g., through MDM2 overexpression or loss of activators such as ARF) to induce a robust p53 response. It is possible that such drugs may, in themselves, be sufficient to induce p53 or, alternatively, may work in concert with other anticancer approaches. Similarly, induction of p53 using MDM2-targeted inhibitors may be of significant value in the development of cyclotherapy-based approaches, i.e., where the activation of p53 in normal tissues causes a reversible cell cycle arrest that can protect normal cells (and thereby minimize side effects) from the action of other anticancer drugs such as antimitotics used to combat p53-mutant cancers. MDM2-targeted drugs may also have additional value independently of p53 status given the increasing range of p53-independent functions associated with MDM2 (discussed above).
The best characterized small molecule inhibitor of MDM2 is Nutlin-3a, a compound that was developed based on the structure of the MDM2-interacting region in the N-terminus of p53. Specifically, Nutlin-3a mimics the presence and spatial location of the three p53 residues (F19, W23, and L26) that fit within the p53-binding pocket of MDM2. It thus acts as a competitive inhibitor of p53 binding.
Stapled peptides have also been developed as inhibitors of the p53/MDM2 interaction. Peptide stapling uses hydrocarbon cross-linkers to stabilize peptide structures with the effect of increasing targeting potency, inhibiting protease susceptibility, and, importantly, providing cell permeability. A key advantage of these reagents over small molecules is that they can be fine-tuned for greater specificity.
There are several other MDM2-targeted molecules that block MDM2/p53 association including benzodiazepine, MI-219, WK298, AM-8553, and RG7112. The compound RITA can also block interaction with MDM2 but does so by binding to p53. There are also novel inhibitors available that act upon dimerization of MDM2 and/or MDM4.
A number of MDM2-targeted compounds are currently undergoing clinical trials.
Fig. 1. MDM2 interacting partners. MDM2 is shown schematically, highlighting the various important functional domains.
The interacting partners shown are those for which the binding site/sites in MDM2 has/have been mapped (in each case represented by the bar/bars)
Fig. 2. X-ray crystal structure of the MDM2 N-terminus in association with an N-terminal peptide of p53. MDM2 is shown in blue/gray. The p53 peptide is shown in salmon with the three important interacting amino acids shown in red. Two separate views are shown. The images were generated using PyMOL from the coordinates in protein database (ID: 1YCR)
Fig. 3. Regulation of MDM2 by multisite phosphorylation. MDM2 is shown schematically, as in Fig. 1, together with the approximate positions of the groups of known sites of phosphorylation. The table shows the specific residues phosphorylated within each cluster (black type), the relevant protein kinase/kinases where known (blue type), the biochemical function regulated by the modification/modifications (red type), and the relevant protein phosphatase/phosphatases where known (green type)
Table 1. Brief summary of protein regulators of MDM2
|ARF||Inhibits E3 ligase function, sequesters MDM2 into nucleolus|
|CtBP2||Cooperates with MDM2 to repress p53-mediated transcription|
|FKBP25||Stimulates auto-ubiquitylation and proteasomal degradation of MDM2|
|Gankyrin||Required for inhibition of MDM2-mediated p53 ubiquitylation by Rb|
|Lats2||Inhibits E3 ligase activity leading to activation of p53|
|Merlin||Promotes MDM2 degradation|
|MTBP||Promotes stabilization of MDM2 and ubiquitylation and degradation of p53|
|Nucleophosmin||Blocks p53/MDM2 interaction|
|Nucleostemin (GNL3)||Inhibition of MDM2|
|p19Ras||Blocks association of MDM2 with p73-beta|
|P300||Required for p53 degradation|
|p53||mRNA Binds to RING finger and impairs E3 ligase activity. It also leads to increased p53-mRNA translation|
|PCAF||Ubiquitylates MDM2 leading to increased turnover|
|PML||Sequesters MDM2 into nucleolus|
|PSME3||Promotes ubiquitylation- and MDM2-dependent proteasomal degradation of p53|
|RASSF1A||Disrupts interactions between MDM2, DAXX, and the deubiquitinase, HAUSP, thereby enhancing the self-ubiquitin ligase activity of MDM2|
|Ribosomal protein L5||Inhibits MDM2-mediated ubiquitylation and degradation of p53|
|Ribosomal protein L11||Binds to zinc finger and inhibits MDM2. Cooperates with Myc to induce p53 and suppress lymphomagenesis|
|Ribosomal protein L23||Inhibits MDM2-induced p53 polyubiquitination and degradation|
|Ribosomal protein S7||Inhibits MDM2 E3 ligase activity, leading to stabilization of MDM2 and p53|
|Ribosomal protein S14||Inhibits E3 ubiquitin ligase activity|
|RYBP||Decreases MDM2-mediated p53 ubiquitylation, leading to stabilization of p53|
|TAF1 (TAFII250)||Downregulates MDM2 auto-ubiquitylation leading to increased ubiquitylation and degradation of p53|
|Yin Yang 1 (YY1)||Facilitates MDM2/p53 interaction and p53 ubiquitylation/degradation|
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