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Molecular and Cellular Biology, July 2003, p. 4939-4947, Vol. 23, No. 14
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.14.4939-4947.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Critical Contribution of the MDM2 Acidic Domain to p53 Ubiquitination

Hidehiko Kawai, Dmitri Wiederschain, and Zhi-Min Yuan*

Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115

Received 3 December 2002/ Returned for modification 22 January 2003/ Accepted 9 April 2003


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ABSTRACT
 
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.


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INTRODUCTION
 
The p53 tumor suppressor gene encodes a sequence-specific transcription factor that controls the expression of a number of genes whose products mediate either cell-cycle arrest or apoptosis (13). Because of its growth inhibitory activity, maintaining p53 at low levels under most physiological conditions is essential to ensure cell survival and proper organism development (2, 14, 16, 17). This is achieved largely at the level of protein, through the ability of MDM2 to target p53 for ubiquitin-dependent proteasomal degradation (10, 12). At the same time, p53 positively regulates the MDM2 gene, the expression of which is often elevated subsequent to the induction of p53 activity, thus forming a negative feedback loop wherein p53 upregulates MDM2 while MDM2 downregulates p53.

The MDM2 gene is conserved through zebra fish, frog, hamster, mouse, and human. Based on sequence similarity, MDMX was cloned from human and mouse. Alignment of the four MDM2 and two MDMX protein sequences highlights three regions of high identity, dubbed CR1, CR2, and CR3. CR1 (residues 42 to 94) is responsible for binding to p53 and inhibiting its transactivation function. In the region between CR1 and CR2 of MDM2, there is a nuclear localization sequence (NLS) and a nuclear export sequence, which are not conserved in MDMX, and an acidic domain (AD). CR2 (residues 301 to 329) codes for a putative zinc-binding domain and partially overlaps with a region required for binding of the retinoblastoma tumor suppressor protein. CR3 (residues 444 to 483) encodes the ring-finger domain (RFD), which binds two Zn atoms and contains a cysteine residue (residue 464) required for ubiquitin conjugation to p53. The region between CR2 and CR3 (designated the CR2 and CR3 linker region, or L2.3) and the acidic region are less conserved between the two proteins. Unlike MDM2, MDMX does not posses the ability to target p53 for destruction. Hence, the relevance of MDMX as a physiological regulator of p53 function has been questioned in the past. Interestingly, MDMX is either amplified or overexpressed in numerous cancers and tumor-derived cell lines where it coexists with elevated levels of wild-type p53 (19). MDMX overexpression increases both p53 and MDM2 levels (20, 22). Importantly, loss of MDMX expression leads to p53-dependent embryonic lethality in mice (6, 15, 18), indicating that MDMX is another essential negative regulator of p53.

MDM2 acts as an E3 ubiquitin ligase for both p53 and itself (5, 11). While recent studies have suggested that the self-ubiquitination activity of MDM2 is separable from its ability to target p53 for degradation (1), the molecular mechanism underlying MDM2-mediated p53 ubiquitination remains elusive. By making use of the fact that MDMX shares substantial structural homology with MDM2 but lacks the E3 ligase activity in vivo (20, 22), we generated a series of MDM2/MDMX chimeric proteins for examining the contribution of each domain of MDM2 to p53 ubiquitination. Here we present compelling evidence that in addition to the p53 binding motif and the RFD, the acidic region is essential to the ability of MDM2 to target p53 for efficient ubiquitination, which in turn is essential for nuclear export and degradation of p53.


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MATERIALS AND METHODS
 
Cell culture and transfection. U2OS (American Type Culture Collection) and p53-/-/MDM2-/- murine embryonic fibroblasts (MEFs) (Carl Maki, Harvard School of Public Health, Boston, Mass.) were maintained in minimal essential medium supplemented with 10% fetal bovine serum. Cells were transfected by the Lipofectamine 2000 (Invitrogen) method according to the manufacturer's instructions.

Plasmid design. p53, MDM2, and MDMX expression plasmids have been described previously (8). Constructs of MDM2/MDMX chimeras were generated by using two-step PCR and primers that encoded 12-nucleotide regions of complementarity between the sequences of MDM2 and MDMX that were to be fused. All constructs were cloned into pCMV-Flag vector or pEGFP-C1 vector by using BamHI and NotI restriction sites.

Preparation of whole-cell extracts and Western blotting. Cells were transfected on 60-mm-diameter plates with 5 µg of DNA and harvested at 24 h posttransfection. Cells were lysed in 200 µl of lysis buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitors) by incubation on ice for 30 min, and the extracts were centrifuged at 18,000 x g for 15 min to remove cell debris. Protein concentrations were determined by using the Bio-Rad protein assay. After the addition of 5x loading buffer, the samples were incubated at 95°C for 5 min and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell) and probed with the indicated antibodies: anti-p53 (Ab-6; Calbiochem), anti-Flag (M5; Sigma), anti-ß-actin (AC-15; Sigma), and anti-green fluorescent protein (anti-GFP; Clontech). Proteins were visualized with an enhanced chemiluminescence detection system (NEN).

GST-protein binding assay. Glutathione S-transferase (GST) constructs of p53 deletion mutants have been generated previously in our laboratory according to published protocols (9). p53-/-/MDM2-/- MEFs transfected with Flag-MDM2 AD (residues 109 to 303) were harvested and lysed in 0.5% Nonidet P-40 lysis buffer for 1 h at 4°C. Cell lysates were incubated with 10 µl of GST beads containing p53 for 3 h at 4°C. Beads were then washed in lysis buffer, 0.1% Nonidet P-40, and protein complexes were liberated by boiling the beads in SDS-PAGE sample buffer for 5 min. Samples were then analyzed by Western blotting. Eluted GST proteins were visualized by using Ponceau S (Sigma) staining.

Immunoprecipitation analysis. Immunoprecipitations were performed as described elsewhere (8). Cell lysates were prepared in 0.5% Triton X-100 lysis buffer and incubated with anti-p53 agarose beads (Ab-6; Calbiochem) for 12 h. Immune complexes and whole lysates were analyzed by Western blotting. The filters were incubated with anti-p53 and anti-Flag antibodies.

Subcellular distribution assay. Cells were grown on chamber slides (Nunc) and transfected with the indicated vector as shown in Fig. 2. Cells were washed with cold phosphate-buffered saline (PBS) 24 h after transfection and fixed with 4% paraformaldehyde (Sigma) for 30 min at 4°C. After being washed with PBS, cells were permeabilized with ice-cold 0.2% Triton X-100 for 5 min, blocked with 0.5% bovine serum albumin for 30 min, and then incubated with the indicated antibody for 1 h. The slides were incubated with secondary antibody (Texas Red X-goat anti-mouse immunoglobulin G; Molecular Probes) and DAPI (4',6'-diamidino-2-phenylindole [10 µg/ml]; Sigma). Following the PBS wash, the slides were mounted with Fluoromount-G (Southern Biotechnology Associates) containing 2.5 mg of n-propyl gallate (Sigma)/ml. Specimens were examined under a fluorescent microscope (Zeiss).



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  		FIG. 2. Subcellular distribution of the MDM2/MDMX complexes. (A) EGFP-MDMX expression vector was cotransfected with the indicated Flag-tagged vector into p53-/-/MDM2-/- MEFs. The transfectants were fixed at 36 h posttransfection and stained with an anti-Flag antibody and secondary antibody (Texas Red X-goat anti-mouse immunoglobulin G). Direct green fluorescence and Texas Red positive staining were visualized under a fluorescent microscope. Cellular nuclei were identified by DAPI staining. Merged images were obtained by superimposing GFP over Texas Red staining. (B) EGFP-MDM2{Delta}NLS expression vector was coexpressed with the indicated Flag vector in p53-/-/MDM2-/- MEFs, and cells were analyzed as described for panel A.


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RESULTS
 
Characterization of the MDM2/MDMX chimeras. The rapid turnover of p53 is primarily mediated by MDM2-dependent ubiquitination (10, 12). The finding that the E3 ligase-containing RFD is necessary but not sufficient for MDM2 to ubiquitinate p53 raises the possibility that another MDM2 region might be required. Given the fact that MDMX shares a high degree of structural homology with MDM2 but lacks the ubiquitin E3 ligase activity in vivo (20, 22), we utilized a domain-swapping approach by replacing each region of MDM2 with the corresponding domain of MDMX to uncover the additional activity of MDM2. We divided the MDM2 and MDMX sequences as shown in Fig. 1A and prepared chimeras by a two-step PCR using primers carrying the 12-nucleotide tail of the parts to be fused. Restriction enzyme digestion and DNA sequencing confirmed the identity of each chimera (data not shown). pCMV-Flag vectors expressing the chimeras were then generated. Each of the vectors was tested for expression by transient transfection into p53-/-/MDM2-/- MEFs and Western blotting with an anti-Flag antibody. The result shows comparable levels of expression achieved for the wild-type proteins and chimeras (Fig. 1B).



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  		FIG. 1. MDM2/MDMX chimeras. (A) MDM2/MDMX chimeras were generated by swapping corresponding domains between MDM2 and MDMX at the indicated positions by using a two-step PCR with primers carrying the 12-nucleotide overlap at the parts to be fused. NES, nuclear export sequence; Nuc., nucleus; Cyto., cytoplasm. (B) cDNAs encoding the indicated chimeras were subcloned into pCMV-Flag expression vector, which was then transfected into p53-/-/MDM2-/- MEFs with enhanced GFP (EGFP)-empty vector (0.2 µg) included as a transfection efficiency control, and the transfectants were harvested at 36 h posttransfection. Whole-cell extracts were prepared, boiled in loading dye, resolved on SDS-PAGE, and transferred onto a nitrocellulose membrane. The membrane was then probed with anti-Flag, anti-GFP, or anti-actin antibody.

We then proceeded to functionally characterize chimeric proteins by first examining their subcellular distribution. Consistent with a previous observation (8), wild-type MDMX was exclusively cytoplasmically localized, in contrast with the predominantly nuclear distribution of the MDM2 protein (Fig. 2A). Analysis of chimera localization revealed that the distribution of each protein is essentially determined by whether it contains the NLS of MDM2 (Fig. 1A). Since MDM2 and MDMX can form hetero- as well as homodimers through the RFD (23), and since binding of MDMX to MDM2 is required for nuclear redistribution of MDMX (8), we made an attempt to assess complex formation between chimeric proteins by analyzing subcellular distribution. As has been shown previously (8), MDMX is redistributed to the nucleus upon binding to MDM2 (Fig. 2A, column 2), indicative of MDM2/MDMX heterocomplex formation. In keeping with the lack of the NLS, coexpression of Flag-MDMX did not result in any significant alteration of the cytoplasmic distribution of GFP-MDMX (Fig. 2A, column 5). However, coexpression of MDMX fusion protein that contained the MDM2 NLS (MDMX/MDM2NLS) induced marked nuclear redistribution of MDMX (Fig. 2A, column 6), demonstrating the formation of an MDMX/MDMX homocomplex. MDM2 with the C464A mutation (MDM2C464A), which cannot bind to MDMX, and MDM2 with the NLS deleted (MDM2{Delta}NLS) were included as controls to demonstrate that both complex formation and the MDM2 NLS are essential for nuclear redistribution of MDMX (Fig. 2A, columns 3 and 4). The MDM2/MDM2 homocomplex formation was demonstrated by nuclear redistribution of a cytoplasmically localized MDM2{Delta}NLS (Fig. 2B, column 1) induced by wild-type MDM2 and MDM2C464A (Fig. 2B, columns 2 and 3). Nuclear relocalization of MDM2{Delta}NLS in the presence of the MDMX/MDM2NLS chimera further demonstrated the process of MDM2/MDMX heterocomplex formation. Together, these data indicate that both MDM2 and MDMX are competent in the formation of either hetero- or homocomplexes, an observation consistent with the results obtained from the yeast two-hybrid analysis (23).

Next, we assessed the ubiquitination activity of chimeric proteins. In addition to targeting p53 for ubiquitination, the E3 ligase activity of MDM2 is capable of promoting self-ubiquitination. This activity is best manifested in the presence of a proteasome inhibitor, as ubiquitinated MDM2 is rapidly degraded via the proteasome pathway. As shown in Fig. 3, treatment of cells with MG132, a proteasome inhibitor, resulted in a marked increase in the abundance of MDM2 as well as the ubiquitinated form of MDM2. In sharp contrast, neither increase of protein levels nor ubiquitination of MDMX was apparent in the presence of the proteasome inhibitor (Fig. 3A, lanes 1 and 2 versus lanes 3 and 4), demonstrating MDMX's distinct stability due to the lack of E3 ligase activity. We then tested the MDMX chimera containing the MDM2 RFD (MDMX/MDM2RFD). Remarkably, swapping the RFD between MDM2 and MDMX resulted in MDMX's gaining the ability to self-ubiquitinate and MDM2's losing such function (Fig. 3A, lanes 5 and 6 versus lanes 7 and 8). This result indicates that the MDM2 RFD is critical for self-ubiquitination activity and, moreover, that this activity can be transferred onto MDMX. Notably, gaining the ability to self-ubiquitinate is associated with a marked increase in susceptibility to proteasome-mediated degradation since the MDM2 RFD converted MDMX into a very unstable protein (Fig. 3A). Therefore, the MDM2 RFD is essential for rendering an MDM2/MDMX chimeric protein susceptible to the proteasome-mediated destruction. A similar conclusion can be drawn from the other chimeric proteins tested (Fig. 3B). Taken together, our results demonstrate that chimeric proteins generated by the domain-swapping approach are functionally competent.



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  		FIG. 3. The MDM2 RFD is necessary and sufficient for self-ubiquitination activity. p53-/-/MDM2-/- MEFs were transfected with the indicated plasmids (2 µg) encoding MDM2, MDMX, or an MDM2/MDMX chimera and EGFP-empty vector (0.2 µg). Cells were treated with MG132 (7.5 µg/ml) or left untreated at 30 h posttransfection and incubated for an additional 6 h before being harvested for Western analysis by using the indicated antibodies. LE, long exposure; SE, short exposure.

The MDM2 RFD is not sufficient for efficient p53 ubiquitination. Having shown that chimeric protein containing the MDM2 RFD is functional in self-ubiquitination, we were interested in assessing the ability of this chimeric protein to target p53 for ubiquitination. The p53-binding motif is highly conserved in MDMX, and it has been shown to bind to p53 with an affinity similar to that of MDM2 (8). If the E3 ligase activity were sufficient for p53 ubiquitination, we would expect MDMX/MDM2RFD protein to efficiently ubiquitinate p53. Plasmids encoding wild-type or chimeric proteins were cotransfected along with p53 into p53-/-/MDM2-/- MEFs. Measurement of cellular p53 abundance by Western analysis at 36 h posttransfection indicated that MDM2, but not MDMX, expression resulted in p53 degradation (Fig. 4A, lane 2 versus lane 3), which was associated with a marked induction of p53 ubiquitination as clearly revealed in MG132-treated cells (Fig. 4A, lane 7). The MDMX chimeric protein containing the MDM2 RFD, however, exhibited neither detectable effect on p53 abundance nor apparent ability to ubiquitinate p53 (Fig. 4A, lanes 4 and 5 and 9 and 10). This result suggests that the MDM2 RFD is not sufficient to target p53 for efficient ubiquitination. To substantiate this finding, we employed p53 nuclear export as an independent readout to assess the ability of the chimeric protein to ubiquitinate p53 since ubiquitination is also required for MDM2 to mediate p53 nuclear export (4, 7). Plasmid encoding GFP-p53 was cotransfected with the indicated Flag-tagged vectors into p53-/-/MDM2-/- MEFs, and immunostaining was performed 36 h posttransfection to examine subcellular distribution of GFP-p53 and Flag-tagged proteins. As expected, MDM2 expression was associated with significant redistribution of the p53 protein to the cytoplasm, whereas no such change in p53 localization was evident when MDMX was coexpressed (Fig. 4B, column 2 versus column 3). Analogous to the ubiquitination-dependent protein degradation, the MDMX/MDM2RFD protein failed to induce significant p53 nuclear export (Fig. 4B, column 4). In comparison with that in MDM2-expressing cells, however, the cytoplasmic concentration of p53 in MDMX/MDM2RFD-expressing cells was slightly increased. Similar to the observation in MDMX-transfected cells, this cytoplasmically localized p53 was not a result of nuclear export but rather a consequence of cytoplasmic sequestration because treatment with leptomycin B, an inhibitor of nuclear export, did not have any apparent effect (8; data not shown). In keeping with the lack of E3 ligase activity in vivo, MDM2 with the RFD of MDMX (MDM2/MDMXRFD) did not induce detectable p53 nuclear export. Together with the results from Western analysis, these data indicate that despite being competent in self-ubiquitination, the MDM2/MDMXRFD protein is defective in promoting p53 ubiquitination. Therefore, our data indicate that the E3 ligase activity and p53 binding are not sufficient for MDM2 to efficiently ubiquitinate p53.



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  		FIG. 4. The MDM2 RFD is not sufficient for p53 ubiquitination. (A) p53 expression vector (1 µg) was cotransfected with the indicated MDM2/MDMX chimera vector (2 µg) into p53-/-/MDM2-/- MEFs. Wild-type MDM2 or MDMX was included as a control. Cells were treated with MG132 (lanes 6 to 10) or left untreated (lanes 1 to 5) for 6 h and then analyzed as described in the legend to Fig. 3. (B) EGFP-p53 vector (1 µg) was cotransfected with the MDM2/MDMX chimera (2 µg) into p53-/-/MDM2-/- MEFs, and cells were analyzed as described in the legend to Fig. 2.

Contribution of each region of MDM2 to its ability to target p53 for ubiquitination. The finding that the MDMX/MDM2RFD chimera binds to p53 and possesses the self-ubiquitination activity but is incompetent in p53 ubiquitination suggests the presence of an additional MDM2 sequence, apart from the RFD, that is essential for targeting of p53 for ubiquitination. We tested this hypothesis by fusing each region of MDM2 into the backbone of MDMX/MDM2RFD and assessing the ability of chimeric proteins to target p53 for ubiquitination.

The first set of chimeric proteins that we tested involved swapping the region of the AD, CR2, L2.3, and the RFD, resulting inMDMX/MDM2CR2.L23.RFD, MDMX/MDM2AD.CR2.L23.RFD, and MDMX/MDM2AD.RFD (Fig. 5A). Western analysis of the lysates prepared from cells coexpressing chimeric proteins along with p53 indicated that in contrast to the MDMX/MDM2CR2.L23.RFD chimera, whose ability to degrade p53 was severely compromised (Fig. 5B, lane 4), chimeras MDMX/MDM2AD.CR2.L23.RFD and MDMX/MDM2AD.RFD efficiently targeted p53 for degradation, similar to wild-type MDM2 (Fig. 5B, lanes 5 and 6). Ubiquitination of p53, as revealed by treatment with MG132, supported the importance of the central AD in MDM2-mediated p53 ubiquitination (Fig. 5B, lanes 7 to 12). When the chimeras were tested for their ability to induce p53 nuclear export, the AD was once again found to be essential for rendering the chimeric protein functionally competent (Fig. 5C).



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  		FIG. 5. The AD of MDM2 is another essential element for p53 ubiquitination. (A) p53-/-/MDM2-/- MEFs were cotransfected with p53 and the indicated chimeras. The cells were analyzed by Western blotting (B) or immunostaining (C).

To substantiate this finding further, a second set of mutants, as shown in Fig. 6A, was tested. Wild-type MDM2 and the MDM2C464A mutant that is deficient in E3 ligase activity were included as positive and negative controls, respectively. Western analysis indicated that in the absence of the MDM2 AD, i.e., with the AD deletion MDM2 mutant (MDM2{Delta}AD) or the MDM2/MDMXAD chimera, the ability of MDM2 to ubiquitinate p53 was significantly impaired (Fig. 6B, lanes 4 and 5). Of note is the difference in the ubiquitination patterns. Consistent with the requirement for the E3 ligase activity, no detectable ubiquitination of p53 was observed in the MDM2C464A-expressing cells (Fig. 6B, lane 3). Interestingly, coexpression of MDM2 mutants lacking the AD was associated with the appearance of a single low-molecular-weight p53 band, which is likely to be the monoubiquitinated form of p53, whereas the high-molecular-weight ubiquitinated p53 ladders, which were prominently featured in the wild-type-MDM2-expressing cells, were almost completely absent (Fig. 6B, lane 2 versus lanes 4 and 5). Again, subcellular distribution analysis demonstrated that the AD of MDM2 is required for MDM2-mediated p53 nuclear export (Fig. 6C). Taken together, our results have uncovered a critical role for the AD of MDM2 in cooperating with the MDM2 RFD to efficiently ubiquitinate p53. Of particular interest is the AD-dependent induction of the high-molecular-weight ubiquitinated p53 species, which seem to symbolize efficient p53 ubiquitination as their appearance was functionally associated with protein degradation and nuclear export of p53.



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  		FIG. 6. Both the AD and the RFD of MDM2 are required for p53 ubiquitination. p53-/-/MDM2-/- MEFs were cotransfected with p53 and the indicated vector as shown in panel A. The cells were analyzed by Western blotting (B) or immunostaining (C).

Characterization of the MDM2 AD-mediated action. A recent study reported that the AD of MDM2 contributes to MDM2-mediated p53 degradation by a mechanism that is independent of either MDM2 E3 ligase activity or MDM2 nucleocytoplasmic shuttling (1), which seems to be inconsistent with our results. To further define the role of the AD in relation to that of the RFD in MDM2-mediated p53 ubiquitination, we tested a pair of chimeras (Fig. 7A) in which the region either N terminal or C terminal to the CR2 domain had been swapped between MDM2 and MDMX for their ability to mediate p53 ubiquitination. When expressed alone, neither chimera was able to mediate significant p53 ubiquitination (Fig. 7B, lanes 7 and 8), reinforcing the notion that neither the RFD nor the AD of MDM2 alone is sufficient for efficient p53 ubiquitination. Interestingly, coexpression of the two chimeras resulted in robust p53 ubiquitination (Fig. 7B, lane 9). Together with the observation that coexpression of the chimeras lacking the AD of MDM2 was not associated with any significant p53 ubiquitination (Fig. 7B, lane 10), it appears that the AD and the RFD of MDM2 are both required and can work in trans to target p53 for ubiquitination. Results obtained from subcellular distribution studies supported this notion (Fig. 7C). To analyze such a trans mode of action further, we tested the MDM2 mutants indicated in Fig. 8A for their ability to functionally rescue the AD deletion mutant of MDM2. Consistent with the requirement for the AD of MDM2, the MDMX/MDM2AD, but not the MDM2/MDMXAD, chimeric protein restored the ability of MDM2{Delta}AD to ubiquitinate p53 (Fig. 8B, lane 5 versus lane 6). Of particular interest is the finding that the mini protein that consisted of the MDM2 AD and an additional region comprising the NLS and the nuclear export sequence was able to ensure the ability of the MDM2{Delta}AD mutant to ubiquitinate p53, albeit to a slightly lesser extent (Fig. 8B, lane 7). Again, a similar conclusion was drawn from p53 nuclear export analysis (Fig. 8C). The ability of the mini protein to functionally complement the MDM2 AD deletion mutant would suggest an association of this small protein with p53. Whereas the GST pull-down assay did not detect any apparent association (Fig. 8D), immunoprecipitation and Western analysis indicated that this small protein is in complex with p53 in vivo (Fig. 8E).



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  		FIG. 7. The AD of MDM2 collaborates with the RFD in p53 ubiquitination and can be provided in trans. (A) The indicated MDM2/MDMX chimeric protein expression vectors were transfected either alone or as a pair into p53-/-/MDM2-/- MEFs. Cells were analyzed by Western blotting (B) or immunostaining (C).



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  		FIG. 8. Functional rescue of the AD-lacking mutant of MDM2 by MDM2 AD-containing chimeras. (A) p53-/-/MDM2-/- MEFs were cotransfected with an AD deletion mutant of MDM2 and with the indicated construct. The cells were analyzed by Western blotting (B) or immunostaining (C). V, vector; LE, long exposure; SE, short exposure. (D) Binding of the MDM2 AD (residues 109 to 303) to p53 was analyzed by incubating lysates from cells expressing Flag-MDM2 AD with the indicated GST-p53 deletion proteins. The adsorbents were resolved by SDS-PAGE and stained with Ponceau S solution (top panel) or Western blotted with an anti-Flag antibody (bottom panel). GST-P300/CH1 was included as a positive control. (E) Anti-p53 immunoprecipitations were performed with cell lysates prepared from the indicated constructs transfected into p53-/-/MDM2-/- MEFs. The whole-cell extracts (WCE) and anti-p53 immunocomplexes were analyzed by immunoblotting (IB) with anti-p53 (top two panels) and anti-Flag (bottom four panels). IP, immunoprecipitate; IgG, immunoglobulin G.


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DISCUSSION
 
Since MDM2-mediated covalent attachment of ubiquitin moieties to p53 is associated with proteasome-dependent proteolysis as well as nuclear export, we made use of these two MDM2 E3 ligase-dependent cellular events as independent readouts to functionally characterize p53 ubiquitination. The MDMX/MDM2 chimeric proteins that we generated by fusion of various distinct regions of MDM2 into the backbone of MDMX enabled us to assess the functional contribution of each domain of MDM2 to p53 ubiquitination. Fusion of the RFD of MDM2 to MDMX converted MDMX into a very unstable protein that was rapidly degraded by ubiquitin-mediated proteolysis, indicating that the E3 ligase activity can be transferred to MDMX and is essential for rendering the resulting chimeric protein capable of self-ubiquitination. The MDMX/MDM2RFD chimeric protein, however, failed to target p53 for ubiquitination despite its E3 ligase activity and binding to p53, consistent with the finding that the RFD is not sufficient to ubiquitinate p53 and that an activity additional to the E3 ligase is required. Indeed, the MDMX/MDM2RFD protein gained full ability to ubiquitinate p53 upon receiving the central AD from MDM2, suggesting a critical role for the AD in cooperating with the RFD to ubiquitinate p53. The finding that chimeric proteins containing the AD of MDM2 were able to functionally complement the AD-deficient mutant of MDM2 further substantiated the requirement for the MDM2 AD in p53 ubiquitination. Interestingly, the AD was essential for MDM2 to induce high-molecular-weight ubiquitinated p53 species, as they were almost completely absent in the AD-deficient MDM2 mutant-expressing cells. While it remains unclear whether these high-molecular-weight p53 ladders are the polyubiquitinated form of p53 or rather represent p53 that is monoubiquitinated at multiple sites, their close correlation with p53 degradation and p53 nuclear export imply their functional significance. Our data therefore demonstrate that the central AD of MDM2 is the region that controls the separation of the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Requirement for an activity that is complementary to the E3 ligase for p53 ubiquitination suggests the possibility that the highly ordered p53 tetramer conformation prevents the RFD from accessing its ubiquitination sites on p53, the exposure of which requires the action of the AD. Consistent with this possibility is the previous finding that MDM2-mediated p53 ubiquitination is in fact a stepwise process involving sequential conformational changes in p53 (9). The AD might interact with a region of p53 distinct from the canonical N-terminal MDM2-binding motif, and this interaction might be required to induce p53 conformational changes so that additional critical lysine residues become exposed for ubiquitination. Interestingly, a recent report revealed a novel MDM2-binding site in the p53 DNA-binding domain, and this interaction seemed to play a regulatory role in modulating MDM2-dependent p53 ubiquitination (21). Alternatively, the AD of MDM2 might associate with additional proteins that induce p53 conformational changes essential for ubiquitination. The trans mode of action exhibited by the MDM2 AD-containing proteins or the MDM2 AD miniprotein is consistent with such a possibility. Additionally, while no direct binding of the MDM2 AD to p53 was detected in the GST pull-down assay, the AD miniprotein was recruited into the p53 complex in vivo, as demonstrated by immunoprecipitation and Western analysis, thus pointing to possible multiple protein assembly. Since it has been reported that ARF, retinoblastoma protein, and p300 all bind to the region near the AD of MDM2, it is therefore of interest to investigate whether these proteins can regulate p53 ubiquitination through interaction with the AD of MDM2. We are currently testing the possibilities.

Significantly, the AD of MDM2 is rich in serine and threonine residues, suggesting the possibility of posttranslational modifications (e.g., phosphorylation and dephosphorylation). Data from a recent report provided convincing evidence to demonstrate that phosphorylation and dephosphorylation of the AD of MDM2 are essential to stress-induced p53 activation (3), supporting a critical role for the MDM2 AD in p53 ubiquitination.

In summary, our data uncover the central AD of MDM2 as an additional essential contributor, apart from the RFD, to MDM2-dependent p53 ubiquitination. Elucidation of the underlying molecular basis of AD involvement could shed new light on the mechanisms by which MDM2 targets p53 for ubiquitination and degradation.


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ACKNOWLEDGMENTS
 
We are grateful to A. G. Jocheshem for providing us with the anti-MDMX antibody.

This work was supported by an NIH grant (RO1CA85679-01) to Z.-M.Y. D.W. is supported by a training grant (T32ES07155) from NIH.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Cancer Cell Biology (Bldg. 1, Room 507), Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Phone: (617) 432-0763. Fax: (617) 432-0377. E-mail: zyuan{at}hsph.harvard.edu. Back


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Molecular and Cellular Biology, July 2003, p. 4939-4947, Vol. 23, No. 14
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.14.4939-4947.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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