MDM2 promotes ubiquitination and degradation of MDMX

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Mitogenic signals activate p53 by induction of ARF expression, which inhibits p53 ubiquitination by MDM2. Recent studies showed that the MDM2 homolog MDMX is also an important regulator of p53. We present evidence that MDM2 promotes MDMX ubiquitination and degradation by the proteasomes. This effect is stimulated by ARF and correlates with the ability of ARF to bind MDM2. Promotion of MDM2-mediated MDMX ubiquitination requires the N-terminal domain of ARF, which normally inhibits MDM2 ubiquitination of p53. An intact RING domain of MDM2 is also required, both to interact with MDMX and to provide E3 ligase function. Increase of MDM2 and ARF levels by DNA damage, recombinant ARF adenovirus infection, or inducible MDM2 expression leads to proteasome-mediated down-regulation of MDMX levels. Therefore, MDMX and MDM2 are coordinately regulated by stress signals. The ARF tumor suppressor differentially regulates the ability of MDM2 to promote p53 and MDMX ubiquitination and activates p53 by targeting both members of the MDM2 family.     

p53蛋白泛素化修饰的研究进展

p53具有抑制肿瘤细胞增殖的作用,但是细胞内p53蛋白的堆积反而加速细胞衰老或凋亡,因此对p53进行严格的调控显得格外重要.泛素化、磷酸化和乙酰化是p53蛋白最主要的几种修饰形式,但近来研究表明泛素化对p53调控发挥着中心作用.MDM2是主要的负调节因子,其具有泛素连接酶的活性,早先的研究认为MDM2的作用主要是特异性结合p53并介导其在蛋白酶作用下降解,但近来的研究发现MDM2还可以介导p53的核-浆交换,这种现象在DNA损伤时尤为明显.推测MDM2介导p53的泛素化在体内可能发挥着多种调控功能.     

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.     

Blockage of MDM2-mediated p53 ubiquitination by yuanhuacine restrains the carcinogenesis of prostate carcinoma cells by suppressing LncRNA LINC00665

Prostate cancer (PCa) is a challenging issue for men's health worldwide due to its uncontrolled proliferation and high metastatic potential. Increasing evidence has supported plant extracts and natural plant derivatives as promising antitumor therapy with less toxic side effects. Yuanhuacine is an active component isolated from Daphne genkwa and can effectively suppress the tumorigenesis of several cancers. However, its role in PCa remains unclear. In this study, yuanhuacine dose-dependently inhibited the proliferation and induced apoptosis of PCa cells. Moreover, yuanhuacine also restrained the invasion and migration of PCa cells. Mechanically, yuanhuacine decreased the ubiquitination and degradation of p53 protein, and ultimately increased p53 levels, which was regulated by inhibiting the phosphorylation and total protein levels of mouse double minute 2 (MDM2). Moreover, elevation of MDM2 reversed the suppressive efficacy of yuanhuacine in PCa cell viability, invasion, and migration. The network pharmacologic and bioinformatics analysis confirmed that MDM2 might be a common target of D. genkwa and LINC00665. Furthermore, yuanhuacine inhibited LINC00665 expression. Upregulation of LINC00665 reversed yuanhuacine-mediated inhibition in MDM2 protein expression and suppressed p53 levels by enhancing its ubiquitination in yuanhuacine-treated cells. Importantly, the inhibitory effects of yuanhuacine on cell viability and metastatic potential were offset after LINC00665 elevation. Together, the current findings highlight that yuanhuacine may possess tumor-suppressive efficacy by inhibiting LINC00665-mediated MDM2/p53 ubiquitination signaling. Therefore, this study indicates that yuanhuacine may be a promising candidate for the treatment of PCa.     

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.     

The MDM2 RING-finger domain is required to promote p53 nuclear export

MDM2 can bind to p53 and promote its ubiquitination and subsequent degradation by the proteasome. Current models propose that nuclear export of p53 is required for MDM2-mediated degradation, although the function of MDM2 in p53 nuclear export has not been clarified. Here we show that MDM2 can promote the nuclear export of p53 in transiently transfected cells. This activity requires the nuclear-export signal (NES) of p53, but not the NES of MDM2. A mutation within the MDM2 RING-finger domain that inhibits p53 ubiquitination also inhibits the ability of MDM2 to promote p53 nuclear export. Finally, inhibition of nuclear export stabilizes wild-type p53 and leads to accumulation of ubiquitinated p53 in the nucleus. Our results indicate that MDM2-mediated ubiquitination, or other activities associated with the RING-finger domain, can stimulate the export of p53 to the cytoplasm through the activity of the p53 NES.     

Critical contribution of the MDM2 acidic domain to p53 ubiquitination

MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Recent studies have shown, however, that the ring-finger domain (RFD) of MDM2, where the ubiquitin E3 ligase activity resides, is necessary but not sufficient for p53 ubiquitination, suggesting that an additional activity of MDM2 might be required. To test this possibility, we generated a series of MDM2/MDMX chimeric proteins to assess the contribution of each domain of MDM2 to the ubiquitination process. MDMX is a close structural homolog of MDM2 that nevertheless lacks the E3 ligase activity in vivo. We demonstrate here that MDMX gains self-ubiquitination activity and becomes extremely unstable upon introduction of the MDM2 RFD, indicating that the RFD is essential for self-ubiquitination. This MDMX chimeric protein, however, is unable to ubiquitinate p53 in vivo despite its E3 ligase activity and binding to p53, separating the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Significantly, fusion of the central acidic domain (AD) of MDM2 to the MDMX chimeric protein renders the protein fully capable of ubiquitinating p53, and p53 ubiquitination is associated with p53 degradation and nuclear export. Moreover, the AD mini protein expressed in trans can functionally rescue the AD-lacking MDM2 mutant, further supporting a critical role for the AD in MDM2-mediated p53 ubiquitination.     

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.     

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.     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.     

C-Terminal Ubiquitination of p53 Contributes to Nuclear Export

The growth inhibitory functions of p53 are controlled in unstressed cells by rapid degradation of the p53 protein. One of the principal regulators of p53 stability is MDM2, a RING finger protein that functions as an E3 ligase to ubiquitinate p53. MDM2 promotes p53 nuclear export, and in this study, we show that ubiquitination of the C terminus of p53 by MDM2 contributes to the efficient export of p53 from the nucleus to the cytoplasm. In contrast, MDM2 did not promote nuclear export of the p53-related protein, p73. p53 nuclear export was enhanced by overexpression of the export receptor CRM1, although no significant relocalization of MDM2 was seen in response to CRM1. However, nuclear export driven by CRM1 overexpression did not result in the degradation of p53, and nuclear export was not essential for p53 degradation. These results indicate that MDM2 mediated ubiquitination of p53 contributes to both nuclear export and degradation of p53 but that these activities are not absolutely dependent on each other.