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.     

Identification of a sequence element from p53 that signals for Mdm2-targeted degradation

The binding of Mdm2 to p53 is required for targeting p53 for degradation. p73, however, binds to Mdm2 but is refractory to Mdm2-mediated degradation, indicating that binding to Mdm2 is not sufficient for degradation. By utilizing the structural homology between p53 and p73, we generated p53-p73 chimeras to determine the sequence element unique to p53 essential for regulation of its stability. We found that replacing an element consisting of amino acids 92 to 112 of p53 with the corresponding region of p73 results in a protein that is not degradable by Mdm2. Removal of amino acids 92 to 112 of p53 by deletion also results in a non-Mdm2-degradable protein. Significantly, the finding that swapping this fragment converts p73 from refractory to sensitive to Mdm2-mediated degradation supports the conclusion that the amino acids 92 to 112 of p53 function as a degradation signal. We propose that the presence of an additional protein recognizes the degradation signal and coordinates with Mdm2 to target p53 for degradation. Our finding opens the possibility of searching for the additional protein, which most likely plays a critical role in the regulation of p53 stability and therefore function.     

Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53

The product of the Mdm2 oncogene directly interacts with p53 and promotes its ubiquitination and proteasomal degradation. Initial biological studies identified nuclear export sequences (NES), similar to that of the Rev protein from the human immunodeficiency virus, both in Mdm2 and p53. The reported phenotypes resulting from mutation of these NESs, together with results obtained using the nuclear export inhibitor leptomycin B (LMB), have led to a model according to which nuclear export of p53 (via either the NES of Mdm2 or its own NES) is required for efficient p53 degradation. In this study we demonstrate that Mdm2 can promote degradation of p53 in the nucleus or in the cytoplasm, provided both proteins are colocalized. We also investigated if nuclear export is an obligate step on the p53 degradation pathway. We find that (1) when proteasome activity is inhibited, ubiquitinated p53 accumulates in the nucleus and not in the cytoplasm; (2) Mdm2 with a mutated NES can efficiently mediate degradation of wild type p53 or p53 with a mutated NES; (3) the nuclear export inhibitor LMB can increase the steady-state level of p53 by inhibiting Mdm2-mediated ubiquitination of p53; and (4) LMB fails to inhibit Mdm2-mediated degradation of the p53NES mutant, demonstrating that Mdm2-dependent proteolysis of p53 is feasible in the nucleus in the absence of any nuclear export. Therefore, given cocompartmentalization, Mdm2 can promote ubiquitination and proteasomal degradation of p53 with no absolute requirement for nuclear to cytoplasmic transport.     

Regulation of Mdm2-Directed Degradation by the C Terminus of p53

The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.     

Gly-Ala repeats induce position- and substrate-specific regulation of 26 S proteasome-dependent partial processing

Partial degradation or regulated ubiquitin proteasome-dependent processing by the 26 S proteasome has been demonstrated, but the underlying molecular mechanisms and the prevalence of this phenomenon remain obscure. Here we show that the Gly-Ala repeat (GAr) sequence of EBNA1 affects processing of substrates via the ubiquitin-dependent degradation pathway in a substrate- and position-specific fashion. GAr-mediated increase in stability of proteins targeted for degradation via the 26 S proteasome was associated with a fraction of the substrates being partially processed and the release of the free GAr. The GAr did not cause a problem for the proteolytic activity of the proteasome, and its fusion to the N terminus of p53 resulted in an increase in the rate of degradation of the entire chimera. Interestingly the GAr had little effect on the stability of EBNA1 protein itself, and targeting EBNA1 for 26 S proteasome-dependent degradation led to its complete degradation. Taken together, our data suggest a model in which the GAr prevents degradation or promotes endoproteolytic processing of substrates targeted for the 26 S proteasome by interfering with the initiation step of substrate unfolding. These results will help to further understand the underlying mechanisms for partial proteasome-dependent degradation.     

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.