MDM2 is an oncogene that is frequently over-expressed in various human cancers, including sarcomas, gliomas, melanomas, and breast cancers.¹ The primary function of MDM2 is to inhibit the activity of the p53 tumor suppressor protein. P53 inhibits cell proliferation in response to DNA damage and other stresses by activating the transcription of genes that mediate either cell-cycle arrest or apoptosis. MDM2 binds the transactivation domain of p53 and inhibits the ability of p53 to activate transcription. Importantly, MDM2 binding also promotes the ubiquitin-mediated degradation of p53, as well as the export of p53 from the nucleus to the cytoplasm. The MDM2 gene itself is transcriptionally activated by p53, thus forming an autoregulatory feedback loop in which p53 promotes the expression of its own inhibitory factor. The ability of MDM2 to promote the degradation and nuclear export of p53 depends on an intact RING finger domain located in the MDM2 C terminus. This chapter will discuss the mechanisms by which MDM2 inhibits p53 function, with an emphasis on the requirement of the MDM2 RING finger domain in p53 inhibition.

Introduction

The MDM2 gene was first discovered in 1987 as a gene that was amplified in spontaneously transformed murine 3T3 fibroblasts.² Subsequent studies revealed that the MDM2 protein interacts strongly with the tumor suppressor protein p53 and inhibits p53 activity.^3,4 It is now well-established that the primary function of MDM2 is to inhibit p53.^5,6 The importance of p53 inhibition by MDM2 is best illustrated through the attempted generation of mice that lack MDM2. These MDM2 “knockout” mice are not viable, dying at an early stage of embryonic development.^7,8 Importantly, however, the concomitant knockout of p53 allows survival of the mice. These results demonstrate that MDM2 normally inhibits p53 function, and that this inhibition is critically important during embryonic development.

Human MDM2 is a 491 amino acid protein that localizes mostly in the nucleus, though it can also localize in the cytoplasm.⁶ The MDM2 gene has been mapped to chromosome 12q13-14 and includes several exons that encode the MDM2 protein. Alternative splicing of the primary mRNA transcript can result in the generation of different MDM2 isoforms, some of which inhibit the activity of the wild-type protein in a dominant-negative fashion.^9,10 Analysis of the MDM2 coding sequence has revealed several structural and functional domains (fig. 1). These include the p53-binding domain located in the N terminus between residues 19-102, the p300-binding domain between residues 217-246, an acidic domain located between residues 222-272, a zinc finger domain located between residues 289-331, and a RING finger domain located in the MDM2 C terminus between residues 438-478. RING fingers are zinc binding domains with a defined octet of cysteine (C) and histidine (H) residues spaced at varying intervals in the coding sequence.^11-14 In the RING H2 subtype, the cysteine and histidines are arranged in the order C₃H₂C₃, while in the RING HC subtype the order is C₃HC₄. The MDM2 RING finger is of the C₃HC₄ subtype and, like other RING fingers, is predicted to bind two zinc atoms. RING finger domains are believed to function in the assembly and architecture of large protein complexes, including enzyme complexes of the ubiquitin system of protein degradation.^15,16 As we will see below, the MDM2 protein can function as an ubiquitin-system E3 ligase that promotes the specific degradation of p53, and this activity requires the MDM2 RING finger domain.

Figure 1

The MDM2 protein. Full-length human MDM2 (also called HDM2) is a 491 amino acid protein. Positions of the nuclear localization signal (NLS), nuclear export signal (NES), and nucleolar localization signal (NoLS) are indicated. Also indicated are the various (more...)

MDM2 Promotes p53 Degradation

In 1997, research groups led by Moshe Oren in Israel and Karen Vousden in Frederick, Maryland were investigating the binding interactions between MDM2 and p53.^17,18 These studies were complicated by the fact that p53 levels were consistently decreased with coexpression of MDM2 (see fig. 2) as an example), which made a quantitative analysis of the p53:MDM2 interaction difficult. The researchers decided to investigate the molecular basis for the decreased levels of p53. Small-molecule inhibitors of the proteasome prevented the decrease in p53 levels observed with MDM2 expression, suggesting that MDM2 was somehow promoting p53 degradation by the proteasome. Moreover, a high molecular weight ladder of p53 species was generated when p53 was coexpressed with MDM2 (see fig. 2) as an example). The size of these p53 species was consistent with the addition of multiple ubiquitin-moieties to p53, and analysis with anti-ubiquitin antibodies identified them as p53:ubiquitin conjugates. These seminal studies provided the first evidence that MDM2 can promote the degradation of p53 through the ubiquitin-proteasome pathway.^17,18

Figure 2

MDM2 promotes p53 ubiquitination and degradation. Saos-2 cells (p53 null) were transfected with DNAs encoding p53, wild-type MDM2, and a RING-finger mutant of MDM2 (C464A). P53 levels were then assessed by immunoblotting with an anti-p53 antibody. In (more...)

MDM2 Is an E3 Ubiquitin-Protein Ligase

The ubiquitin-proteasome system is the major pathway in the cell for selective protein degradation.^19,20 The covalent attachment of multiple ubiquitin molecules to lysine residues of a target protein serves to signal its recognition and rapid degradation by the 26S proteasome. Ubiquitination of a protein substrate requires the concerted action of three classes of enzymes designated E1, E2, and E3. The ubiquitin activating enzyme E1 initially activates ubiquitin in an ATP-dependent reaction through the formation of a thiol-ester bond between the carboxyl terminus of ubiquitin and the thiol group of a specific cysteine residue of E1. Ubiquitin is then transferred to a specific cysteine residue on one of several ubiquitin-conjugating enzymes (Ubcs or E2s). E2 enzymes in turn may transfer the ubiquitin either directly to a substrate or to the final class of enzymes known as ubiquitin protein ligases (or E3s). The E3 enzymes catalyze the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and the ε-amino group of lysine residues on the target protein. A substrate may then be multiply ubiquitinated through the attachment of additional ubiquitin molecules to specific lysine residues of ubiquitin itself. In many cases the E1, E2, and E3 enzymes involved form large, multi-protein complexes. This increases the efficiency of the process by allowing the rapid thiol-ester transfer of ubiquitin molecules between proteins.

In vitro studies with purified E1 enzyme, E2 enzyme, MDM2, and p53 showed that MDM2 could form a direct thiol-ester linkage with ubiquitin at Cys464 in the MDM2 RING finger domain, and could transfer the ubiquitin to p53.²¹ Removal of either the E1 or E2 enzymes prevented formation of the thiol-ester bond between ubiquitin and MDM2, and also prevented the transfer of ubiquitin to p53. Moreover, mutations that converted Cys464 to Ala464 (C464A) also inhibited the transfer of ubiquitin to p53. Taken together, these data support a model in which MDM2 functions as an E3 ubiquitin ligase that can accept activated ubiquitin from an E2 enzyme, and then transfer the ubiquitin to p53 (fig. 3).

Figure 3

MDM2 is an E3 ubiquitin-ligase for p53. Current models suggest that MDM2 accepts activated ubiquitin from an E2 enzyme and transfers the ubiquitin to multiple lysine residues in the p53 C terminus.

The nature of p53 ubiquitination by MDM2 has not been fully clarified. MDM2 can stimulate the formation of a “ladder” of p53:ubiquitin conjugates in cells transiently expressing both proteins (see fig. 2 as an example). This ladder could result from the addition of single ubiquitin molecules to multiple lysine residues in p53, or from the formation of poly-ubiquitin chains of varying lengths on one or only a few lysines. Mutational analysis of p53, as well as in vitro studies using ubiquitin-aldehyde, support the notion that MDM2 mono-ubiquitinates p53, transferring single ubiquitin moieties to multiple lysine residues located in the extreme C terminus of p53.^22,23 However, the fact that formation of poly-ubiquitin chains is necessary to target a protein to the proteasome²⁴ indicates that a so called “E4” enzyme must exist that can promote p53 poly-ubiquitination. Recent studies suggest p300 may be such a factor. P300 is a large (approx. 300 kilodalton) protein that can bind the central region of MDM2 (fig. 1), as well as two separate regions of p53. While purified MDM2 can promote p53 mono-ubiquitination in an in vitro system, addition of p300 stimulates the poly-ubiquitination of p53,²⁵ probably through the formation of a p53:MDM2:p300 trimeric complex. These and other studies indicate that p300 can participate with MDM2 to promote p53 degradation by promoting p53 poly-ubiquitination^25-27 (fig. 4 Left). However, it is important to note that p300 can also function as an activator of p53 by promoting p53 acetylation at one or more lysine residues in the p53 C terminus (Fig 4 Right). This acetylation increases the DNA binding activity of p53, thus leading to an activation of p53-responsive genes, and will be discussed in more detail below. Regulatory interactions between p53, MDM2, and p300 remain both interesting and complex.

Figure 4

Dual roles for p300 in regulating p53 levels and activity. Left MDM2 promotes the addition of single ubiquitin moieties to multiple lysine residues in p53. p300 is believed to act in conjunction with MDM2 to promote the poly-ubiquitination of p53, and (more...)

P53 Is Stabilized and Its Levels Increase in Response to Stress

P53 levels increase in response to multiple genotoxic and nongenotoxic stresses, including DNA damage, hypoxia, ribonucleotide depletion, and the abnormal activation of oncogene signaling pathways, among others.^28-30 The effect of increasing p53 is to inhibit cell proliferation through either cell cycle arrest or apoptosis (fig. 5). These effects are mediated by p53-responsive genes, such as p21 and bax. The importance of signaling either a cell cycle arrest or apoptosis in response to these stresses is abundantly clear. DNA damaging stresses have the potential to damage chromosomes, alter genomic integrity, and cause mutations that may activate oncogenes and/or inhibit tumor suppressors. By triggering a cell cycle arrest or apoptosis in response to DNA damaging stress, p53 prevents the propagation of cells that may have an altered genome or acquired potentially cancer-promoting mutations. Similarly, the mutational activation of oncogenes is an early step in the development of most or all human cancers. By triggering an arrest or apoptosis in response to the mutational activation of oncogenes, p53 prevents the growth of cells that would otherwise be prone to becoming cancerous. It is worth noting that many chemotherapeutic drugs used in cancer treatment in some way modify DNA, resulting in the generation of single or double-stranded breaks. In cancers that retain wild-type p53, these DNA breaks trigger an increase in p53 levels, and a resulting activation of p53-induced cell death pathways.^31,32 In contrast, the same thing does not occur in cancers that express mutant p53 or in which the wild-type p53 gene has been deleted.^31,32 This explains the increased sensitivity to chemotherapeutics observed in cancers with wild-type p53 compared with cancers that lack wild-type p53 expression.

Figure 5

p53 levels increase in response to diverse stresses, resulting in cell cycle arrest or apoptosis.

The increase in p53 levels following stress results from a stabilization of the p53 protein.^33,34 This is illustrated in (fig. 6.) RKO cells (human colon cancer cells that express wild-type p53) were untreated, or exposed to UV or ionizing (IR) radiation. Six hours after radiation exposure, the cells were treated with cyclohexamide (CHX) to inhibit de novo protein synthesis, and p53 steady state levels were monitored at various time points after CHX addition. The rate at which p53 levels decrease under these conditions is a measure of protein stability. In untreated cells, p53 levels decrease rapidly following CHX treatment, with a half-life (T_1/2) of approximately 30 min. In contrast, p53 levels are elevated in the UV and IR-treated cells and remain elevated for up to 2 hours following CHX treatment. The half-life (T_1/2) of p53 is increased following UV or IR-treatment to about 100 minutes or greater than 2 hours, respectively. These results demonstrate that the stress-induced increase in p53 levels is due, at least in part, to an increase in p53 protein stability.

Figure 6

p53 is stabilized in response to DNA damaging stress. RKO cells (wild-type p53) were untreated or exposed to either ionizing radiation (IR) or UV radiation. 6 hrs after treatment, cells were treated with cyclohexamide (CHX) for the indicated amount of (more...)

Stress Inhibits MDM2-dependent p53 Degradation

Given that MDM2 promotes p53 ubiquitination and degradation, it is perhaps not surprising that the stress-induced stabilization of p53 results from an inhibition of these MDM2 activities.³⁴ Agents that damage or modify DNA, such as IR and UV radiation, DNA cross-linkers, and chemotherapeutic drugs, activate a signal transduction cascade that culminates in the phosphorylation of p53 at multiple sites, including sites within or near the N-terminal MDM2-binding domain (fig.7).^28-30,35 Phosphorylation of Ser15 and Ser20 in p53 is critical for the stabilization and activation of p53 in response to DNA damage,^36-38 and kinases that can phosphorylate p53 at these sites have been identified. One of these kinases, referred to as the ataxia telangiectasia mutated (ATM) kinase, is activated in response to DNA strand breaks, such as those that might occur in response to IR, and can directly phosphorylate p53 at Ser15 and other residues ^28-30,35 (fig. 7). A second kinase that is related to ATM and referred to as the ATM and Rad3-related (ATR) kinase, is activated in response to stalled replication forks, such as might occur in response to UV radiation. ATR can also phosphorylate p53 at Ser15. P53 phosphorylation at Ser20 requires ATM or ATR, but is not phosphorylated by either of these kinases directly. Instead, ATM and ATR activate other kinases referred to as Chk1 (activated by ATR) and Chk2 (activated by ATM) that can directly phosphorylate p53 at Ser20.³⁹ Most current models propose that Ser20 phosphorylation stabilizes p53 by blocking the interaction between p53 and MDM2, whereas the phosphorylated Ser15 residue is believed to activate p53 transcriptional activity by recruiting p300 or CBP.^40,41 As mentioned earlier, p300 activates p53 promoting its acetylation at C-terminal lysine residues. This acetylation is believed to increase the DNA binding affinity of p53, thus leading to an activation of p53 responsive genes. It is important to note that ATM can also phosphorylate MDM2 (Fig. 7), and this phosphorylation has been reported to diminish MDM2-mediated degradation and nuclear export of p53.^42,43

Figure 7

Mechanism for p53 stabilization in response to DNA damage. DNA damage activates two related kinases, ATM and ATR. These kinases can phosphorylate p53 directly at serines-15 and -37. ATM has been reported to also phosphorylate MDM2. ATM activates the kinase (more...)

The stabilization of p53 in response aberrant oncogene signaling occurs through a pathway different from that which stabilizes p53 following DNA damage.^44,45 In this case, activated oncogenes signal an increase in the transcription of a tumor suppressor protein referred to as either p14 or Arf (referred to as p19/Arf in the mouse). P14 forms a direct complex with MDM2 and inhibits the MDM2-mediated ubiquitination of p53 (Fig. 8). Most current models suggest that p14 inactivates MDM2 by sequestering it in the nucleolus,^44-47 though there was some suggestion that p14 binding with MDM2 in the nucleoplasm is enough to inhibit MDM2 activity. Regardless, the effect of inhibiting MDM2 is to stabilize p53 and thus increase p53 levels. The tumor suppressor PML has also been implicated in the oncogene-mediated activation of p53. PML is the major component in multi-protein sub-nuclear complexes termed PML-nuclear bodies (PML-NBs).^48-49 PML can interact with several different proteins and recruit them to PML-NBs, including p53⁵⁰ and p300/CBP.⁵¹ Further, PML can stimulate p53 transcriptional activity in transiently transfected cells, and this requires recruitment of p53 to PML-NBs.⁵⁰ Studies from the laboratories of Pier Pandolfi at Memorial Sloan Kettering in New York and Scott Lowe at the Cold Spring Harbor Laboratories in New York have provided compelling evidence that the oncogene-mediated activation of p53 requires PML. In these studies, p53 activity was measured in PML^+/+ and PML^-/- mouse embryo fibroblasts (MEFs) infected with retroviruses expressing an activated ras oncogene (Ras V12). Ras V12 expression in PML^+/+ cells resulted in the activation of p53, recruitment of p53 into PML-NBs, and the induction of premature senescence. In contrast, p53 was neither activated nor recruited into PML-NBs in PML^-/- cells, and the cells were resistant to ras-induced senescence.^52,53 From these studies emerge a model in which p14/Arf stabilizes p53 in response to aberrant oncogene signaling by inhibiting the activity of MDM2, whereas the activation of p53 involves recruitment of p53 to PML-NBs, and its subsequent acetylation by p300/CBP^52-54 (fig. 8).

Figure 8

Oncogene-mediated activation of p53. Activated oncogenes stimulate expression of the tumor suppressor protein p14/Arf, which stabilizes p53 by sequestering MDM2 in the nuclelous. P53 may then be recruited to PML-nuclear bodies (PML-NBs) through its association. (more...)

MDM2 Promotes p53 Nuclear Export

In addition to promoting p53 degradation, MDM2 can also promote the export of p53 from the nucleus to the cytoplasm, and this is expected to further inhibit the transcriptional activity of p53. p53 contains two leucine-rich nuclear export signals (NESs), one located in the N-terminal MDM2-binding domain of p53, and the second located in the C terminus within the tetramerization domain.^55,56 Both of these NESs can support nuclear export when fused to a heterologous, nuclear protein. Disruption of the C-terminal NES causes p53 to display a more pronounced nuclear localization;⁵⁵ however, a similar finding has not been demonstrated for the N-terminal NES. Despite the identification of these NESs, the molecular mechanisms that control p53 nuclear export and the role of MDM2 in this process have remained a matter of debate. Initial studies found that endogenous wild-type p53 accumulated in the nucleus and was stabilized in cells exposed to leptomycin B (LMB), a specific inhibitor of nuclear export.^55,57 This suggested that nuclear export is required for p53 degradation. Further studies reported that MDM2 contains an NES within its sequence, and that blocking nuclear export could inhibit the ability of MDM2 to promote p53 degradation.^57-59 Based on these findings a model was proposed in which MDM2 transports p53 from the nucleus to the cytoplasm for p53 to be degraded, and this transport depends on a functional NES within MDM2. In conflict with this model were reports that p53 contains its own NES, and that this NES is sufficient to promote p53 nuclear export even in cells that lack MDM2 expression.⁵⁵ The NES identified in these studies was the C-terminal NES located within the p53 tetramerization domain, and structural analyses indicated that this NES would be inaccessible to the export machinery when p53 is a tetramer, but would be exposed and accessible when p53 is in either a dimeric or monomeric form. Taken together, these latter findings supported a second model in which p53 is exported from the nucleus via its own NES and independent of MDM2, and that this export may be regulated by changes in p53 tetramerization.

In an attempt to clarify the role of MDM2 in p53 nuclear export, cultured cells were transiently transfected with DNAs encoding p53 and MDM2, and localization of p53 and MDM2 was then monitored by immunofluorescence staining.^60,61 As shown in (Figs. 9 and 10), p53 displayed a predominantly nuclear localization when expressed alone, but was relocalized to the cytoplasm in cells coexpressing MDM2. These results are consistent with the hypothesis that MDM2 can promote p53 nuclear export. Importantly, MDM2 remained in the nucleus despite the fact that p53 was relocalized to the cytoplasm, suggesting that nuclear MDM2 can promote p53 nuclear export. Mutants of p53 and MDM2 deficient in their nuclear export signals (NES mutants) were also tested in these experiments. The MDM2 NES mutant promoted the nuclear export of p53 to an extent similar to wild-type MDM2 (fig.10), indicating that the MDM2 NES was not required for this effect. In contrast, the p53 NES- mutant was nuclear when expressed alone, and remained nuclear when coexpressed with wild-type MDM2 (figs. 9 and 10). These results indicate that the MDM2-mediated nuclear export of p53 requires the NES of p53, but not the NES of MDM2. Finally, to assess whether the RING-finger domain of MDM2 has a function in p53 nuclear export, p53 localization was monitored in cells cotransfected with wild-type p53 and the MDM2 C464A mutant. As mentioned earlier, the C464A mutation in the MDM2 RING-finger domain inhibits the ability of MDM2 to promote p53 ubiquitination. As shown in (Figs. 9 and 10), MDM2 C464A was unable to promote the nuclear export of p53, indicating that an intact MDM2 RING finger domain is required for p53 nuclear export. It is important to note that the C464A mutation did not affect the ability of MDM2 to bind p53.^60,61 Taken together, these results support the current model for p53 nuclear export (Fig. 11). According to this model, MDM2 can bind p53 and promote its ubiqutination in the nucleus, and this leads to the export of p53 from the nucleus to the cytoplasm. This export requires the p53 NES located in p53s C terminus. Based on the structural studies mentioned earlier, it is proposed that the addition of ubiquitin exposes the C-terminal NES, probably by causing dissolution of p53 tetramers into p53 monomers or dimers. This allows recognition of the C-terminal NES by the export factor CRM1. This model was further supported by reports demonstrating that mutations in specific ubiquitination sites in p53's C terminus can inhibit the nuclear export of p53 that is mediated by MDM2.^62,63

Figure 9

MDM2 promotes p53 nuclear export. Cells were transfected with DNAs encoding the indicated forms of p53 and MDM2. Localization of p53 and MDM2 was then monitored by immunofluorescence staining. Representative pictures are shown. Wild-type p53 localizes (more...)

Figure 10

Quantification of p53 localization in transfected cells. The localization pattern for p53 in cells expressing the indicated p53 and MDM2 DNAs was scored for 150 cells in 3 separate experiments. Graph shows the percentage of cells with the indicated p53 (more...)

Figure 11

A model for MDM2-dependent nuclear export of p53. According to this model, MDM2 binds p53 in the nucleus and promotes p53 ubiquitination. Addition of ubiquitin moieties to the p53 C terminus are predicted to cause dissolution of p53 tetramers into either (more...)

Several studies have supported the notion that nuclear export is an important component of p53 regulation. For example, various tumor types have been described in which p53 is functionally inactivated due to its abnormal sequestration in the cytoplasm, including inflammatory breast carcinoma, undifferentiated neuroblastoma, colorectal carcinoma, and retinoblastoma.^64-68 Sequestration of p53 in the cytoplasm is believed to contribute to the generation of these cancers by inhibiting p53 activity. In some cases, treatments that either block nuclear export (exposure to the nuclear export inhibitor leptomycin B), or inhibit endogenous MDM2 expression, result in the redistribution of p53 into the nucleus.^55,69 These findings suggest that the cytoplasmic localization of p53 in these cancers results, at least in part, from excessive nuclear export that is mediated by MDM2.

Interactions Between MDM2 and Other Proteins

Notwithstanding its effects on p53, there is abundant evidence that MDM2 has p53-independent activities that may contribute to its oncogenic properties. This evidence includes the following: First, rare cancers bearing both p53 mutations and MDM2 gene amplifications have been described and are more aggressive than those with alterations of either gene alone.⁷⁰ This suggests that MDM2 over-expression may accelerate tumor growth even in the absence of functional p53. Second, MDM2 over-expression has been reported to transform cells in culture in the absence of a functioning p53.⁷¹ Finally, targeted MDM2 over-expression in the mammary glands of mice leads to abnormal cell proliferation and mammary hypertrophy to a similar extent in both a p53 ^+/+ and p53 ^-/- background.⁷² Accordingly, considerable effort has been aimed at identifying cell-cycle regulatory proteins other than p53 with which MDM2 can interact. A number of such factors have been identified, including the p53-related protein p73, the MDM2-related protein MDMX, the tumor suppressor proteins pRB and PML, the ribosomal protein L5, the transcription factor E2F1, and the neuronal differentiation factor Numb, among others⁷³ (fig. 12). In most cases, the effect of these interactions on cell growth has not been completed clarified. In the paragraphs below, we briefly discuss the interactions of MDM2 with these other proteins.

Figure 12

MDM2 interacts with multiple different proteins other than p53. The region of MDM2 involved in binding the indicated protein is shown.

Conclusions

MDM2 is an oncoprotein that is frequently overexpressed in various human cancers. The primary function of MDM2 is to inhibit the tumor suppressor protein p53. MDM2 promotes the ubiquitination of p53 and its degradation by the proteasome, as well as the export of p53 from the nucleus to the cytoplasm. Both of these activities require the RING finger domain located in the MDM2 C terminus. DNA damage and other stresses stabilize p53 through an inhibition of MDM2-mediated degradation. MDM2 interacts with a variety of other proteins in addition to p53, and these interactions may contribute to the oncogenic potential of MDM2.

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