MDM2 (Murine Double Minute 2)
Structure and Functions
The RING-finger domain and the zinc-finger domain are both required for MDM2’s E3 ubiquitin ligase activity (Honda and Yasuda 2000). The RING-finger domain of MDM2 is also vital for its ability to interact with the RING-finger domain of MDM4. Through heterodimerization, MDM4 stabilizes MDM2 by preventing MDM2 ubiquitination and enhances MDM2 E3 ligase activity (Stad et al. 2001; Singh et al. 2007). Deficiency in MDM2-MDM4 heterodimerization leads to embryonic lethality in mice, but this phenotype can be rescued by codeletion of p53 or by hypomorphic p53 alleles (Itahana et al. 2007; Huang et al. 2011; Pant et al. 2011). These results indicate that MDM2-MDM4 heterodimers play a critical role in preventing lethal p53 activation during embryogenesis. Importantly, mouse genetic studies demonstrated that the MDM2-MDM4 heterodimer, but not MDM2 E3 ligase activity, is required to suppress p53 activity during embryogenesis (Itahana et al. 2007; Tollini et al. 2014).
MDM2 activity is tightly regulated by various mechanisms. First, MDM2 is regulated at the level of protein turnover; MDM2 has a short protein half-life (for review, see Li and Kurokawa 2015). Moreover, genotoxic stress further accelerates MDM2 protein degradation, which promotes p53 activation. The E3 ubiquitin ligases that facilitate MDM2 ubiquitination and degradation include the p300-CBP-associated factor (PCAF) (Linares et al. 2007), β-TrCP (Inuzuka et al. 2010), the anaphase-promoting complex/cyclosome (APC/C) (He et al. 2014), the F-box protein FBXO31 (Malonia et al. 2015), and MDM2 itself (Stommel and Wahl 2004). On the contrary, several deubiquitinating enzymes (deubiquitinases) have been found to stabilize the MDM2 protein by removing ubiquitin molecules on MDM2. These enzymes include USP2a (Stevenson et al. 2007), USP7 (also known as HAUSP) (Cummins et al. 2004), and USP15 (Zou et al. 2014). It appears that the stability of MDM2 protein is regulated by the net balance between the various E3 ubiquitin ligases and deubiquitinases. The molecular mechanism of how MDM2 stability is regulated in response to p53-activating stimuli remains to be fully elucidated.
MDM2 activity is also regulated by MDM2-binding proteins, such as ARF and ribosomal proteins. Expression of the tumor suppressor protein ARF is induced in response to oncogenic stimuli, such as activation of MYC or RAS. Once expressed, ARF promotes p53 activation by suppressing MDM2 (for review, see Sherr 2006). Through directly binding to MDM2, ARF inhibits the catalytic activity of MDM2 and sequesters MDM2 in the nucleolus (Honda and Yasuda 1999; Tao and Levine 1999b; Weber et al. 1999). Likewise, free forms of ribosomal proteins are released from the S40 and S60 ribosome subunits upon nucleolar stress, metabolic stress, and protein synthesis inhibition, all of which lead to ribosomal biogenesis perturbation (Zhang and Lu 2009). In the nucleus, ribosomal proteins bind to MDM2 and inhibit its function, resulting in p53 stabilization and activation. MDM2C305F mutant cannot bind to two ribosomal proteins, RPL5 and RPL11. Transgenic mice expressing this mutant Mdm2 exhibit impaired fatty acid oxidation regulated by p53 target genes and develop hepatosteatosis under fasting conditions, indicating that MDM2 regulation by ribosomal proteins plays a critical role in the metabolic regulation of p53 (Liu et al. 2014).
Another mechanism of MDM2 regulation is phosphorylation. MDM2 is a target of various kinases and can be phosphorylated at multiple sites, especially in response to genotoxic stress (Fig. 2) (for review, see Riley and Lozano 2012). For instance, DNA-activated Protein Kinase (DNA-PK) impairs the ability of MDM2 to bind p53 by phosphorylating the Ser17 residue near the p53-binding domain of MDM2 (Mayo et al. 1997; Shieh et al. 1997). Likewise, in response to DNA damage, Ataxia Telangiecstasia Mutated (ATM) kinase phosphorylates MDM2 at multiple Serine residues, including Ser395 (Ser394 in mice) in the RING-finger domain (Cheng et al. 2009). ATM phosphorylation impairs MDM2 dimerization, which significantly diminishes MDM2’s ability to suppress p53 (Cheng et al. 2011). In contrast, the phosphatase Wip1 dephosphorylates Ser395 and stabilizes MDM2 protein, indicating that ATM phosphorylation may also promote proteasomal degradation of MDM2 (Lu et al. 2007). Importantly, transgenic mice that express the Mdm2S394A mutant are highly resistant to p53 activation induced by DNA damage and are predisposed to spontaneous tumor development (Gannon et al. 2012). This clearly highlights the importance of Ser395 phosphorylation in MDM2 regulation. Other DNA damage-activated kinases, such as Ataxia Telangiectasia and Rad3 related (ATR) and Abelson murine leukemia viral oncogene homolog 1 (c-ABL), also phosphorylate MDM2 at Ser407 and Tyr394, respectively, suppressing its activity toward p53 (Goldberg et al. 2002; Shinozaki et al. 2003). The serine/threonine kinases Casein Kinase 1 and 2 (CK1 and CK2) also phosphorylate multiple sites in the acidic domain of MDM2. Of note, CK1 phosphorylation leads to MDM2 protein degradation by β-TrCP (Inuzuka et al. 2010).
In the absence of DNA damage, MDM2 is also regulated by multiple kinases. Glycogen synthase kinase 3 (GSK3) phosphorylates MDM2 at two serine residues in the acidic domain, which limits p53 activation (Blattner et al. 2002). The prosurvival kinase AKT often plays a key role in cancer development. An earlier study has shown that AKT phosphorylates MDM2 at Ser166 and Ser186, which promotes MDM2 nuclear localization (Zhou et al. 2001). Later, it was found that AKT phosphorylation also stabilizes MDM2 protein by inhibiting its self-ubiquitination (auto-ubiquitination) (Feng et al. 2004).
MDM2 and Cancers
The tumor suppressor p53 plays a critical role in the prevention of cancer. In 50% of cancers, p53 is genetically mutated or deleted. In cancers with two wild-type p53 alleles, however, the tumor suppressor function of p53 is inhibited through various other mechanisms (Moll and Petrenko 2003). In this regard, MDM2, a major inhibitor of p53, is often overexpressed or amplified in cancers. For example, MDM2 amplification is often seen in sarcomas, especially in well-differentiated liposarcomas (Dei Tos et al. 2000; Oliner et al. 1992). MDM2 gene amplification is also associated with gliomas (Watanabe et al. 1994) as well as lymphomas, particularly B-cell non-Hodgkin’s lymphoma and advanced B-cell chronic lymphocytic leukemia (Watanabe et al. 1996; Korkolopoulou et al. 1997). Moreover, transgenic mice that overexpress Mdm2 are prone to spontaneous tumorigenesis, particularly lymphoma and sarcoma (Jones et al. 1998).
MDM2 overexpression in cancer can also be ascribed to a “T-to-G” single nucleotide polymorphism (SNP) in the MDM2 gene promoter (Bond et al. 2004). As opposed to wild-type T/T alleles, the homozygous G/G alleles result in enhanced MDM2 expression and are frequently associated with early onset or progression of leukemogenesis (Phillips et al. 2010; Zhuo et al. 2012). Moreover, SNP309 (G/G) has also been implicated in poor therapeutic response as well as high risk of developing acute myeloid leukemia (Phillips et al. 2010; Post et al. 2010).
Given the critical role of MDM2 in p53 inhibition and the frequency of MDM2 overexpression in cancer cells, it is tempting to speculate that inhibition of MDM2 would lead to robust p53 activation and kill cancer cells. Towards this end, compounds that can inhibit MDM2 have been studied extensively.
The nature of the direct interaction between MDM2 and p53 is dependent upon a deep hydrophobic N-terminus domain, or “pocket,” on MDM2 that binds the N-terminal transactivation domain of p53. Several small molecules have been developed to effectively inhibit the interaction between MDM2 and p53 by displacing p53 from its respective hydrophobic binding pocket on MDM2 (for review, see Burgess et al. 2016). The most well-known of these inhibitors falls under the cis-imidazoline family of compounds, commonly referred to as Nutlin-3 (Vassilev et al. 2004). Importantly, animal models and cell culture studies show that this class of compounds effectively kills cancer cells with wild-type p53 but not normal healthy cells (Vassilev et al. 2004). Currently, some of the Nutlin-like compounds (e.g., RG7112) are being tested in clinical trials for their efficacy and toxicity (Burgess et al. 2016).
Besides disrupting MDM2-p53 interaction, compounds that directly inhibit E3 ligase activity of MDM2 have also been identified. A high-throughput screening discovered two small molecules, MEL23 and MEL24, to be inhibitors of the ligase activity of the MDM2-MDMX heterodimeric complex (Herman et al. 2011). Because the heterodimer is crucial in p53 degradation, MEL23 and MEL24 are shown to stabilize the expression of p53. Likewise, a screen for inhibitors of MDM2 self-ubiquitination discovered that 5-deazaflavin analogs bind to the MDM2 RING domain and suppress its E3 ligase activity (Roxburgh et al. 2012).
p53-Independent Roles of MDM2
Although much effort has been focused on determining the role of MDM2 in p53 regulation, accumulating evidence indicates that MDM2 has many p53-independent functions (for review, see Ganguli and Wasylyk 2003; Biderman et al. 2012). MDM2 regulates the stability and/or activity of several transcription factors, including E2F1, thereby controlling cell growth and apoptosis independently of p53. MDM2 also interacts with p73, a p53 homologue that can induce cell cycle arrest and apoptosis, and inhibits its transcriptional activity. Furthermore, MDM2 directly regulates mRNA stability and translation, including p53 mRNA. Recently, we have also shown that MDM2 can target another E3 ligase, HUWE1, for degradation, which in turn triggers the accumulation of HUWE1 substrates, such as the anti-apoptotic BCL2 family protein MCL-1 (Kurokawa et al. 2013).
The oncoprotein MDM2 was initially identified in mice as a gene product that can cause cellular transformation. Later, it was found that MDM2 is the major inhibitor of the p53 tumor suppressor. MDM2 suppresses the transcriptional activity of p53 via physical inhibition and also promotes the degradation of p53 protein. p53, in turn, induces MDM2 gene expression, thus establishing a negative feedback loop. In normal healthy cells, MDM2 activity is tightly regulated by inhibitory proteins (e.g., ARF and ribosomal proteins), phosphorylation, and rapid protein turnover. MDM2 overexpression or amplification is often associated with cancers, including sarcomas and lymphomas. Development of MDM2 inhibitors is underway to treat patients with wild-type p53 in clinical settings. Although the role of MDM2 as an inhibitor of p53 is most well established, MDM2 also has various p53-independent functions. The physiological implications of these p53-independent functions have yet to be fully elucidated but nonetheless, such functions suggest that MDM2 is a multifaceted and indispensable protein.
The authors acknowledge support from an NCI Career Development Award R00 CA140948 (to M.K.), American Cancer Society Institutional Research Grant IRG-82-003-30 (to M.K.), a Norris Cotton Cancer Center Prouty Grant (to M.K.), a Norris Cotton Cancer Center Leukemia/Lymphoma Fund (to M.K.), and Waterhouse Research Award (to Y.Y.C.).
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