Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms

The p53 protein maintains genomic integrity through its ability to induce cell cycle arrest or apoptosis in response to various forms of stress. Substantial regulation of p53 activity occurs at the level of protein stability, largely determined by the activity of the Mdm2 protein. Mdm2 targets both p53 and itself for ubiquitylation and subsequent proteasomal degradation by acting as an ubiquitin ligase, a function that needs an intact Mdm2 RING finger. For efficient degradation of p53 nuclear export appears to be required. The Mdmx protein, structurally homologous to Mdm2, does not target p53 for degradation, but even stabilizes both p53 and Mdm2, an activity most likely mediated by heterodimerization of the RING fingers of Mdm2 and Mdmx. Here we show that Mdmx expression leads to accumulation of ubiquitylated, nuclear p53 but does not significantly affect the Mdm2-mediated ubiquitylation of p53. In contrast, Mdmx stabilizes Mdm2 by inhibiting its self-ubiquitylation.     

Proteasome activator PA28 gamma regulates p53 by enhancing its MDM2-mediated degradation

Downregulation of p53 by MDM2-mediated proteasomal degradation makes cells resistant to apoptosis. The MDM2-p53 interaction is well characterized, but the mechanisms that regulate the interaction are not well understood. Here, we show that PA28gamma, a proteasome activator that inhibits apoptosis and promotes cell cycle progression through unknown mechanisms, exerts an effect as a cofactor in the MDM2-p53 interaction. The polymer form of PA28gamma interacts with both MDM2 and p53 proteins and facilitates their physical interaction. This promotes ubiquitination- and MDM2-dependent proteasomal degradation of p53, limiting its accumulation and resulting in inhibited apoptosis after DNA damage. Elimination of endogenous PA28gamma in human cancer cells abrogates MDM2-mediated p53 degradation, increases the activity of p53, and enhances apoptosis. These findings reveal the mechanism by which PA28gamma affects apoptosis and proliferation. Manipulation of the level of PA28gamma, an approach that would regulate the cellular content of p53, may improve the efficacy of current cancer therapies.     

MDM2 Promotes Proteasomal Degradation of p21Waf1 via a Conformation Change

MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21^Waf1. The present study was designed to characterize the mechanism(s) by which MDM2 induces p21^Waf1 degradation. We first determined the regions of MDM2 required for p21^Waf1 degradation using pulldown assays and Western blotting and then examined the mechanisms using limited proteolysis and fluorescence resonance energy transfer assays. We found that the MDM2-p21^Waf1 interaction depended on the central domain of MDM2 and that nuclear localization of both proteins was necessary for p21^Waf1 degradation. Specifically, amino acids 226–250 of MDM2 were required for p21^Waf1 binding and degradation, and amino acids 251–260 were necessary for p21^Waf1 degradation. The latter region induced a conformation change in p21^Waf1, increasing its interaction with the C8 subunit of the proteasome, leading to its degradation. When MDM2 lacked either segment (aa 226–250 or aa 251–260), its capacity to promote p21^Waf1 degradation and cell cycle progression was significantly reduced. In summary, the present study elucidated a previously unknown mechanism by which MDM2 promotes the degradation of an intact protein (p21^Waf1) through an ubiquitin-independent proteasomal degradation pathway. Because MDM2 also increases the degradation of other proteins in a ubiquitin-independent manner, this mechanism may underlie part of its tumorigenic properties.     

Mdm2 regulates p53 mRNA translation through inhibitory interactions with ribosomal protein L26

Mdm2 regulates the p53 tumor suppressor by promoting its proteasome-mediated degradation. Mdm2 and p53 engage in an autoregulatory feedback loop that maintains low p53 activity in nonstressed cells. We now report that Mdm2 regulates p53 levels also by targeting ribosomal protein L26. L26 binds p53 mRNA and augments its translation. Mdm2 binds L26 and drives its polyubiquitylation and proteasomal degradation. In addition, the binding of Mdm2 to L26 attenuates the association of L26 with p53 mRNA and represses L26-mediated augmentation of p53 protein synthesis. Under nonstressed conditions, both mechanisms help maintain low cellular p53 levels by constitutively tuning down p53 translation. In response to genotoxic stress, the inhibitory effect of Mdm2 on L26 is attenuated, enabling a rapid increase in p53 synthesis. The Mdm2-L26 interaction thus represents an additional important component of the autoregulatory feedback loop that dictates cellular p53 levels and activity.     

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.     

Ubiquitin- and MDM2 E3 ligase-independent proteasomal turnover of nucleostemin in response to GTP depletion

Nucleostemin (NS) is a nucleolar GTP-binding protein essential for ribosomal biogenesis, proliferation, and animal embryogenesis. It remains largely unclear how this protein is regulated. While working on its role in suppression of MDM2 and activation of p53, we observed that NS protein (but not mRNA) levels decreased drastically in response to GTP depletion. When trying to further elucidate the molecular mechanism(s) underlying this unusual phenomenon, we found that NS was degraded independently of ubiquitin and MDM2 upon GTP depletion. First, depletion of GTP by treating cells with mycophenolic acid decreased the level of NS without apparently affecting the levels of other nucleolar proteins. Second, mutant NS defective in GTP binding and exported to the nucleoplasm was much less stable than wild-type NS. Although NS was ubiquitinated in cells, its polyubiquitination was independent of Lys-48 or Lys-63 in the ubiquitin molecule. Inactivation of E1 in E1 temperature-sensitive mouse embryonic fibroblast (MEF) cells failed to prevent the proteasomal degradation of NS. The proteasomal turnover of NS was also MDM2-independent, as its half-life in p53/MDM2 double knock-out MEF cells was the same as that in wild-type MEF cells. Moreover, NS ubiquitination was MDM2-independent. Mycophenolic acid or doxorubicin induced NS degradation in various human cancerous cells regardless of the status of MDM2. Hence, these results indicate that NS undergoes a ubiquitin- and MDM2-independent proteasomal degradation when intracellular GTP levels are markedly reduced and also suggest that ubiquitination of NS may be involved in regulation of its function rather than stability.     

MDM2 is a negative regulator of p21WAF1/CIP1, independent of p53

The MDM2 oncogene has both p53-dependent and p53-independent activities. We have previously reported that antisense MDM2 inhibitors have significant anti-tumor activity in multiple human cancer models with various p53 statuses (Zhang, Z., Li, M., Wang, H., Agrawal, S., and Zhang, R. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 11636-11641). We have also provided evidence that MDM2 has a direct role in the regulation of p21, a cyclin-dependent kinase inhibitor. Here we provide evidence supporting functional interaction between MDM2 and p21 in vitro and in vivo. The inhibition of MDM2 with anti-MDM2 antisense oligonucleotide or Short Interference RNA targeting MDM2 significantly elevated p21 protein levels in PC3 cells (p53 null). In contrast, overexpression of MDM2 diminished the p21 level in the same cells by shortening the p21 half-life, an effect reversed by MDM2 antisense inhibition. MDM2 facilitates p21 degradation independent of ubiquitination and the E3 ligase function of MDM2. Instead, MDM2 promotes p21 degradation by facilitating binding of p21 with the proteasomal C8 subunit. The physical interaction between p21 and MDM2 was demonstrated both in vitro and in vivo with the binding region in amino acids 180-298 of the MDM2 protein. In summary, we provide evidence supporting a physical interaction between MDM2 and p21. We also demonstrate that, by reducing p21 protein stability via proteasome-mediated degradation, MDM2 functions as a negative regulator of p21, an effect independent of both p53 and ubiquitination.     

The acetylase activity of p300 is dispensable for MDM2 stabilization

It has been shown that p300 binds to MDM2 and leads to down-regulation of the p53 function. However, it remains unclear whether the acetylase activity of p300 is necessary for regulating MDM2 stability. In this study, we address this issue. First, p300 did not acetylate MDM2 in solution and in cells. Second, overexpression of p300 in cells increased the level of the MDM2 protein but not its mRNA. Similarly, the acetylase-defective p300 AT2 mutant stabilized the MDM2 protein as well. Consistently, the deacetylase inhibitor, trichostatin A, did not significantly affect the half-life of the endogenous MDM2 protein, whereas p300 enhanced the half-life of MDM2. Finally, both wild type and acetylase-defective mutant p300 proteins associated with MDM2 in nuclear body-like structures where MDM2 might be protected from proteasomal degradation. Thus, these results suggest that p300 appears to stabilize MDM2 by retaining this protein in a specific nuclear structure rather than by acetylating it.     

C-terminal modifications regulate MDM2 dissociation and nuclear export of p53

p53 functions to prevent malignant progression, in part by inhibiting proliferation or inducing the death of potential tumour cells. One of the most important regulators of p53 is MDM2, a RING domain E3 ligase that ubiquitinates p53, leading to both proteasomal degradation and relocation of p53 from the nucleus to the cytoplasm. Previous studies have suggested that although polyubiquitination is required for degradation, monoubiquitination of p53 is sufficient for nuclear export. Using a p53-ubiquitin fusion protein we show that ubiquitination contributes to two steps before export: exposure of a carboxy-terminal nuclear export sequence (NES), and dissociation of MDM2. Monoubiquitination can directly promote further modifications of p53 with ubiquitin-like proteins and MDM2 promotes the interaction of the SUMO E3 ligase PIASy with p53, enhancing both sumoylation and nuclear export. Our results suggest that modifications such as sumoylation can regulate the strength of the p53-MDM2 interaction and participate in driving the export of p53.     

UBE4B,a ubiquitin chain assembly factor,is required for MDM2-mediated p53 polyubiquitination and degradation

Although MDM2 is known to be a critical negative regulator of p53, MDM2 only catalyzes p53 mono- or multiple monoubiquitination in vitro and in vivo, which is insufficient for the initiation of proteasomal degradation. MDM2 does not polyubiquitinate p53 in vitro, however, which indicates that the activity of other ubiquitin ligase(s) or cofactor(s) is required for MDM2-mediated p53 polyubiquitination and degradation. In our recent study, we demonstrated that UBE4B, an E3 and E4 ubiquitin ligase with a U-box domain, interacts physically with both p53 and MDM2. Our findings revealed that UBE4B negatively regulates the level of p53 and inhibits p53-dependent transactivation and apoptosis. We propose that inhibition of MDM2 binding to UBE4B may provide another approach to inhibit MDM2 E3 ligase activity for tumor suppressor p53. It could lead to novel anticancer therapies, with the possibility of reducing the public health burden from cancer.Key words: ubiquitination, MDM2, UBE4B, p53, degradation     

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis

BackgroundP53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells.PurposeTo explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation.Study design and methodsDifferent mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53.ResultsThe triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation.ConclusionOur study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.