Ubiquitination and degradation of mutant p53

While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. In this study, we show that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild-type p53, our data suggest that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. Our initial attempts to identify ubiquitin ligases that are responsible for the ubiquitination of mutant p53 have suggested a role for the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein), although other unidentified ubiquitin ligases also appear to contribute. The contribution of Mdm2 to the degradation of mutant p53 may reflect the ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome.     

Mdm2 inhibits the apoptotic function of p53 mainly by targeting it for degradation

The ability of Mdm2 to inhibit the activities of a C-terminal truncated p53 mutant, p53-Delta30, which can bind Mdm2 but is resistant to Mdm2-mediated protein degradation was investigated. The inhibitory function of an Mdm2 mutant, Mdm2-Delta(222-437), which can bind p53 but is defective in targeting p53 for degradation was also studied. We have demonstrated that targeting p53 for degradation is the most effective way for Mdm2 to inhibit the apoptotic function of p53. However, we have also shown that Mdm2 can inhibit the transactivation function of p53 without targeting it for degradation, although Mdm2 releases the transrepression ability of p53 mainly by targeting it for degradation. The ability of Mdm2 to inhibit the apoptotic function of p53 was linked to its ability to inhibit the transrepression but not the transactivation function of p53. Furthermore, we have demonstrated that the transrepression function of p53 was specific to p53-induced apoptosis and was not simply a result of cell death.     

Mdm2 and Mdm4 loss regulates distinct p53 activities

Mutational inactivation of p53 is a hallmark of most human tumors. Loss of p53 function also occurs by overexpression of negative regulators such as MDM2 and MDM4. Deletion of Mdm2 or Mdm4 in mice results in p53-dependent embryo lethality due to constitutive p53 activity. However, Mdm2(-/-) and Mdm4(-/-) embryos display divergent phenotypes, suggesting that Mdm2 and Mdm4 exert distinct control over p53. To explore the interaction between Mdm2 and Mdm4 in p53 regulation, we first generated mice and cells that are triple null for p53, Mdm2, and Mdm4. These mice had identical survival curves and tumor spectrum as p53(-/-) mice, substantiating the principal role of Mdm2 and Mdm4 as negative p53 regulators. We next generated mouse embryo fibroblasts null for p53 with deletions of Mdm2, Mdm4, or both; introduced a retrovirus expressing a temperature-sensitive p53 mutant, p53A135V; and examined p53 stability and activity. In this system, p53 activated distinct target genes, leading to apoptosis in cells lacking Mdm2 and a cell cycle arrest in cells lacking Mdm4. Cells lacking both Mdm2 and Mdm4 had a stable p53 that initiated apoptosis similar to Mdm2-null cells. Additionally, stabilization of p53 in cells lacking Mdm4 with the Mdm2 antagonist nutlin-3 was sufficient to induce a cell death response. These data further differentiate the roles of Mdm2 and Mdm4 in the regulation of p53 activities.     

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.     

Glycogen synthase kinase 3-dependent phosphorylation of Mdm2 regulates p53 abundance

The Mdm2 oncoprotein regulates abundance and activity of the p53 tumor suppressor protein. For efficient degradation of p53, Mdm2 needs to be phosphorylated at several contiguous residues within the central conserved domain. We show that glycogen synthase kinase 3 (GSK-3) phosphorylated the Mdm2 protein in vitro and in vivo in the central domain. Inhibition of GSK-3 rescued p53 from degradation in an Mdm2-dependent manner while its association with Mdm2 was not affected. Likewise, inhibition of GSK-3 did not alter localization of p53 and Mdm2 or the interaction of Mdm2 and MdmX. Ionizing radiation, which leads to p53 accumulation, directed phosphorylation of GSK-3 at serine 9, which preceded and overlapped with the increase in p53 levels. Moreover, expression of a GSK-3 mutant where serine 9 was replaced with an alanine reduced the accumulation of p53 and induction of its target p21(WAF-1). We therefore conclude that inhibition of GSK-3 contributes to hypophosphorylation of Mdm2 in response to ionizing rays, and in consequence to p53 stabilization.     

The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation

The cellular level of the tumor suppressor p53 is tightly regulated through induced degradation via the ubiquitin/proteasome system. The ubiquitin ligase Mdm2 plays a pivotal role in stimulating p53 turnover. However, recently additional ubiquitin ligases have been identified that participate in the degradation of the tumor suppressor. Apparently, multiple degradation pathways are employed to ensure proper destruction of p53. Here we show that the chaperone-associated ubiquitin ligase CHIP is able to induce the proteasomal degradation of p53. CHIP-induced degradation was observed for mutant p53, which was previously shown to associate with the chaperones Hsc70 and Hsp90, and for the wild-type form of the tumor suppressor. Our data reveal that mutant and wild-type p53 transiently associate with molecular chaperones and can be diverted onto a degradation pathway through this association.     

p53-Independent Down-Regulation of Mdm2 in Human Cancer Cells Treated with Adriamycin

Mdm2 is a nuclear phosphoprotein which functions as a negative feedback regulator of the p53 tumor suppressor gene. In this study, we investigated the alteration of Mdm2 and p53 in three human cancer cell lines containing either a wild-type or mutant p53 gene after treatment with Adriamycin (doxorubicin, ADR), a DNA damaging agent. We found that human breast cancer MCF-7 cells containing wild-type p53 were much more susceptible to ADR compared to human breast cancer MDA-MB-231 and human prostate cancer Du-145 cells which contain mutant p53. ADR resulted in a significant dose-dependent accumulation of p53 protein in MCF-7 cells, whereas little or no influence was observed on p53 protein of the two mutant p53 cell lines. However, a significant down-regulation of Mdm2 at protein and mRNA levels was observed in these three cell lines following ADR treatment. Moreover, the decrease of Mdm2 was in both a dose- and time-dependent manner. It is interestingly noted that 5 μM is a critical dose for significant down-regulation of the Mdm2 protein. Selected proteasome inhibitors did not rescue the ADR-caused decline in the expression of Mdm2 protein. Therefore, our present results reveal that ADR can induce a down-regulation of Mdm2 via a p53-independent pathway in human cancer cells and the ubiquitin-proteasome degradation mechanism may not be involved in the decreased expression of Mdm2 protein.     

Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2.