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An essential function of the extreme C-terminus of MDM2 can be provided by MDMX

2006; Springer Nature; Volume: 26; Issue: 1 Linguagem: Inglês

10.1038/sj.emboj.7601469

1460-2075

Stjepan Uldrijan, Willem‐Jan Pannekoek, Karen H. Vousden,

Microtubule and mitosis dynamics

Article14 December 2006free access An essential function of the extreme C-terminus of MDM2 can be provided by MDMX Stjepan Uldrijan Stjepan Uldrijan The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Willem-Jan Pannekoek Willem-Jan Pannekoek The Beatson Institute for Cancer Research, Glasgow, UKPresent address: Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Karen H Vousden Corresponding Author Karen H Vousden The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Stjepan Uldrijan Stjepan Uldrijan The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Willem-Jan Pannekoek Willem-Jan Pannekoek The Beatson Institute for Cancer Research, Glasgow, UKPresent address: Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Karen H Vousden Corresponding Author Karen H Vousden The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Author Information Stjepan Uldrijan1, Willem-Jan Pannekoek1 and Karen H Vousden 1 1The Beatson Institute for Cancer Research, Glasgow, UK *Corresponding author. The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK. Tel.: +44 141 330 2424; Fax: +44 141 943 0372; E-mail: [email protected] The EMBO Journal (2007)26:102-112https://doi.org/10.1038/sj.emboj.7601469 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info MDM2 (HDM2) is a ubiquitin ligase that can target the p53 tumor suppressor protein for degradation. The RING domain is essential for the E3 activity of MDM2, and we show here that the extreme C-terminal tail of MDM2 is also critical for efficient E3 activity. Loss of E3 function in MDM2 mutants deleted of the C-terminal tail correlated with a failure of these mutants to oligomerize with MDM2, or with the related protein MDMX (HDMX). However, MDM2 containing point mutations within the C-terminus that inactivated E3 function retained the ability to oligomerize with the wild-type MDM2 RING domain and MDMX, and our results indicate that oligomers containing both wild-type MDM2 and a C-terminal mutant protein retain E3 function both in auto-degradation and degradation of p53. Interestingly, the E3 activity of C-terminal point mutants of MDM2 can also be supported by interaction with wild-type MDMX, suggesting that MDMX can directly contribute to E3 function. Introduction The p53 protein functions to inhibit the outgrowth of cells with malignant potential through the induction of cell cycle arrest and apoptosis in cells that have been stressed by genotoxic and other types of damage (Vogelstein et al, 2000). These activities are key to p53's ability to act as a tumor suppressor, and their loss is an important step in the development of most malignancies. Although these anti-proliferative activities of p53 are beneficial in tumor suppression, they are extremely deleterious to normal growth and development, and several mechanisms exist to keep p53 activity in check during normal proliferation (Ryan et al, 2001). One of these is the control of p53 protein stability, which allows for the maintenance of low levels of p53 protein in unstressed cells and the rapid increase in p53 levels owing to stabilization in response to stress. The degradation of p53 is principally through the proteasome, and a number of ubiquitin ligases have been described that can target the degradation of p53 through this pathway (Brooks and Gu, 2006). One of the most important ubiquitin ligases is MDM2 (also called HDM2), a RING domain E3 that targets a number of proteins, including p53, for degradation. The importance of MDM2 as a regulator of p53 has been demonstrated in a number of systems, where deletion of MDM2 leads to the stabilization and activation of p53 (Mendrysa et al, 2003; Grier et al, 2006). Almost all tumor cells show defects in the p53 pathway, either through mutations within the p53 gene itself or by perturbations in the mechanisms that allow for the activation of p53 in response to stress (Vousden, 2002). Most p53-activating signals result in the stabilization of p53 through inhibition of MDM2, and in several tumor types failure to activate p53 has been associated with a failure to properly inactivate MDM2. This can result from amplification and overexpression of MDM2 (Momand et al, 1998), loss of kinases that phosphorylate p53 (Brooks and Gu, 2003) or MDM2 (Meek and Knippschild, 2003) to inhibit their interaction or function, or from defects in negative regulators of MDM2, such as p14ARF (Sharpless, 2005). These observations suggest that small molecule inhibitors of MDM2 might be beneficial in the treatment of cancers that retain wild-type p53 but cannot properly activate it (Buolamwini et al, 2005). The successful development of such drugs will depend on a clear understanding of how MDM2 functions to target p53 for degradation. MDM2 belongs to the RING domain family of E3 ligases, and mutations in the core metal-coordinating residues within the RING domain result in the complete loss of MDM2's E3 function (Fang et al, 2000). In addition to p53, several other proteins are targeted for degradation by MDM2, including MDM2 itself and the MDM2-related protein MDMX (Mdm4 in mice) (de Graaf et al, 2003; Kawai et al, 2003a; Pan and Chen, 2003). While the isolated RING domain can be shown to possess some E3 activity, the ability to degrade p53 also depends on the integrity of other regions of MDM2, such as the central acidic domain (Argentini et al, 2001; Kawai et al, 2003b; Meulmeester et al, 2003). It has been shown that the ability of MDM2 to degrade itself can be separated from the ability to degrade p53, and some forms of genotoxic damage function to stabilize p53 by promoting the auto-degradation of MDM2, and so shifting the balance between the levels of the two proteins (Stommel and Wahl, 2004). Unlike MDM2, MDMX possesses no intrinsic E3 activity, despite a strong similarity between the two proteins within the RING domains (Jackson and Berberich, 2000; Stad et al, 2001). However, MDMX can form an interaction with MDM2 through the RING domain (Sharp et al, 1999; Tanimura et al, 1999), and whereas at high levels of expression this interaction can inhibit the ability of MDM2 to degrade p53 (Jackson and Berberich, 2000; Stad et al, 2000), at physiological levels MDMX has been shown to be important for the negative regulation of p53 (Parant et al, 2001; Migliorini et al, 2002b). Clearly, the ability of MDMX to bind directly to p53 and inhibit p53's transcriptional activity plays a major role in the negative regulation of p53 by MDMX (Shvarts et al, 1996), and this function of MDMX is independent of MDM2 (Francoz et al, 2006; Xiong et al, 2006). The current data most strongly support a model in which MDM2 regulates p53 by targeting it for degradation, whereas MDMX functions by directly inhibiting the transcriptional activity of p53 by binding to its N-terminal transactivation domain (Marine et al, 2006). However, in addition to these independent and synergistic activities of MDM2 and MDMX, there is evidence that interactions between these two proteins also play an important role in their mutual regulation, and thereby the regulation of the p53 response. A large part of this interconnection reflects the regulation of MDMX stability by MDM2. In response to DNA damage, phosphorylation of MDMX enhances the degradation of MDMX by MDM2 (Chen et al, 2005)—an effect that may be related to reduced binding to the deubiquitinating enzyme HAUSP (Meulmeester et al, 2005) or an increase in 14-3-3 binding (Okamoto et al, 2005; Lebron et al, 2006)—leading to the stabilization and activation of p53. MDM2 also promotes the nuclear accumulation of MDMX (Stad et al, 2001; Li et al, 2002; Migliorini et al, 2002a), although MDM2-independent nuclear localization of MDMX is also seen in response to DNA damage (Li et al, 2002; Lebron et al, 2006). In addition to the ability of MDM2 to regulate MDMX, there is some evidence that MDMX can function to enhance the ability of MDM2 to degrade p53, either by promoting the E3 activity of MDM2 against both itself and p53 (Linares et al, 2003) or by inhibiting the auto-degradation of MDM2, so allowing sufficient MDM2 accumulation to degrade p53 (Stad et al, 2001; Gu et al, 2002). Despite these observations, the overall contribution of MDMX to p53 or MDM2 stability remains unclear. Studies in mice have indicated that an effect of MDMX on p53 stability, through the modulation of MDM2, can be seen in some but not all tissues (Marine et al, 2006). It is evident that there is a complex and dynamic relationship between MDM2 and MDMX, which is key to the regulation of p53 stability and function. Understanding the functions of these proteins, and how they might interact with each other, will be critical for exploiting them as potential therapeutic targets. Here, we identify a new region in the extreme C-terminus of MDM2 and MDMX that plays an important role in regulating MDM2 E3 activity. Results Contribution of the C-terminal tail of MDM2 to p53 degradation The RING domain of MDM2 is located very close to its C-terminus, with the last cysteine of the zinc coordination motif 14 amino acids from the end of the protein. A comparison of the sequence of this C-terminal tail showed that this region is highly conserved through evolution (Figure 1A). In order to assess any role that this region might have in MDM2 activity, we tested two MDM2 mutants, deleting the C-terminal 9 or 12 amino acids (MDM2Δ9 and MDM2Δ12), for their ability to degrade and ubiquitylate p53 (Figure 1B). In cotransfection experiments, both mutants were defective for the degradation of p53 compared with wild-type MDM2, and both mutants were expressed at higher levels, suggesting a failure to auto-degrade (Figure 1B). This defect in p53 degradation was also reflected in an inability of the MDM2 mutants to ubiquitylate p53 in an in vitro assay (Figure 1C). Loss of the C-terminal tail also prevented the enhanced ubiquitylation of p53 seen following expression of MDM2 in cells (Figure 1D), similar to the effect of a much larger C-terminal deletion that also removes the RING domain (MDM2ΔRING). Figure 1.C-terminal tail of MDM2 is required for MDM2-mediated p53 degradation and ubiquitylation. (A) C-terminal tail sequences of MDM2 proteins were aligned using BOXSHADE 3.21 software at http://www.ch.embnet.org/software/BOX_form.html. (B) MDM2 C-terminal deletions are not able to target p53 for degradation. U2OS cells were transiently cotransfected with FLAG-p53, GFP and MDM2 C-terminal deletions and analyzed by Western blotting. (C) MDM2 C-terminal tail deletions prevent efficient p53 ubiquitylation in vitro. Bacterially expressed wild-type and mutant GST-MDM2, in vitro-translated p53, recombinant ubiquitin, E1 and E2 enzymes were incubated in the reaction buffer at 37°C for 2 h. A negative control reaction was prepared without the E2 enzyme. Reaction products were resolved by SDS–PAGE and analyzed by Western blotting with anti-p53 DO-1. (D) MDM2 C-terminal deletions prevent efficient p53 ubiquitylation in vivo. U2OS cells, transiently cotransfected with FLAG-p53 (0.2 μg) and MDM2 C-terminal deletion mutants (1.5 μg), were treated with 10 μM MG132 24 h after transfection and analyzed by Western blotting. Download figure Download PowerPoint The C-terminal region of MDM2 contains threonine (serine in mouse Mdm2) and tyrosine residues at amino acids 488 and 489 that are potential targets for phosphorylation. Although these residues do not lie within predicted consensus sequences for kinase recognition sites, we made mutants of MDM2 carrying substitutions of these amino acids to non-phosphorylatable (T488A, Y489F) and phospho-mimetic (T488D, Y489D) alternatives (Figure 2A). Mutation of threonine 488 to alanine (T488A) or tyrosine 489 to phenylalanine (Y489F) did not affect the ability of MDM2 to degrade p53, compared with the wild-type protein (Figure 2B). However, mutation to a phospho-mimetic amino acid (T488D and Y489D) resulted in a loss of p53-degrading activity, comparable to that seen in an MDM2 mutant carrying a substitution of one of the key RING domain cysteines (C464A). Interestingly, each of the MDM2 mutants that failed to degrade p53 also showed evidence for increased stability, suggesting that these mutants also failed to target themselves for degradation. Although these results suggest that phosphorylation of either threonine 488 or tyrosine 489 may inhibit the ability of MDM2 to degrade p53, a double mutant substituting alanine at both positions (TY488-9AA) also lost the ability to degrade p53 (Figure 2B), suggesting that the retention of an aromatic residue at position 489 (either tyrosine or phenylalanine) is important for MDM2 activity. We made a number of further mutations affecting the C-terminal tail and examined their ability to degrade p53 (Figure 2C). As predicted, substitution of tyrosine for alanine (Y489A) destroyed the p53-degrading activity, whereas a double substitution conserving the aromatic nature of the residue at position 489 (TY488-9AF) retained this function. In support of the suggestion that the aromatic residues in this region of MDM2 are important for activity, substitution of the phenylalanine at position 490 to alanine (F490A) also prevented p53 degradation. Deletion of the last amino acid (MDM2Δ1) did not affect the ability to degrade p53 (Figure 2C). Substitutions of the two conserved hydrophobic amino acids within the C-terminus of MDM2 (IV485-6AA) also prevented the function of MDM2 in degrading p53 (Figure 2C), further supporting the importance of the integrity of the C-terminal tail for MDM2 function. Figure 2.Point mutations within the C-terminal tail inhibit MDM2 function. (A) Location of mutants generated within MDM2 C-terminal tail. (B) MDM2 T488D and Y489D phospho-mimicking mutants do not degrade p53. U2OS cells were transiently cotransfected with FLAG-p53, wild-type or mutant MDM2 and pEGFP-N1 for 30 h and analyzed by Western blotting. (C) Aromatic residue at position 489 of MDM2 is required for p53 degradation. U2OS cells were cotransfected with FLAG-p53 and wild-type or C-terminal tail mutants of MDM2 for 30 h, then analyzed by Western blotting. (D) Aromatic residue at position 489 of MDM2 is required for p53 ubiquitylation in vitro. Bacterially expressed wild-type and mutant GST-MDM2, in vitro-translated p53, recombinant ubiquitin, E1 and E2 enzymes were incubated in the reaction buffer at 37°C for 2 h. Reactions containing Y489 mutants were performed in duplicate. (E) C-terminal tail is not required for MDM2 binding to p53. HEK293 cells, transiently cotransfected with FLAG-p53 and wild-type or C-terminal tail mutants of MDM2 for 36 h, were treated with proteasome inhibitor MG132 for 4 h and p53-MDM2 protein complexes were immunoprecipitated with anti-FLAG antibody M2. Immunoprecipitated proteins were analyzed by Western blotting. Download figure Download PowerPoint To confirm that the loss of function of some of these C-terminal MDM2 mutants directly reflected a loss of E3 activity, we tested the effect of mutations of tyrosine 489 on the ability of MDM2 to ubiquitinate p53 in an in vitro assay. In agreement with the degradation results, mutation of the tyrosine to phenylalanine (Y489F) did not affect E3 function, whereas substitution of alanine at this position (Y489A) destroyed this activity (Figure 2D). Contribution of the C-terminal tail of MDM2 to p53 binding Although the p53-binding region of MDM2 has been clearly mapped to the N-terminus of the protein, recent studies have shown that the central region of MDM2 also provides another interaction site for p53 (Yu et al, 2006), and it is possible that alterations in the C-terminus of the full-length MDM2 protein might affect the p53 interaction. However, we were unable to detect any defect in their ability to bind p53 (Figure 2E), indicating that failure of some of these mutants to degrade p53 is not the result of a defect in the ability to interact with p53. Oligomerization of C-terminal MDM2 mutants Previous studies have shown that MDM2 can oligomerize through RING/RING interactions (Tanimura et al, 1999; Dang et al, 2002; Linares et al, 2003), and that this interaction may be important for MDM2's E3 activity. We therefore tested whether the defect in E3 activity exhibited by the C-terminal MDM2 mutants was a result of a failure to homo-oligomerize. Initial co-precipitation experiments showed no significant difference in the ability of the C-terminal MDM2 mutants to interact with a GFP-RING domain (data not shown). However, it has been shown that MDM2 can form interactions through both the RING and the acidic domains (Dang et al, 2002), and it is possible that binding between the RING and acidic domains was masking differences in the RING/RING interactions. Therefore, in order to look only at the interaction of MDM2 through the RING domains, we examined the ability of a GFP-tagged MDM2 RING only protein (GFP-RING) to bind an MDM2 protein deleted of the acidic domain (amino acids 245–295; MDM2ΔAD) and carrying the C-terminal mutations. Either co-precipitation through the GFP-RING protein or the reciprocal co-precipitation through the MDM2ΔAD protein (Figure 3A) showed a clear reduction in the interaction between the wild-type RING domain of the MDM2Δ9 deletion mutant. However, there was no detectable defect in the binding of the wild-type RING to the point mutants Y489A or Y489F. Interestingly, a very similar pattern of interaction was seen following coexpression of the C-terminal mutants (in the context of MDM2ΔAD) with a GFP-RING protein carrying the TY488-9AA double mutant (Figure 3B). These results therefore show that although the loss of p53 degradation activity shown by MDM2Δ9 may be a reflection of an inability to oligomerize through the RING domain, such a defect in oligomerization is not obviously the cause of the lack of p53 degradation activity of the Y489A mutant. To further confirm the oligomerization capacities of the MDM2 mutants, we examined their interaction in cells by immunofluorescence (Figure 3C). A GFP-RING protein, which lacks the MDM2 nuclear localization signals (NLS), localized diffusely in the cell nucleus and cytoplasm. However, following oligomerization with coexpressed MDM2ΔAD protein (which retains the NLS), the GFP-RING protein was also relocalized to the nucleus. Using this assay, we were able to confirm that point mutations in the C-terminal tail of MDM2 (Y489A or TY488-489AA) did not affect homo-oligomerization through the RING domain, whereas this ability was lost with the deletion of the C-terminal tail (MDM2Δ9). Figure 3.Point mutants of MDM2 within the C-terminal tail retain the ability to homo-oligomerize through RING–RING interactions. C-terminal tail mutants in the context of MDM2 lacking the acidic domain (MDM2ΔAD) were transiently transfected into HEK293 cells together with GFP-RING (A) or GFP-RING:TY488-9AA (B). Cells were treated 36 h post-transfection with proteasome inhibitor MG132 for 4 h, lysed and immunoprecipitated with anti-MDM2 (Ab-1) and anti-GFP (7.1/13.1) antibodies, then analyzed by Western blotting. (C) Wild-type RING domains of MDM2 can homo-oligomerize in vivo with the MDM2 C-terminal tail point mutants, but not with the C-terminal tail deletion mutants. U2OS cells were cotransfected with constructs coding for GFP-tagged MDM2 RING (lacks nuclear localization signal (NLS); diffuse pattern of subcellular localization) and MDM2ΔAD (contains NLS; nuclear protein) with wild-type or mutant C-terminal tail. MDM2ΔAD-induced translocation of GFP-RING into the nucleus was used as an indicator of the interaction between the two MDM2 proteins. Download figure Download PowerPoint As the Y489A mutant fails to target p53 for degradation, but retains the ability to oligomerize with the wild-type MDM2 RING domain, we were interested in determining whether this mutant might function as a dominant negative, and so inhibit the p53-degrading activity of wild-type MDM2. Interestingly, coexpression of the Y489A or Y489D mutants with wild-type MDM2 resulted in an efficient rate of p53 degradation (Figure 4A). A reduction in the degradation of p53 is not apparent until a high ratio of mutant to wild-type MDM2 is expressed, and only when mutant MDM2 is expressed alone is a complete failure to degrade p53 apparent. These results suggest that the Y489A and Y489D mutants do not function as dominant negatives, and that although a homo-oligomer of these mutant MDM2 proteins is inactive in the degradation of p53, a hetero-oligomer containing wild-type and mutant proteins is still functional. To compare the activities of different MDM2 mutants, we carried out a similar experiment using the MDM2Δ9 mutant (Figure 4B). Unlike either the Y489A or IV485-6AA mutants, which did not impede degradation of p53 by wild-type MDM2, coexpression of the MDM2Δ9 mutant was able to block p53 degradation in the presence of wild-type MDM2. This inhibition of wild-type MDM2 by the MDM2Δ9 mutant, which shows a defect in the RING/RING interaction, presumably results from the acidic domain interaction or by competing for p53 binding, and the extent of inhibition was dependent on the ratios of wild-type and MDM2Δ9 expressed. Taken together, these results suggest that the Y489A mutant can retain some function in p53 degradation when oligomerized with wild-type MDM2. Figure 4.C-terminal tail point mutants can function in p53 degradation if oligomerized with wild-type MDM2. (A) U2OS cells were transiently transfected with FLAG-p53, GFP and different ratios of wild-type MDM2 to Y489A or Y489D mutants (to give a constant total amount of transfected MDM2 plasmid of 1.6 μg) and analyzed by Western blotting. (B) FLAG-p53 was transiently cotransfected into U2OS cells with wild-type MDM2 and C-terminal tail mutants in a 1:1 ratio. Download figure Download PowerPoint Contribution of the C-terminal tail of MDM2 to MDMX degradation Each of the C-terminal MDM2 mutants that was defective for p53 degradation also showed elevated expression, suggesting that they are also defective for auto-degradation. This effect is similar to that seen with RING domain mutants and might suggest that these mutations completely inactivate the E3 activity of the MDM2 protein. To examine this more closely, we tested the MDM2 mutants for their ability to drive the degradation of MDMX, another MDM2 target protein. Surprisingly, none of the MDM2 C-terminal point mutants showed any reduction in the ability to degrade MDMX (Figure 5A), although the MDM2Δ9 deletion mutant, like the RING domain mutant C464A, lost this activity. Therefore, despite their defects in auto-degradation and p53 degradation, the IV485-6AA, Y489A, TY488-9AA and F490A mutants retained the ability to target the degradation of MDMX. Figure 5.C-terminal tail of MDM2 contributes to MDMX degradation. (A) MDM2 C-terminal point mutants retain the ability to degrade MDMX. U2OS cells were transfected with Myc-MDMX and MDM2 C-terminal tail mutants and analyzed by Western blotting. (B, C) Reactivation of MDM2 C-terminal tail mutants by MDMX. U2OS cells were transfected with FLAG-p53 and MDM2 mutants and Myc-MDMX in a 1:1 ratio and analyzed by Western blotting. Download figure Download PowerPoint Reactivation of MDM2 mutants by MDMX Previous studies have suggested that MDMX can enhance the ability of MDM2 to degrade p53, possibly by preventing the auto-degradation of MDM2. As we had shown that some of the inactive C-terminal point mutants of MDM2 appear to retain function as part of a complex with wild-type MDM2, we examined whether these mutant MDM2 proteins showed any activity with MDMX (Figure 5B and C). Although high levels of ectopic MDMX expression can inhibit MDM2 activity, at lower relative expression levels, MDMX does not clearly affect p53 degradation by wild-type MDM2 or the active MDM2 mutants Y489F and T488A. As expected, the RING domain MDM2 mutant C464A failed to degrade p53 both in the absence and presence of MDMX (Figure 5B and C). Surprisingly, the MDM2 mutants T488D, Y489A, Y489D and a double mutant TY488-9DD, which have all lost the ability to degrade p53, regained p53 degradation function when coexpressed with MDMX. However, MDM2 carrying a complete deletion of the C-terminal tail (MDM2Δ9) was not reactivated for p53 degradation following coexpression of MDMX (Figure 5B). These results show that MDM2 C-terminal point mutants that retain the ability to degrade MDMX (Figure 5A) also regain the ability to degrade p53 in the presence of MDMX (Figure 5B). These unexpected results suggested that MDMX might be able to restore the E3 activity of the inactive MDM2 point mutants. To test this possibility, we performed an in vivo p53 ubiquitylation assay in two different cell lines DKO and U2OS (Figure 6A). As expected, MDMX was inactive in this assay and MDM2 mutants Y489A and Y489D alone were also unable to efficiently ubiquitylate p53. However, the strong increase in p53 ubiquitylation upon coexpression of both MDMX and the MDM2 mutants confirmed that MDMX is indeed capable of restoring the ubiquitin ligase activity of MDM2 C-terminal tail point mutants (Figure 6A). Figure 6.(A) MDMX restores p53 ubiquitylation by MDM2 C-terminal tail point mutants in vivo. DKO or U2OS cells, transiently cotransfected with FLAG-p53, HA-ubiquitin, MDM2 Y489 mutants and Myc-MDMX for 24 h, were treated with proteasome inhibitor MG132 for 3 h before lysis. Following immunoprecipitation of p53 with DO-1 antibody, ubiquitin-conjugated p53 was detected by Western blotting using anti-HA tag rabbit polyclonal antibody Y11 and p53 levels in the input for IP using DO-1 antibody. (B) Loss of the MDM2 C-terminal tail, but not point mutation of Y489, interferes with MDMX binding. HEK293 cells, transiently cotransfected with Myc-MDMX and MDM2 C-terminal tail mutants for 36 h, were treated with MG132 for 4 h, lysed and protein complexes containing wild-type or mutant MDM2 and Myc-MDMX were immunoprecipitated with anti-MDM2 antibody Ab-1 and anti-Myc antibody 9E10, resolved by SDS–PAGE and analyzed by Western blotting with anti-MDM2 (Ab-1) and anti-Myc (9E10) antibodies. Download figure Download PowerPoint We have shown that the C-terminal point mutants, but not the C-terminal deletion mutants, retain the ability to interact with the MDM2 RING (Figure 3). In light of the differential activity of these two classes of mutants following coexpression with MDMX, we examined their ability to interact with MDMX. In co-immunoprecipitation experiments, wild-type MDM2 associated with MDMX (Figure 6B), as previously described (Sharp et al, 1999; Tanimura et al, 1999). Deletion of the C-terminal tail of MDM2 (MDM2Δ9) significantly reduced the interaction with MDMX, whereas the point mutants (Y489A and Y489F) retained binding (Figure 6B). However, the Y489A mutant (which is inactive for p53 degradation) showed some variability in the strength of interaction with MDMX (e.g., Figure 6B, RHS). Therefore, to confirm the significance of this interaction, we tested the ability of the MDM2 mutants to translocate MDMX from the cytoplasm to the nucleus, a function that has been shown to be dependent on the RING domains of both MDM2 and MDMX (Gu et al, 2002; Migliorini et al, 2002a). These studies confirmed that wild-type MDM2 can drive nuclear localization of MDMX, which is cytoplasmic in the absence of additional MDM2 (Figure 7). As expected, the RING domain MDM2 mutant (C464A) and the C-terminal deletion (MDM2Δ9), which fail to interact with MDMX, also failed to translocate MDMX. However, all the C-terminal point mutants (TY488-9AA, Y489A and Y489D) allowed translocation of MDMX to the nucleus (Figure 7), confirming the significance of the interaction of these mutants with MDM2. Figure 7.Subcellular translocation of MDMX by coexpression of MDM2 C-terminal tail mutants. U2OS cells were cotransfected with Myc-tagged MDMX and MDM2 C-terminal tail mutants, then proteins detected by immunofluorescence using a mixture of anti-Mdm2 mouse monoclonal IF2 (Ab-1, Calbiochem) and anti-Myc rabbit polyclonal (A14, Santa Cruz Biotechnology) antibodies. Download figure Download PowerPoint The C-terminal tail of MDMX is important for the cooperation with MDM2 The ability of MDMX to cooperate with C-terminal MDM2 mutants in the degradation of p53 suggests that a hetero-oligomer of MDM2 and MDMX, which is capable of degrading MDMX, can also target the degradation of p53. Although MDMX does not have E3 activity by itself, it contains a RING domain and C-terminal region that is similar to MDM2 (Figure 8A). Using immunofluorescence to examine the ability of MDM2 to relocalize MDMX mutants to the nucleus, we found that as with MDM2, a point mutation within the MDMX C-terminal tail in MDMX(F488A) did not prevent the interaction with MDM2, and so, relocalization (Figure 8B). However, deletion of the last five amino acids of the C-terminal tail of MDMX in MDMX(Δ5) prevented the interaction and relocalization with MDM2. Figure 8.C-terminal tail of MDMX participates in the MDM2–MDMX interaction. (A) C-termi