MdmX protects p53 from Mdm2-mediated degradation

The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.     

Regulation of p63 function by Mdm2 and MdmX.

p63, a p53-related protein, has been shown to activate p53-responsive genes and induce apoptosis in certain cell types. In this study, we examined the effects of Mdm2 and MdmX proteins on p63 transactivation, apoptosis, and protein levels. The isoforms of p63 most structurally similar to p53, p63gamma (p51A) and p63alpha (p51B), were chosen for study. Our results confirm earlier reports demonstrating that although both p63 isoforms can transactivate p53-responsive promoters and induce apoptosis, p63gamma has a stronger transactivation potential and is a more potent inducer of apoptosis than is p63alpha. In addition, both Mdm2 and MdmX were able to inhibit the transactivation induced by p63gamma and p63alpha. However, only Mdm2 overexpression led to a detectable decrease in p63-induced apoptosis. Although Mdm2 binding to p53 triggers ubiquitin-mediated proteosome degradation, p63 protein levels were unaltered by association with either Mdm2 or MdmX. Finally, immunofluorescence experiments showed that both p63 isoforms were localized in the nucleus and could be exported when coexpressed with Mdm2 but not with MdmX. These findings suggest that both Mdm2 and MdmX can downregulate p63 transactivation potential; however, only Mdm2 is capable of inhibiting the apoptotic function of p63 by removing it from the nucleus.     

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.     

Analysis of JNK, Mdm2 and p14(ARF) contribution to the regulation of mutant p53 stability

Identification of Mdm2 and JNK as proteins that target degradation of wt p53 prompted us to examine their effect on mutant p53, which exhibits a prolonged half-life. Of five mutant p53 forms studied for association with the targeting molecules, two no longer bound to Mdm2 and JNK. Three mutant forms, which exhibit high expression levels, showed lower affinity for association with Mdm2 and JNK in concordance with greater affinity to p14(ARF), which is among the stabilizing p53 molecules. Monitoring mutant p53 stability in vitro confirmed that, while certain forms of mutant p53 are no longer affected by either JNK or Mdm2, others are targeted for degradation by JNK/Mdm2, albeit at lower efficiency when compared with wt p53. Expression of wt p53 in tumor cells revealed a short half-life, suggesting that the targeting molecules are functional. Forced expression of mutant p53 in p53 null cells confirmed pattern of association with JNK/Mdm2 and prolonged half-life, as found in the tumor cells. Over-expression of Mdm2 in either tumor (which do express endogenous functional Mdm2) or in p53 null cells decreased the stability of mutant p53 suggesting that, despite its expression, Mdm2/JNK are insufficient (amount/affinity) for targeting mutant p53 degradation. Based on both in vitro and in vivo analyses, we conclude that the prolonged half-life of mutant p53 depends on the nature of the mutation, which either alters association with targeting molecules, ratio between p53 and targeting/stabilizing molecules or targeting efficiency.     

C-terminal region of human p53 attenuates buffalo p53 N-terminal-specific transactivation of p21 promoter by modulating tetramerization of the protein

Here, we have studied in p53 null H1299 lung carcinoma cells, the dominant-negative effect of human p53 (h-p53) on buffalo p53 (b-p53) induced nuclear transactivation-dependent function. Recently, we have isolated and cloned the full-length cDNA of buffalo p53. Buffalo and human p53 proteins exhibit a high degree of structural and functional similarities. In transiently transfected H1299 cell line b-p53 appeared to be more sensitive to Mdm2-mediated degradation as compared to h-p53, although its ability to transactivate p21 promoter was stronger than that of the human counterpart. This higher transactivation ability of b-p53 was lost in the presence of h-p53 suggesting, a dominant-negative effect of h-p53 on b-p53’s transactivation of p21 promoter. Both human and buffalo p53 proteins could hetero-oligomerize but the b-p53 could tetramerize much faster than the h-p53. A chimeric cDNA construct of human p53 was made where the 1–260 bp N-terminus was replaced with buffalo p53 counterpart and expressed in H1299 cell line. The tetramerization ability of the chimeric p53 protein was comparable to that of h-p53. Properties of b-p53 like stronger p21 transactivation and super sensitivity to Mdm2 mediated degradation were lacking in the chimeric protein. Thus, it is suggested that faster ability of tetramerization as well as higher transactivation property of buffalo p53 is determined by the interplay of N- and C-terminal domains through macromolecular interactions.     

Hetero-oligomerization with MdmX rescues the ubiquitin/Nedd8 ligase activity of RING finger mutants of Mdm2

Mdm2 is a member of the RING finger family of ubiquitin ligases and is best known for its role in targeting the tumor suppressor p53 for ubiquitination and degradation. Mdm2 can bind to itself and to the structurally related protein MdmX, and these interactions involve the RING finger domain of Mdm2 and MdmX, respectively. In this study, we performed a mutational analysis of the RING finger domain of Mdm2, and we identified several amino acid residues that are important for Mdm2 to exert its ubiquitin ligase function. Mutation of some of these residues interfered with the Mdm2-Mdm2 interaction indicating that a homomeric complex represents the active form of Mdm2. Mutation of other residues did not detectably affect the ability of Mdm2 to interact with itself but reduced the ability of Mdm2 to interact with UbcH5. Remarkably, MdmX efficiently rescued the ubiquitin ligase activity of the latter Mdm2 mutants in vitro and within cells. Because the interaction of Mdm2 with MdmX is more stable than the Mdm2-Mdm2 interaction, this suggests that Mdm2-MdmX complexes play a prominent role in p53 ubiquitination in vivo. Furthermore, we show that, similar to Mdm2, the Mdm2-MdmX complex has Nedd8 ligase activity and that all mutations that affect the ubiquitin ligase activity of Mdm2 also affect its Nedd8 ligase activity. From a mechanistic perspective, this suggests that the actual function of Mdm2 and Mdm2-MdmX, respectively, in p53 ubiquitination and in p53 neddylation is similar for both processes.     

MdmX binding to ARF affects Mdm2 protein stability and p53 transactivation

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity.     

Hypophosphorylation of Mdm2 augments p53 stability

The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.     

Regulation of p53 mediated transactivation by the beta-subunit of protein kinase CK2

The growth suppressor protein p53 plays a main part in cellular growth control. Two of its key functions are sequence specific DNA binding and transactivation. Functions of p53 in growth control are regulated at least in part by its interaction with protein kinases. p53 binds to protein kinase CK2, formerly known as casein kinase 2, and it is phosphorylated by this enzyme. CK2 is composed of two regulating beta-subunits and two catalytic alpha- or alpha'-subunits and the interaction with p53 is mediated by the regulatory beta-subunit of CK2. Recently we showed that the beta-subunit could inhibit the sequence specific DNA binding activity of p53 in vitro. Based on this finding, we asked if a coexpression of the beta-subunit of CK2 with p53 in mammalian cells could inhibit the DNA binding activity of p53 in a physiological context. We found that the coexpression of the beta-subunit showed the same inhibitory effect as in the previous assays with purified proteins. Then, we investigated the effects of the coexpression of the beta-subunit of CK2 on the transactivation and transrepression activity of p53. We found that transactivation of the mdm2, p21(WAF1/CIP1) and cyclin G promoter was inhibited in three different cell lines whereas transactivation of the bax promoter was not affected in COS1 cells but down-regulated in MCO1 and SaosS138V21 cells. p53 mediated transrepression of the fos promoter was not influenced by coexpression of the CK2 beta-subunit. Taken together we propose a cell type dependent fine regulation of the p53 transactivation function by the CK2 beta-subunit in vivo, which does not affect p53 mediated transrepression.     

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.