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Hdmx Recruitment into the Nucleus by Hdm2 Is Essential for Its Ability to Regulate p53 Stability and Transactivation

Hdmx Recruitment into the Nucleus by Hdm2 Is Essential for Its Ability to Regulate p53 Stability... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 9, Issue of March 1, pp. 7318 –7323, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Hdmx Recruitment into the Nucleus by Hdm2 Is Essential for Its Ability to Regulate p53 Stability and Transactivation* Received for publication, September 12, 2001, and in revised form, November 7, 2001 Published, JBC Papers in Press, December 13, 2001, DOI 10.1074/jbc.M108795200 Domenico Migliorini‡, Davide Danovi‡, Emanuela Colombo‡§, Roberta Carbone‡, Pier Giuseppe Pelicci‡¶, and Jean-Christophe Marine‡** From the ‡Department of Experimental Oncology, European Institute of Oncology, 435 Via Ripamonti, 20141 Milan, Italy and the ¶FIRC Institute of Molecular Oncology, 20 Via Serio, 20139 Milan, Italy The Hdmx gene product is related to the Hdm2 onco- function of Hdm2 and p53. From a genetic standpoint, the close relationship between the murine ortholog of Hdm2, Mdm2, and protein, both of which interact with and regulate p53 stability and function. Like Hdm2, Hdmx is able to in- p53 is further supported by the striking observation that a hibit p53 transactivation; however, at variance with p53-null state can completely rescue the early-embryonic-le- Hdm2, which promotes ubiquitination, nuclear export, thal phenotype associated with Mdm2 deficiency (8, 9). and degradation of p53, Hdmx increases p53 stability. Hdmx is structurally related to Hdm2, and a significant We report here (i) that overexpressed Hdmx is cytoplas- similarity is especially notable in the p53-binding domain and mic and Hdm2 recruits Hdmx into the nucleus and (ii) in the C-terminal RING-finger domain (10). Hdmx has been that nuclear Hdmx blocks Hdm2-mediated nuclear ex- shown to bind p53 and inhibit p53 transactivation in specific port of p53 and down-regulates p53-dependent tran- cellular contexts but, unlike Hdm2, is unable to induce p53 scription. Furthermore we showed that Hdmx inhibits degradation (10, 11). Similar conclusions were drawn when Hdm2-mediated p53 ubiquitination. It appears, there- using the Hdmx murine ortholog Mdmx (12, 13). Moreover, fore, that a regulatory loop exists in which Hdm2 regu- Mdmx and Mdm2 form stable heterodimers through their C- lates the intracellular localization of Hdmx, and nuclear terminal RING-finger domains, and this interaction results in Hdmx regulates several functions of Hdm2 (ubiquitin a substantial increase in the steady-state levels of Mdm2 (14, ligase activity and p53 nuclear export). 15). Finally it has been reported that both Mdmx (13) and Hdmx (11), through a yet unknown mechanism, are able to interfere with Mdm2-mediated p53 degradation, resulting in p53 is the most frequently inactivated tumor suppressor an increase in p53 protein levels while maintaining suppres- gene in human cancer irrespective of the tumor type, site, and sion of p53 transactivation. It was therefore proposed that patient age. In cells undergoing DNA damage or responding to Hdmx (and Mdmx) secures a nuclear pool of transcriptionally certain forms of stress (replicative senescence, hypoxia, oxida- incompetent p53, which upon stress can be quickly activated. tive stress, and oncogene overexpression) the ordinarily short- Finally the first genetic evidences confirming the role of Mdmx lived p53 protein is stabilized, rapidly accumulates, and under- as a critical regulator of p53 function in vivo are now available. goes several activating post-translational modifications. In this Mdmx-mutant mice die around midgestation because of loss of context, activated p53, via its ability to regulate gene transcrip- cell proliferation, while loss of p53 completely rescues this tion, elicits either a cell cycle arrest or apoptosis (1). As it would embryonic lethality (16). These new exciting data raise the be expected, p53 actions are largely regulated by post-transla- possibility that increased Mdmx levels and the resulting inhi- tional events, including direct binding and occlusion of the bition of p53 transcriptional activity contribute to development transactivation domain, ubiquitination and degradation by the of human tumors. proteosome, and control of subcellular localization. The Hdm2 We show here that exogenous Hdmx is mainly localized in protein has been implicated in all three of these processes, most the cytoplasm of transfected cells by indirect immunofluores- recently as mediator of the export of p53 from the nucleus, cence, while when co-expressed with Hdm2, Hdmx was then which enhances the degradation of p53 in cytoplasmic proteo- found almost exclusively in the nucleus. We show that this somes (2–5). Hdm2 functions as an E3 ubiquitin ligase for p53, effect is dependent on the integrity of the RING-finger domain and the C-terminal RING-finger domain of Hdm2 is critical for of both Hdmx and Hdm2 proteins. Furthermore, we demon- ligase activity (6, 7). Interestingly the Hdm2 gene itself is a strate that the Hdm2-dependent nuclear recruitment of Hdmx transcriptional target of p53. Thus, there is evidence for an is indispensable for its ability to exert its two previously re- autoregulatory feedback loop involving the expression and ported activities, regulation of p53 steady state levels and block of p53 transcriptional ability. * This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro and European Community. The costs of EXPERIMENTAL PROCEDURES publication of this article were defrayed in part by the payment of page Plasmids—DNAs encoding wild-type human p53 and Hdm2 in charges. This article must therefore be hereby marked “advertisement” pCMV and the p53 reporter plasmid pG13Luc were a gift from B. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Vogelstein. The HA-tagged wild-type p53, p53 K305N (nuclear localiza- § Recipient of a fellowship from the Giancarla Vollaro Foundation. tion signal (NLS)) (17), and p53 173L (DNA binding-defective) mutants To whom correspondence may be addressed. Tel.: 39-0257489834; Fax: 39-0257489851; E-mail: [email protected]. were provided to us by K. Maki and K. Vousden; Hdm2 NLS and Hdm2 ** Recipient of a fellowship from the European Community (Marie G58A mutants were obtained from A. Levine. The pcDNA3.1 Myc- Curie Fellowship). To whom correspondence may be addressed. Tel.: tagged Hdmx constructs (wild type and N-terminal truncation mutant 39-0257489834; Fax: 39-0257489851; E-mail: [email protected]. The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; NLS, nuclear localization signal; GFP, green fluo- D. Migliorini, D. Danovi, E. Lazzerini-Denchi, A. Jochemsen, M. rescent protein. Capilo, A. Gobbi, K. Helin, P. G. Pelicci, and J.-C. Marine, submitted. 7318 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Regulation of p53 Stability and Activity by Nuclear Hdmx 7319 (amino acids 101– 490), also designated Hdmxp53bs) were described nology) was used to detect Hdm2, and an anti-Myc (9E10) monoclonal previously (15) and kindly provided by D. George. Full-length Hdmx antibody was used to detect the Myc-tagged Hdmx proteins. and the N-terminal truncated (amino acids 101– 490) Hdmx fragment were released from the above constructs using a BamHI-XhoI restric- RESULTS tion digest and subcloned into the pcDNA3X()MyEGFP to generate Hdm2 Recruits Hdmx into the Nucleus—Determination of Myc-GFP-tagged Hdmx and Myc-GFP-Hdmx-(101– 490). The tagged the subcellular localization of transfected Hdmx and/or Mdmx Hdmx (R-NLS (amino acids 2– 430) and R (amino acids 2– 451)) in different cellular contexts has led to conflicting observations deletion mutants, deleted either of the entire RING-finger region including the putative NLS or most of it to leave the putative NLS (13, 18 –21). To revisit this issue, we determined the localiza- intact, were generated by PCR amplification of the corresponding tion of exogenous Myc-tagged (N-terminal) Hdmx and GFP- fragments using a 5-primer including a BamHI site and 3-primers Hdmx in U2OS, Soas-2, and NIH-3T3 cell lines by immunoflu- including XhoI sites. The PCR products were subcloned into orescence. In all cell types analyzed, both Hdmx-tagged pcDNA3.1Myc and pcDNA3X()MyEGFP to generate the Myc-tagged proteins were mainly found in the cytoplasm (Fig. 1, a– d). The Hdmx (R-NLS) and the Myc-tagged Hdmx (R), and the Myc-GFP- intensity and the frequency of the nuclear staining slightly tagged Hdmx (R-NLS) and the Myc-GFP-tagged Hdmx (R), respec- tively. Hdmx NLS mutants (KRPRD 3 ATPLD) were generated varied from one cell line to another but always appeared much using the QuikChange mutagenesis kit (Stratagene). The following less frequent (5% of cells) and less intense than the cytoplas- oligonucleotide and its complement were used for the mutagenesis in mic staining (Fig. 1h). the context of the Myc-tagged Hdmx and Myc-GFP-tagged Hdmx Sequence analysis of Hdmx reveals a single putative NLS in constructs: 5-tgtgaggcaacaccactagacggg. the C-terminal RING-finger domain (KRPRD). Point mutations Tissue Culture and Immunofluorescence—U2OS, Soas-2, NIH-3T3, in this motif (KTLLD) had no major effect on the cellular and Phoenix (derived from the 293T cell line) cell lines were grown in distribution of Hdmx (Fig. 1, e and h). Deletion of the C- Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bo- vine serum, and 100 g/ml penicillin and streptomycin. Transfections terminal RING domain instead resulted in both nuclear and were carried out using the calcium phosphate method or the FuGENE cytoplasmic localization of Hdmx (Hdmx R-NLS and R, car- transfection reagent (Roche Molecular Biochemicals). For immunofluo- rying deletions of either the entire RING domain, including the rescence staining, cells were transfected at 80% confluency on glass putative NLS, or most of it to leave the putative NLS intact) coverslips in six-well dishes and fixed 24 h after addition of the DNA (Fig. 1, f– h). It appears, therefore, that the putative NLS of mixtures. Fixed coverslips were washed twice in phosphate-buffered Hdmx is not functional and that its C-terminal RING-finger saline, permeabilized in 0.15% Triton X-100 for 5 min, and incubated in blocking buffer (phosphate-buffered saline  2% bovine serum albumin) motif is an important determinant of the cellular distribution of for 30 min. Cells were than incubated in blocking buffer containing Hdmx. primary antibody for1has indicated below and then washed exten- Since Hdm2 is a nuclear cytoplasmic shuttling protein (2, 3) sively in phosphate-buffered saline before incubation with the appro- and Mdm2 (the mouse ortholog of Hdm2) forms stable hetero- priate fluorochrome-conjugated secondary antibody for 1 h. Stained oligomers with Mdmx (14, 15), we investigated whether Hdm2 cells were mounted on glass slides and examined using standard im- levels affect Hdmx localization. Strikingly, GFP-Hdmx was munofluorescence or confocal microscopy. Alexa or Jackson Immunore- entirely nuclear when co-expressed with Hdm2 in U2OS (Fig. search secondary antibodies were used for confocal or standard micros- copy, respectively. 2, c– e and o) or Saos-2 cells (not shown). Similar results were Staining of p53 was carried out using the anti-p53 polyclonal FL-393 obtained using a Hdmx-Myc tagged construct (not shown). This or anti-p53 monoclonal DO-1 (Santa Cruz Biotechnology), or, to detect effect did not depend on the ability of Hdm2 to bind p53 since HA-p53, the anti-HA polyclonal antibody HA.11 (Babco) was used. the p53 binding-defective mutant, Hdm2 G58A (22), was still Mdm2 staining was carried out using the anti-Mdm2 monoclonal anti- able to recruit Hdmx into the nucleus (Fig. 2o). body SMP14 or the polyclonal antibody N20 (Santa Cruz Biotechnolo- More importantly this effect depends on the ability of Hdm2 gy). The Myc-tagged Hdmx proteins were detected using an anti-Myc to interact with Hdmx. Indeed, deletion of the RING domains of (9E10) monoclonal or polyclonal (Upstate Biotechnology) antibody and/or the previously described anti-Hdmx mouse monoclonal antibody Hdm2 (Hdm2R) or Hdmx (Hdmx R and R-NLS) abrogated 6B1A (11) kindly provided by A. Jochemsen. their ability to form heterodimers (Fig. 2p) and the nuclear Luciferase Assay—U2OS cells were grown on six-well plates and recruitment of Hdmx by co-expressed Hdm2 (Fig. 2, f– k and o). transfected as outlined above. Luciferase analyses were performed with Moreover, the putative NLS motif of Hdmx appears to be a commercial kit (Promega), and activity was measured in a luminom- dispensable for its recruitment into the nucleus (Fig. 2o), eter. To correct for transfection efficiency, 1 g of CMVlacZ was co- whereas this effect is dependent on the integrity of the Hdm2 transfected in all cases, and luciferase values were corrected for -ga- lactosidase activity. NLS (Fig. 2, l–n and o) (3) further supporting that the observed Immunoprecipitation and Western Blotting—Cells were rinsed with effect on Hdmx localization is mediated by a direct interaction phosphate-buffered saline and scraped in the lysis buffer (50 mM Hepes, with Hdm2. M NaCl, 1 mM EDTA, 2.5 mM EGTA, 2 mM dithiothreitol, pH 7.5, 150 m Since p53 binds Hdmx and possesses a nuclear cytoplasmic 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride, 0.4 units/ml apro- shuttling activity independent of Hdm2 (23, 24), we tested tinin, 10 mM -glycerophosphate, 1 mM NaF, 0.1 mM NaVO )  10% whether p53 can also recruit Hdmx in the nucleus. As shown, glycerol and sonicated 2  7 s (Sonicator, Ultrasonic processor XL, Heat overexpression of wild-type p53 had a minor, yet reproducible, Systems, 12–14% power). Debris were removed by sedimentation at 4 °C (20 min at 14,000 rpm). For immunoprecipitation assays, cell delocalization effect on Hdmx (Fig. 2o). Expression of a p53 lysates (250 g of protein) were incubated with 1–3 g of the appro- DNA binding-defective mutant (173L) was unable to alter the priate antibody (9E10 or SMP14) for2hat4 °C. Following the addition Hdmx localization pattern (Fig. 2o), thus suggesting that the l of a protein A-Sepharose beads mixture, the reactions were of 30 slight nuclear accumulation of Hdmx following p53 overexpres- incubated for1hat4 °C; the beads were then washed three times in the sion is caused by the increased expression of a p53 target gene. lysis buffer and resuspended in 30 l of SDS sample loading buffer. The Based on the data above, we propose that this effect is medi- immunoprecipitates or the protein extracts (for straight Western blot analysis) were resolved by SDS-PAGE and transferred to nitrocellulose ated by the transcriptional activation of endogenous Hdm2. membrane (Schleicher & Schuell). The proteins of interest were de- Hdmx Increases Hdm2 Protein Levels and Inhibits Hdm2- tected by incubation of the membrane with primary specific antibody as mediated p53 Ubiquitination—It is generally accepted that indicated below in 5% milk followed by a horseradish peroxidase- Hdm2-mediated ubiquitination of p53 is essential for its deg- conjugated anti-mouse or anti-rabbit antibody and a chemiluminescent radation. Since binding of both Mdmx and Hdmx to Mdm2 substrate (ECL, Amersham Biosciences, Inc.). The membrane was probed increases the steady-state level of Mdm2 without affecting p53 with either an anti-HA monoclonal antibody (HA.11 from Babco) to detect stability (13, 15), one obvious possibility is that Hdmx binding the transfected p53 proteins (wild type and K305N) or the anti-p53 mono- clonal antibody (DO-1). The Mdm2 antibody SMP14 (Santa Cruz Biotech- inhibits the Hdm2 E3 ligase function. This is supported by the 7320 Regulation of p53 Stability and Activity by Nuclear Hdmx finding that Hdmx contacts Hdm2 in its RING-finger domain, a region essential for its ubiquitin ligase activity (6, 7). We therefore investigated the levels of p53 ubiquitination in the presence of overexpressed Hdm2 and Hdmx by immuno- blotting analysis (Fig. 3). As expected, overexpressed Hdm2 promoted ubiquitination of endogenous p53. This effect was dose-dependent (Fig. 3, lanes 2 and 3) and based on the ability of Hdm2 to bind p53 (G58A mutant, Fig. 3, lane 4). Expression of Hdmx instead had no measurable effect on p53 ubiquitina- tion (Fig. 3, lane 5), indicating that Hdmx by itself does not have ubiquitin ligase activity toward p53. Co-transfection of Hdmx and Hdm2 resulted in stabilization of the Hdm2 protein, as expected, that was dependent on the integrity of the Hdmx RING domain (Fig. 3, compare lanes 2 with 6, 7 and 2 with 8, 9). In these conditions, Hdmx did not detectably change the overall pattern of Hdm2-dependent ubiquitination of p53. How- ever, in evaluating the effect of Hdmx on Hdm2-dependent p53 ubiquitination, one should consider the property of Hdmx to increase the protein levels of Hdm2. To this end, we set up experimental conditions that allowed the same amount of Hdm2 protein to be expressed even in the presence of increas- ing Hdmx protein concentrations (Fig. 3, lanes 10 and 11). Under these conditions, a significant reduction of p53 ubiquiti- nation was observed in the presence of high Hdmx protein levels. Notably, expression of the HdmxR mutant did not significantly interfere with Hdm2-mediated p53 ubiquitination (Fig. 3, lanes 8 and 9). Similar results were obtained analyzing the status of ubiquitination of exogenous p53 in different cel- lular contexts such as in U2OS, Soas-2, and p53xMdm2 knock- out murine embryonic fibroblasts (HA-p53) (not shown). To- gether these data indicate that Hdmx has the potential to inhibit Hdm2 ligase activity. However, the concomitant ability of Hdmx to stabilize the Hdm2 protein suggests that, under physiological conditions, inhibition of the Hdm2 ligase activity by Hdmx is not sufficient to account for the inhibition of Hdm2- mediated p53 degradation by Hdmx. Hdmx Blocks Hdm2-mediated Nuclear Export of p53—It is well documented that wild-type p53 must reach the cytoplasm to be efficiently degraded (2, 4, 5, 24, 25). An alternative ex- planation for the stabilization of p53 is, therefore, that Hdmx interferes with the Hdm2-dependent p53 nuclear export. To test this possibility, we investigated the reciprocal effects of Hdm2, Hdmx, and p53 on their subcellular localization. Overexpressed p53 was almost exclusively nuclear and, when expressed with Hdm2, was partly relocalized to the cy- toplasm as expected (4, 5) (Fig. 4, a–f and s). Strikingly, co- expression of Hdmx inhibited the Hdm2-induced cytoplasmic delocalization of p53 (Fig. 4, g–j and s), indicating that nuclear Hdmx can block the Hdm2-dependent nuclear export of p53. p53 export, however, was not efficiently perturbed by Hdmx when co-expressed with the Hdm2 NLS mutant. Importantly we have shown earlier that the nuclear recruitment of Hdmx by Hdm2 NLS is only partial. Mutants of Hdmx unable to bind Hdm2, such as Hdmx R and R-NLS, were incapable of revers- FIG.1. Cellular localization of exogenous Hdmx wild type and mutants in NIH-3T3, Saos-2, and U2OS cells. a– c, U2OS, Saos-2, ing the Hdm2-induced export of p53 (Fig. 4, k–n and s) even if and NIH-3T3 cells were transfected with a plasmid expressing Myc- a significant fraction of these mutants are found in the nucleus tagged Hdmx wild type alone. d– g, U2OS cells were transfected with a (see also Fig. 1, f– h). Together these observations indicate that plasmid expressing GFP-Hdmx wild type (d), an NLS mutant (e), and the nuclear localization of Hdmx is necessary but not sufficient two C-terminal truncated mutants, R (f) and R-NLS (g). After 24 h, cells were fixed and stained for Hdmx (red) using the anti-Myc mono- to reverse Hdm2-induced nuclear export and that this effect is clonal antibody 9E10 (a– c) by indirect immunofluorescence. Cells were dependent on the ability of Hdmx to interact with Hdm2. examined for Cy3 fluorescence (a– c) and for GFP-Hdmx fluorescence Finally it has been shown that nuclear export of p53 is not (green)(d– g). DNA (blue) is stained with 4,6-diamidino-2-phenylindole required for its ubiquitination and that a p53 cytoplasmic mu- (a– g). h, the Hdmx staining pattern in cells (Saos-2 and U2OS) trans- fected with the indicated Hdmx constructs was scored for more than 100 tant (K305N) can be ubiquitinated and degraded by Hdm2 NLS cells in two separate experiments; values are means  S.D. Cells were (26). Our data suggest that stabilization of p53 by Hdmx is the scored as having fluorescence exclusively in the nucleus (N) that was consequence of its ability to block p53 nuclear export. There- stronger in the nucleus (N/{C}), equal in the nucleus and cytoplasm fore, Hdmx should be unable to reverse the Hdm2-dependent (N/C), or stronger in the cytoplasm ({N}/C  C). Regulation of p53 Stability and Activity by Nuclear Hdmx 7321 FIG.3. Effects of Hdmx on Mdm2-mediated p53 ubiquitination. Phoenix cells were transfected with DNAs encoding Hdm2 wild type and mutants alone (lanes 2, 3, and 4) or in the presence of either Myc-GFPHdmx wild type (lanes 6, 7, 10, and 11) or Myc-GFPHdmxR (lanes 8 and 9). Cells were also transfected with DNA expressing Myc- GFPHdmx alone (lane 5). The amount of DNA (g) transfected is indicated. Lanes 1 correspond to nontransfected cells. The protein ex- tracts were prepared 24 h later and examined by immunoblot analysis with the p53 monoclonal antibody DO-1. The ladder of bands indicated is endogenous ubiquitinated p53 ((Ub)n-p53). Hdm2 and Hdmx protein levels were monitored in the transfected cells by stripping the blot and reprobing with the Mdm2 monoclonal antibody SMP14 and the anti- Myc monoclonal antibody 9E10. degradation of cytoplasmic p53. To test this hypothesis, we examined the levels of p53 and p53(K305N) following overex- pression of Hdm2 or Hdm2 NLS, either alone or in the presence of Hdmx, in the U2OS (Fig. 5, a and b) or Soas-2 cells and in the p53xMdm2 knockout murine embryonic fibroblasts. In the ab- sence of Hdmx, Hdm2 and Hdm2 NLS induced ubiquitination (not shown) and subsequent degradation of p53 or p53(K305N), respectively. However, co-expression of Hdmx appeared to sta- bilize wild-type p53 but had no effect on the p53 cytoplasmic mutant, indicating that the ability of Hdmx to inhibit Hdm2 function is restricted to the nuclear pool of Hdmx. In contrast, Hdmx expression resulted in the stabilization of both Hdm2 and Hdm2 NLS. This last observation suggests that the Hdmx- dependent stabilization of Hdm2 and p53 are achieved through different mechanisms. Nuclear Hdmx Down-regulates p53-dependent Transcrip- tion—Overexpression of Hdmx results in a modest, yet consist- ent, decrease of the p53 transcriptional regulation ability (11– 13, 20). At first analysis, this effect of Hdmx on p53-dependent transcription contrasts with our observation that overex- for Hdm2 (red) using the anti-Mdm2 monoclonal antibody SMP14 by indirect immunofluorescence. Cells were examined for Cy3 fluorescence and for GFP-Hdmx fluorescence (green). DNA (blue) is stained with 4,6-diamidino-2-phenylindole. o, the GFPHdmx pattern in U2OS cells transfected with the indicated GFPHdmx, Hdm2, and p53 constructs was scored for more than 100 cells in three separate experiments; values are means  S.D. Cells were scored as having fluorescence exclusively in the nucleus (N), stronger in the nucleus (N/{C}), equal in the nucleus and cytoplasm (N/C), or stronger in the cytoplasm ({N}/C C). Only cells expressing both Hdmx and Hdm2 proteins were consid- ered for the analysis. (For I, 0.5 g of GFPHdmx was transfected with 0.25 g of Hdm2, and for all the other transfection conditions, 0.5 gof GFPHdmx-expressing constructs was transfected with 1 g of Hdm2- or p53-expressing constructs.) p, co-immunoprecipitation of Hdmx species with wild-type or mutant Hdm2 proteins. Lysates from U2O cells trans- fected with the indicated constructs were treated with anti-Myc anti- FIG.2. Cellular redistribution of exogenous Hdmx wild type body (9E10) and protein A beads. Immunoprecipitates were analyzed by and mutants upon Hdm2 or p53 co-expression. a–f, U2OS cells Western blotting using anti-Hdm2 antibody (upper panel). Comparable were transfected with a plasmid expressing GFPHdmx (a) and Hdm2 Hdmx and Hdm2 transfection efficiencies were confirmed by Western (b) wild type alone or with plasmids expressing GFPHdmx and Hdm2 blotting of lysate samples using anti-Myc antibody (middle panel) and (c– e), GFPHdmx and Hdm2R (f– h), GFPHdmxR and Hdm2 (i– k), or anti-Hdm2 antibody (lower panel). The Hdm2 protein levels are in- GFPHdmx and Hdm2 NLS (l–n). After 24 h, cells were fixed and stained creased in the presence of wild-type Hdmx. Ip, immunoprecipitation. 7322 Regulation of p53 Stability and Activity by Nuclear Hdmx FIG.5. Hdmx does not reverse Hdm2NLS-mediated degrada- tion of cytoplasmic p53(K305N). a, U2OS cells were transfected with 2 g of HA-p53 alone or together with 5 g of Hdm2 in the absence or presence of either 5 g of Myc-GFPHdmx or 5 g of Myc-GFPHdmxR. b, U2OS cells were transfected with 2 g of HA-p53(K305N) alone or together with 5 g of Hdm2NLS in the absence or presence of either 5 g of Myc-GFPHdmx or 5 g of Myc-GFPHdmxR, all in combination with 1 g of Myc-EGFP. Cell lysates were analyzed by immunoblot analysis with the anti-HA monoclonal antibody (HA.11) to detect p53 levels, with the anti-Hdm2 SMP14 antibody to detect the Hdm2 pro- teins, and with the anti-Myc antibody (9E10) to detect the Hdmx pro- teins. Equal transfection efficiencies were confirmed using the anti-Myc antibody (9E10) for the detection of Myc-EGFP. FIG.4. Hdm2 does not relocalize p53 in the presence of Hdmx. a–r, U2OS cells were transfected with a plasmid expressing p53 alone (a– c) or with plasmids expressing p53 and Hdm2 (d–f); p53, Hdm2, and GFPHdmx (g–j); p53, Hdm2, and GFPHdmxR (k–n); or p53, Hdm2R, and GFPHdmx (o–r). After 24 h, cells were fixed and stained for p53 (red) Hdm2 (blue) using the anti-p53 polyclonal antibody FL393 and the anti-Mdm2 monoclonal antibody SMP14 by indirect immunofluores- FIG.6. Hdm2-dependent nuclear recruitment of Hdmx is nec- cence. Cells were examined for Cy3 and Alexa (blue) fluorescence and essary for efficient inhibition of p53 transcriptional activity. for GFP-Hdmx fluorescence (green). s, the staining pattern for p53 in U2OS cells were transfected with 400 ng of the p53 reporter construct U2OS cells expressing the indicated GFPHdmx, Hdm2, and p53 con- (pG13Luc) and DNAs expressing Hdm2 wild type and p53 binding- structs were scored for more than 100 cells in three separate experi- defective mutant G58A (300 ng), GFP-Hdmx wild type, p53 binding- ments; values are means  S.D. Cells were scored as described in Fig. defective mutant (Hdmxp53bs), and RING domain-deleted mutant 2o. Only cells expressing the three proteins were considered for the (HdmxR) alone (800 ng) or a combination of both wild-type proteins as analysis. Hdmx does not prevent Hdm2NLS-induced p53(K305N) deg- outlined in the transfection scheme. Luciferase activity is shown as -fold radation. Dapi, 4,6-diamidino-2-phenylindole. induction compared with no transfection of the reporter with the S.D. of triplicates. pressed Hdmx is mainly localized to the cytoplasm. However, in all reported experiments but one (12), the effect of Hdmx was HdmxR, which is partly localized in the nucleus (see Fig. 1, f investigated by transient transfection using various p53-defi- and h), slightly, yet reproducibly, reduced the p53 transcrip- cient cell lines (Hep3B, H1299, Calu, and p53/ murine em- tional ability. Strikingly, simultaneous expression of Hdm2 bryonic fibroblasts), overexpressed p53, and various reporter and Hdmx resulted in an almost complete repression of p53 constructs (14, 15). In these experimental conditions, overex- pG13Luc transcription. These findings demonstrate that regu- pressed p53 might trigger recruitment of Hdmx into the nu- lation of Hdmx by Hdm2 is an absolute requirement for the cleus indirectly through its effect on the endogenous Hdm2 efficient regulation of p53 activity by Hdmx and suggest that promoter. To test this possibility, we have transfected U2OS only the nuclear fraction of Hdmx regulates p53-dependent cells, which express endogenous p53, with the reporter con- transcription. struct pG13Luc containing 13 copies of the p53 consensus se- DISCUSSION quence upstream of a minimal promoter driving transcription of the luciferase gene. As expected, endogenous p53 is able to We have shown here that exogenous Hdmx, when expressed sustain a significant basal level of expression of the reporter alone, is mainly cytoplasmic, while, in the presence of Hdm2, it construct (Fig. 6). Overexpression of Hdm2, but not of Hdmx, becomes mainly nuclear. Nuclear relocalization of Hdmx by significantly inhibited pG13Luc transcription. Expression of overexpressed Hdm2 depends on their reciprocal interaction as Regulation of p53 Stability and Activity by Nuclear Hdmx 7323 suggested by the finding that this phenomenon requires the Hdmx, however, remains to be elucidated. Recent reports, in- integrity of the RING domain of both proteins. Surprisingly, cluding observations presented here, indicate that stabilized however, RING mutants of Hdmx (R and R-NLS) were found p53 protein is not able to activate transcription, and it was in both the nucleus and cytoplasm even when expressed alone proposed that Hdmx secures the presence of a pool of inactive suggesting that Hdmx binds other cellular factor(s) that might p53 that can be instantly activated when needed. Overex- sequester Hdmx in the cytoplasm in the absence of Hdm2. pressed Hdmx, however, is an artificial situation, and it is not Since Hdm2 binds Hdmx in the same RING-finger region, it known whether or not it corresponds to any physiological sit- might compete for binding and displace this putative interac- uations. Indeed, Hdmx protein levels seem to be very low in all tion. Alternatively, formation of Hdmx oligomers might occur primary cells tested. On the other hand, increased Hdmx levels in the cytoplasm and inhibit the nuclear import of Hdmx by, for were found in a significant fraction of tumor cell lines when instance, masking its putative NLS, which is located in the compare with normal cells (28). In general Hdmx expression in RING-finger motif of the protein. In this context, Hdm2, which these tumor cell lines correlates with the presence of wild-type forms stable heterodimers with Hdmx (14), might disrupt the p53, suggesting that deregulated expression of Hdmx plays a formation of Hdmx oligomers. role in carcinogenesis as an alternative way to inactivate p53. We then investigated the mechanisms by which nuclear High levels of Hdmx may also explain the efficient response to Hdmx inhibits Hdm2-induced p53 degradation. Since this ef- radiation and chemotherapy displayed by a subset of tumors, fect was previously reported to be mediated by the Hdmx including germ-cell tumors, which express high levels of wild- RING-finger and to involve hetero-oligomerization with the type p53 and Hdm2 (29, 30). The now available mouse models Hdm2 RING-finger, we hypothesized that Hdmx inhibits the further support the notion that, at least during embryogenesis, ubiquitin ligase activity of Hdm2. Our data, however, are not the primary function of Hdmx is to inhibit p53 function (16). entirely consistent with this model. Indeed, as it was demon- ARF Acknowledgments—We thank A. Jochemsen, K. Helin, S. Minucci, strated earlier for p19 (27), we showed that the interaction and M. Faretta for many helpful discussions and/or critically reviewing with Hdmx reduces the ubiquitin ligase activity of Hdm2. How- this manuscript. ever, this effect is counteracted in vivo by the ability of Hdmx to stabilize Hdm2 suggesting that Hdmx stabilizes p53 through REFERENCES other mechanisms. And in fact, Hdmx is able to rescue Hdm2- 1. Amundson, S. A., Myers, T. G., and Fornace, A. J., Jr. (1998) Oncogene 17, 3287–3299 mediated degradation even in those experimental conditions 2. Roth, J., Dobbelstein, M., Freedman, D. A, Shenk, T., and Levine, A. J. (1998) where the overall levels of p53 ubiquitination are not affected EMBO J. 17, 554 –564 by Hdmx. 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The mechanism for this suppression is unknown. 29. Guillou, L., Estreicher, A., Chaubert, P., Hurlimann, J., Kurt, A. M., Metthez, G., Iggo, R., Gray, A. C., Jichlinski, P., Leisinger, H. J., and Benhattar, J. The formation of a trimeric complex could block crucial inter- (1996) Am. J. Pathol. 149, 1221–1228 actions with proteins of the transcriptional machinery. 30. Riou, G., Barrois, M., Prost, S., Terrier, M. J., Theodore, C., and Levine, A. J. The biological significance of these particular functions of (1995) Mol. Carcinog. 12, 124 –131 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Hdmx Recruitment into the Nucleus by Hdm2 Is Essential for Its Ability to Regulate p53 Stability and Transactivation

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 9, Issue of March 1, pp. 7318 –7323, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Hdmx Recruitment into the Nucleus by Hdm2 Is Essential for Its Ability to Regulate p53 Stability and Transactivation* Received for publication, September 12, 2001, and in revised form, November 7, 2001 Published, JBC Papers in Press, December 13, 2001, DOI 10.1074/jbc.M108795200 Domenico Migliorini‡, Davide Danovi‡, Emanuela Colombo‡§, Roberta Carbone‡, Pier Giuseppe Pelicci‡¶, and Jean-Christophe Marine‡** From the ‡Department of Experimental Oncology, European Institute of Oncology, 435 Via Ripamonti, 20141 Milan, Italy and the ¶FIRC Institute of Molecular Oncology, 20 Via Serio, 20139 Milan, Italy The Hdmx gene product is related to the Hdm2 onco- function of Hdm2 and p53. From a genetic standpoint, the close relationship between the murine ortholog of Hdm2, Mdm2, and protein, both of which interact with and regulate p53 stability and function. Like Hdm2, Hdmx is able to in- p53 is further supported by the striking observation that a hibit p53 transactivation; however, at variance with p53-null state can completely rescue the early-embryonic-le- Hdm2, which promotes ubiquitination, nuclear export, thal phenotype associated with Mdm2 deficiency (8, 9). and degradation of p53, Hdmx increases p53 stability. Hdmx is structurally related to Hdm2, and a significant We report here (i) that overexpressed Hdmx is cytoplas- similarity is especially notable in the p53-binding domain and mic and Hdm2 recruits Hdmx into the nucleus and (ii) in the C-terminal RING-finger domain (10). Hdmx has been that nuclear Hdmx blocks Hdm2-mediated nuclear ex- shown to bind p53 and inhibit p53 transactivation in specific port of p53 and down-regulates p53-dependent tran- cellular contexts but, unlike Hdm2, is unable to induce p53 scription. Furthermore we showed that Hdmx inhibits degradation (10, 11). Similar conclusions were drawn when Hdm2-mediated p53 ubiquitination. It appears, there- using the Hdmx murine ortholog Mdmx (12, 13). Moreover, fore, that a regulatory loop exists in which Hdm2 regu- Mdmx and Mdm2 form stable heterodimers through their C- lates the intracellular localization of Hdmx, and nuclear terminal RING-finger domains, and this interaction results in Hdmx regulates several functions of Hdm2 (ubiquitin a substantial increase in the steady-state levels of Mdm2 (14, ligase activity and p53 nuclear export). 15). Finally it has been reported that both Mdmx (13) and Hdmx (11), through a yet unknown mechanism, are able to interfere with Mdm2-mediated p53 degradation, resulting in p53 is the most frequently inactivated tumor suppressor an increase in p53 protein levels while maintaining suppres- gene in human cancer irrespective of the tumor type, site, and sion of p53 transactivation. It was therefore proposed that patient age. In cells undergoing DNA damage or responding to Hdmx (and Mdmx) secures a nuclear pool of transcriptionally certain forms of stress (replicative senescence, hypoxia, oxida- incompetent p53, which upon stress can be quickly activated. tive stress, and oncogene overexpression) the ordinarily short- Finally the first genetic evidences confirming the role of Mdmx lived p53 protein is stabilized, rapidly accumulates, and under- as a critical regulator of p53 function in vivo are now available. goes several activating post-translational modifications. In this Mdmx-mutant mice die around midgestation because of loss of context, activated p53, via its ability to regulate gene transcrip- cell proliferation, while loss of p53 completely rescues this tion, elicits either a cell cycle arrest or apoptosis (1). As it would embryonic lethality (16). These new exciting data raise the be expected, p53 actions are largely regulated by post-transla- possibility that increased Mdmx levels and the resulting inhi- tional events, including direct binding and occlusion of the bition of p53 transcriptional activity contribute to development transactivation domain, ubiquitination and degradation by the of human tumors. proteosome, and control of subcellular localization. The Hdm2 We show here that exogenous Hdmx is mainly localized in protein has been implicated in all three of these processes, most the cytoplasm of transfected cells by indirect immunofluores- recently as mediator of the export of p53 from the nucleus, cence, while when co-expressed with Hdm2, Hdmx was then which enhances the degradation of p53 in cytoplasmic proteo- found almost exclusively in the nucleus. We show that this somes (2–5). Hdm2 functions as an E3 ubiquitin ligase for p53, effect is dependent on the integrity of the RING-finger domain and the C-terminal RING-finger domain of Hdm2 is critical for of both Hdmx and Hdm2 proteins. Furthermore, we demon- ligase activity (6, 7). Interestingly the Hdm2 gene itself is a strate that the Hdm2-dependent nuclear recruitment of Hdmx transcriptional target of p53. Thus, there is evidence for an is indispensable for its ability to exert its two previously re- autoregulatory feedback loop involving the expression and ported activities, regulation of p53 steady state levels and block of p53 transcriptional ability. * This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro and European Community. The costs of EXPERIMENTAL PROCEDURES publication of this article were defrayed in part by the payment of page Plasmids—DNAs encoding wild-type human p53 and Hdm2 in charges. This article must therefore be hereby marked “advertisement” pCMV and the p53 reporter plasmid pG13Luc were a gift from B. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Vogelstein. The HA-tagged wild-type p53, p53 K305N (nuclear localiza- § Recipient of a fellowship from the Giancarla Vollaro Foundation. tion signal (NLS)) (17), and p53 173L (DNA binding-defective) mutants To whom correspondence may be addressed. Tel.: 39-0257489834; Fax: 39-0257489851; E-mail: [email protected]. were provided to us by K. Maki and K. Vousden; Hdm2 NLS and Hdm2 ** Recipient of a fellowship from the European Community (Marie G58A mutants were obtained from A. Levine. The pcDNA3.1 Myc- Curie Fellowship). To whom correspondence may be addressed. Tel.: tagged Hdmx constructs (wild type and N-terminal truncation mutant 39-0257489834; Fax: 39-0257489851; E-mail: [email protected]. The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; NLS, nuclear localization signal; GFP, green fluo- D. Migliorini, D. Danovi, E. Lazzerini-Denchi, A. Jochemsen, M. rescent protein. Capilo, A. Gobbi, K. Helin, P. G. Pelicci, and J.-C. Marine, submitted. 7318 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Regulation of p53 Stability and Activity by Nuclear Hdmx 7319 (amino acids 101– 490), also designated Hdmxp53bs) were described nology) was used to detect Hdm2, and an anti-Myc (9E10) monoclonal previously (15) and kindly provided by D. George. Full-length Hdmx antibody was used to detect the Myc-tagged Hdmx proteins. and the N-terminal truncated (amino acids 101– 490) Hdmx fragment were released from the above constructs using a BamHI-XhoI restric- RESULTS tion digest and subcloned into the pcDNA3X()MyEGFP to generate Hdm2 Recruits Hdmx into the Nucleus—Determination of Myc-GFP-tagged Hdmx and Myc-GFP-Hdmx-(101– 490). The tagged the subcellular localization of transfected Hdmx and/or Mdmx Hdmx (R-NLS (amino acids 2– 430) and R (amino acids 2– 451)) in different cellular contexts has led to conflicting observations deletion mutants, deleted either of the entire RING-finger region including the putative NLS or most of it to leave the putative NLS (13, 18 –21). To revisit this issue, we determined the localiza- intact, were generated by PCR amplification of the corresponding tion of exogenous Myc-tagged (N-terminal) Hdmx and GFP- fragments using a 5-primer including a BamHI site and 3-primers Hdmx in U2OS, Soas-2, and NIH-3T3 cell lines by immunoflu- including XhoI sites. The PCR products were subcloned into orescence. In all cell types analyzed, both Hdmx-tagged pcDNA3.1Myc and pcDNA3X()MyEGFP to generate the Myc-tagged proteins were mainly found in the cytoplasm (Fig. 1, a– d). The Hdmx (R-NLS) and the Myc-tagged Hdmx (R), and the Myc-GFP- intensity and the frequency of the nuclear staining slightly tagged Hdmx (R-NLS) and the Myc-GFP-tagged Hdmx (R), respec- tively. Hdmx NLS mutants (KRPRD 3 ATPLD) were generated varied from one cell line to another but always appeared much using the QuikChange mutagenesis kit (Stratagene). The following less frequent (5% of cells) and less intense than the cytoplas- oligonucleotide and its complement were used for the mutagenesis in mic staining (Fig. 1h). the context of the Myc-tagged Hdmx and Myc-GFP-tagged Hdmx Sequence analysis of Hdmx reveals a single putative NLS in constructs: 5-tgtgaggcaacaccactagacggg. the C-terminal RING-finger domain (KRPRD). Point mutations Tissue Culture and Immunofluorescence—U2OS, Soas-2, NIH-3T3, in this motif (KTLLD) had no major effect on the cellular and Phoenix (derived from the 293T cell line) cell lines were grown in distribution of Hdmx (Fig. 1, e and h). Deletion of the C- Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bo- vine serum, and 100 g/ml penicillin and streptomycin. Transfections terminal RING domain instead resulted in both nuclear and were carried out using the calcium phosphate method or the FuGENE cytoplasmic localization of Hdmx (Hdmx R-NLS and R, car- transfection reagent (Roche Molecular Biochemicals). For immunofluo- rying deletions of either the entire RING domain, including the rescence staining, cells were transfected at 80% confluency on glass putative NLS, or most of it to leave the putative NLS intact) coverslips in six-well dishes and fixed 24 h after addition of the DNA (Fig. 1, f– h). It appears, therefore, that the putative NLS of mixtures. Fixed coverslips were washed twice in phosphate-buffered Hdmx is not functional and that its C-terminal RING-finger saline, permeabilized in 0.15% Triton X-100 for 5 min, and incubated in blocking buffer (phosphate-buffered saline  2% bovine serum albumin) motif is an important determinant of the cellular distribution of for 30 min. Cells were than incubated in blocking buffer containing Hdmx. primary antibody for1has indicated below and then washed exten- Since Hdm2 is a nuclear cytoplasmic shuttling protein (2, 3) sively in phosphate-buffered saline before incubation with the appro- and Mdm2 (the mouse ortholog of Hdm2) forms stable hetero- priate fluorochrome-conjugated secondary antibody for 1 h. Stained oligomers with Mdmx (14, 15), we investigated whether Hdm2 cells were mounted on glass slides and examined using standard im- levels affect Hdmx localization. Strikingly, GFP-Hdmx was munofluorescence or confocal microscopy. Alexa or Jackson Immunore- entirely nuclear when co-expressed with Hdm2 in U2OS (Fig. search secondary antibodies were used for confocal or standard micros- copy, respectively. 2, c– e and o) or Saos-2 cells (not shown). Similar results were Staining of p53 was carried out using the anti-p53 polyclonal FL-393 obtained using a Hdmx-Myc tagged construct (not shown). This or anti-p53 monoclonal DO-1 (Santa Cruz Biotechnology), or, to detect effect did not depend on the ability of Hdm2 to bind p53 since HA-p53, the anti-HA polyclonal antibody HA.11 (Babco) was used. the p53 binding-defective mutant, Hdm2 G58A (22), was still Mdm2 staining was carried out using the anti-Mdm2 monoclonal anti- able to recruit Hdmx into the nucleus (Fig. 2o). body SMP14 or the polyclonal antibody N20 (Santa Cruz Biotechnolo- More importantly this effect depends on the ability of Hdm2 gy). The Myc-tagged Hdmx proteins were detected using an anti-Myc to interact with Hdmx. Indeed, deletion of the RING domains of (9E10) monoclonal or polyclonal (Upstate Biotechnology) antibody and/or the previously described anti-Hdmx mouse monoclonal antibody Hdm2 (Hdm2R) or Hdmx (Hdmx R and R-NLS) abrogated 6B1A (11) kindly provided by A. Jochemsen. their ability to form heterodimers (Fig. 2p) and the nuclear Luciferase Assay—U2OS cells were grown on six-well plates and recruitment of Hdmx by co-expressed Hdm2 (Fig. 2, f– k and o). transfected as outlined above. Luciferase analyses were performed with Moreover, the putative NLS motif of Hdmx appears to be a commercial kit (Promega), and activity was measured in a luminom- dispensable for its recruitment into the nucleus (Fig. 2o), eter. To correct for transfection efficiency, 1 g of CMVlacZ was co- whereas this effect is dependent on the integrity of the Hdm2 transfected in all cases, and luciferase values were corrected for -ga- lactosidase activity. NLS (Fig. 2, l–n and o) (3) further supporting that the observed Immunoprecipitation and Western Blotting—Cells were rinsed with effect on Hdmx localization is mediated by a direct interaction phosphate-buffered saline and scraped in the lysis buffer (50 mM Hepes, with Hdm2. M NaCl, 1 mM EDTA, 2.5 mM EGTA, 2 mM dithiothreitol, pH 7.5, 150 m Since p53 binds Hdmx and possesses a nuclear cytoplasmic 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride, 0.4 units/ml apro- shuttling activity independent of Hdm2 (23, 24), we tested tinin, 10 mM -glycerophosphate, 1 mM NaF, 0.1 mM NaVO )  10% whether p53 can also recruit Hdmx in the nucleus. As shown, glycerol and sonicated 2  7 s (Sonicator, Ultrasonic processor XL, Heat overexpression of wild-type p53 had a minor, yet reproducible, Systems, 12–14% power). Debris were removed by sedimentation at 4 °C (20 min at 14,000 rpm). For immunoprecipitation assays, cell delocalization effect on Hdmx (Fig. 2o). Expression of a p53 lysates (250 g of protein) were incubated with 1–3 g of the appro- DNA binding-defective mutant (173L) was unable to alter the priate antibody (9E10 or SMP14) for2hat4 °C. Following the addition Hdmx localization pattern (Fig. 2o), thus suggesting that the l of a protein A-Sepharose beads mixture, the reactions were of 30 slight nuclear accumulation of Hdmx following p53 overexpres- incubated for1hat4 °C; the beads were then washed three times in the sion is caused by the increased expression of a p53 target gene. lysis buffer and resuspended in 30 l of SDS sample loading buffer. The Based on the data above, we propose that this effect is medi- immunoprecipitates or the protein extracts (for straight Western blot analysis) were resolved by SDS-PAGE and transferred to nitrocellulose ated by the transcriptional activation of endogenous Hdm2. membrane (Schleicher & Schuell). The proteins of interest were de- Hdmx Increases Hdm2 Protein Levels and Inhibits Hdm2- tected by incubation of the membrane with primary specific antibody as mediated p53 Ubiquitination—It is generally accepted that indicated below in 5% milk followed by a horseradish peroxidase- Hdm2-mediated ubiquitination of p53 is essential for its deg- conjugated anti-mouse or anti-rabbit antibody and a chemiluminescent radation. Since binding of both Mdmx and Hdmx to Mdm2 substrate (ECL, Amersham Biosciences, Inc.). The membrane was probed increases the steady-state level of Mdm2 without affecting p53 with either an anti-HA monoclonal antibody (HA.11 from Babco) to detect stability (13, 15), one obvious possibility is that Hdmx binding the transfected p53 proteins (wild type and K305N) or the anti-p53 mono- clonal antibody (DO-1). The Mdm2 antibody SMP14 (Santa Cruz Biotech- inhibits the Hdm2 E3 ligase function. This is supported by the 7320 Regulation of p53 Stability and Activity by Nuclear Hdmx finding that Hdmx contacts Hdm2 in its RING-finger domain, a region essential for its ubiquitin ligase activity (6, 7). We therefore investigated the levels of p53 ubiquitination in the presence of overexpressed Hdm2 and Hdmx by immuno- blotting analysis (Fig. 3). As expected, overexpressed Hdm2 promoted ubiquitination of endogenous p53. This effect was dose-dependent (Fig. 3, lanes 2 and 3) and based on the ability of Hdm2 to bind p53 (G58A mutant, Fig. 3, lane 4). Expression of Hdmx instead had no measurable effect on p53 ubiquitina- tion (Fig. 3, lane 5), indicating that Hdmx by itself does not have ubiquitin ligase activity toward p53. Co-transfection of Hdmx and Hdm2 resulted in stabilization of the Hdm2 protein, as expected, that was dependent on the integrity of the Hdmx RING domain (Fig. 3, compare lanes 2 with 6, 7 and 2 with 8, 9). In these conditions, Hdmx did not detectably change the overall pattern of Hdm2-dependent ubiquitination of p53. How- ever, in evaluating the effect of Hdmx on Hdm2-dependent p53 ubiquitination, one should consider the property of Hdmx to increase the protein levels of Hdm2. To this end, we set up experimental conditions that allowed the same amount of Hdm2 protein to be expressed even in the presence of increas- ing Hdmx protein concentrations (Fig. 3, lanes 10 and 11). Under these conditions, a significant reduction of p53 ubiquiti- nation was observed in the presence of high Hdmx protein levels. Notably, expression of the HdmxR mutant did not significantly interfere with Hdm2-mediated p53 ubiquitination (Fig. 3, lanes 8 and 9). Similar results were obtained analyzing the status of ubiquitination of exogenous p53 in different cel- lular contexts such as in U2OS, Soas-2, and p53xMdm2 knock- out murine embryonic fibroblasts (HA-p53) (not shown). To- gether these data indicate that Hdmx has the potential to inhibit Hdm2 ligase activity. However, the concomitant ability of Hdmx to stabilize the Hdm2 protein suggests that, under physiological conditions, inhibition of the Hdm2 ligase activity by Hdmx is not sufficient to account for the inhibition of Hdm2- mediated p53 degradation by Hdmx. Hdmx Blocks Hdm2-mediated Nuclear Export of p53—It is well documented that wild-type p53 must reach the cytoplasm to be efficiently degraded (2, 4, 5, 24, 25). An alternative ex- planation for the stabilization of p53 is, therefore, that Hdmx interferes with the Hdm2-dependent p53 nuclear export. To test this possibility, we investigated the reciprocal effects of Hdm2, Hdmx, and p53 on their subcellular localization. Overexpressed p53 was almost exclusively nuclear and, when expressed with Hdm2, was partly relocalized to the cy- toplasm as expected (4, 5) (Fig. 4, a–f and s). Strikingly, co- expression of Hdmx inhibited the Hdm2-induced cytoplasmic delocalization of p53 (Fig. 4, g–j and s), indicating that nuclear Hdmx can block the Hdm2-dependent nuclear export of p53. p53 export, however, was not efficiently perturbed by Hdmx when co-expressed with the Hdm2 NLS mutant. Importantly we have shown earlier that the nuclear recruitment of Hdmx by Hdm2 NLS is only partial. Mutants of Hdmx unable to bind Hdm2, such as Hdmx R and R-NLS, were incapable of revers- FIG.1. Cellular localization of exogenous Hdmx wild type and mutants in NIH-3T3, Saos-2, and U2OS cells. a– c, U2OS, Saos-2, ing the Hdm2-induced export of p53 (Fig. 4, k–n and s) even if and NIH-3T3 cells were transfected with a plasmid expressing Myc- a significant fraction of these mutants are found in the nucleus tagged Hdmx wild type alone. d– g, U2OS cells were transfected with a (see also Fig. 1, f– h). Together these observations indicate that plasmid expressing GFP-Hdmx wild type (d), an NLS mutant (e), and the nuclear localization of Hdmx is necessary but not sufficient two C-terminal truncated mutants, R (f) and R-NLS (g). After 24 h, cells were fixed and stained for Hdmx (red) using the anti-Myc mono- to reverse Hdm2-induced nuclear export and that this effect is clonal antibody 9E10 (a– c) by indirect immunofluorescence. Cells were dependent on the ability of Hdmx to interact with Hdm2. examined for Cy3 fluorescence (a– c) and for GFP-Hdmx fluorescence Finally it has been shown that nuclear export of p53 is not (green)(d– g). DNA (blue) is stained with 4,6-diamidino-2-phenylindole required for its ubiquitination and that a p53 cytoplasmic mu- (a– g). h, the Hdmx staining pattern in cells (Saos-2 and U2OS) trans- fected with the indicated Hdmx constructs was scored for more than 100 tant (K305N) can be ubiquitinated and degraded by Hdm2 NLS cells in two separate experiments; values are means  S.D. Cells were (26). Our data suggest that stabilization of p53 by Hdmx is the scored as having fluorescence exclusively in the nucleus (N) that was consequence of its ability to block p53 nuclear export. There- stronger in the nucleus (N/{C}), equal in the nucleus and cytoplasm fore, Hdmx should be unable to reverse the Hdm2-dependent (N/C), or stronger in the cytoplasm ({N}/C  C). Regulation of p53 Stability and Activity by Nuclear Hdmx 7321 FIG.3. Effects of Hdmx on Mdm2-mediated p53 ubiquitination. Phoenix cells were transfected with DNAs encoding Hdm2 wild type and mutants alone (lanes 2, 3, and 4) or in the presence of either Myc-GFPHdmx wild type (lanes 6, 7, 10, and 11) or Myc-GFPHdmxR (lanes 8 and 9). Cells were also transfected with DNA expressing Myc- GFPHdmx alone (lane 5). The amount of DNA (g) transfected is indicated. Lanes 1 correspond to nontransfected cells. The protein ex- tracts were prepared 24 h later and examined by immunoblot analysis with the p53 monoclonal antibody DO-1. The ladder of bands indicated is endogenous ubiquitinated p53 ((Ub)n-p53). Hdm2 and Hdmx protein levels were monitored in the transfected cells by stripping the blot and reprobing with the Mdm2 monoclonal antibody SMP14 and the anti- Myc monoclonal antibody 9E10. degradation of cytoplasmic p53. To test this hypothesis, we examined the levels of p53 and p53(K305N) following overex- pression of Hdm2 or Hdm2 NLS, either alone or in the presence of Hdmx, in the U2OS (Fig. 5, a and b) or Soas-2 cells and in the p53xMdm2 knockout murine embryonic fibroblasts. In the ab- sence of Hdmx, Hdm2 and Hdm2 NLS induced ubiquitination (not shown) and subsequent degradation of p53 or p53(K305N), respectively. However, co-expression of Hdmx appeared to sta- bilize wild-type p53 but had no effect on the p53 cytoplasmic mutant, indicating that the ability of Hdmx to inhibit Hdm2 function is restricted to the nuclear pool of Hdmx. In contrast, Hdmx expression resulted in the stabilization of both Hdm2 and Hdm2 NLS. This last observation suggests that the Hdmx- dependent stabilization of Hdm2 and p53 are achieved through different mechanisms. Nuclear Hdmx Down-regulates p53-dependent Transcrip- tion—Overexpression of Hdmx results in a modest, yet consist- ent, decrease of the p53 transcriptional regulation ability (11– 13, 20). At first analysis, this effect of Hdmx on p53-dependent transcription contrasts with our observation that overex- for Hdm2 (red) using the anti-Mdm2 monoclonal antibody SMP14 by indirect immunofluorescence. Cells were examined for Cy3 fluorescence and for GFP-Hdmx fluorescence (green). DNA (blue) is stained with 4,6-diamidino-2-phenylindole. o, the GFPHdmx pattern in U2OS cells transfected with the indicated GFPHdmx, Hdm2, and p53 constructs was scored for more than 100 cells in three separate experiments; values are means  S.D. Cells were scored as having fluorescence exclusively in the nucleus (N), stronger in the nucleus (N/{C}), equal in the nucleus and cytoplasm (N/C), or stronger in the cytoplasm ({N}/C C). Only cells expressing both Hdmx and Hdm2 proteins were consid- ered for the analysis. (For I, 0.5 g of GFPHdmx was transfected with 0.25 g of Hdm2, and for all the other transfection conditions, 0.5 gof GFPHdmx-expressing constructs was transfected with 1 g of Hdm2- or p53-expressing constructs.) p, co-immunoprecipitation of Hdmx species with wild-type or mutant Hdm2 proteins. Lysates from U2O cells trans- fected with the indicated constructs were treated with anti-Myc anti- FIG.2. Cellular redistribution of exogenous Hdmx wild type body (9E10) and protein A beads. Immunoprecipitates were analyzed by and mutants upon Hdm2 or p53 co-expression. a–f, U2OS cells Western blotting using anti-Hdm2 antibody (upper panel). Comparable were transfected with a plasmid expressing GFPHdmx (a) and Hdm2 Hdmx and Hdm2 transfection efficiencies were confirmed by Western (b) wild type alone or with plasmids expressing GFPHdmx and Hdm2 blotting of lysate samples using anti-Myc antibody (middle panel) and (c– e), GFPHdmx and Hdm2R (f– h), GFPHdmxR and Hdm2 (i– k), or anti-Hdm2 antibody (lower panel). The Hdm2 protein levels are in- GFPHdmx and Hdm2 NLS (l–n). After 24 h, cells were fixed and stained creased in the presence of wild-type Hdmx. Ip, immunoprecipitation. 7322 Regulation of p53 Stability and Activity by Nuclear Hdmx FIG.5. Hdmx does not reverse Hdm2NLS-mediated degrada- tion of cytoplasmic p53(K305N). a, U2OS cells were transfected with 2 g of HA-p53 alone or together with 5 g of Hdm2 in the absence or presence of either 5 g of Myc-GFPHdmx or 5 g of Myc-GFPHdmxR. b, U2OS cells were transfected with 2 g of HA-p53(K305N) alone or together with 5 g of Hdm2NLS in the absence or presence of either 5 g of Myc-GFPHdmx or 5 g of Myc-GFPHdmxR, all in combination with 1 g of Myc-EGFP. Cell lysates were analyzed by immunoblot analysis with the anti-HA monoclonal antibody (HA.11) to detect p53 levels, with the anti-Hdm2 SMP14 antibody to detect the Hdm2 pro- teins, and with the anti-Myc antibody (9E10) to detect the Hdmx pro- teins. Equal transfection efficiencies were confirmed using the anti-Myc antibody (9E10) for the detection of Myc-EGFP. FIG.4. Hdm2 does not relocalize p53 in the presence of Hdmx. a–r, U2OS cells were transfected with a plasmid expressing p53 alone (a– c) or with plasmids expressing p53 and Hdm2 (d–f); p53, Hdm2, and GFPHdmx (g–j); p53, Hdm2, and GFPHdmxR (k–n); or p53, Hdm2R, and GFPHdmx (o–r). After 24 h, cells were fixed and stained for p53 (red) Hdm2 (blue) using the anti-p53 polyclonal antibody FL393 and the anti-Mdm2 monoclonal antibody SMP14 by indirect immunofluores- FIG.6. Hdm2-dependent nuclear recruitment of Hdmx is nec- cence. Cells were examined for Cy3 and Alexa (blue) fluorescence and essary for efficient inhibition of p53 transcriptional activity. for GFP-Hdmx fluorescence (green). s, the staining pattern for p53 in U2OS cells were transfected with 400 ng of the p53 reporter construct U2OS cells expressing the indicated GFPHdmx, Hdm2, and p53 con- (pG13Luc) and DNAs expressing Hdm2 wild type and p53 binding- structs were scored for more than 100 cells in three separate experi- defective mutant G58A (300 ng), GFP-Hdmx wild type, p53 binding- ments; values are means  S.D. Cells were scored as described in Fig. defective mutant (Hdmxp53bs), and RING domain-deleted mutant 2o. Only cells expressing the three proteins were considered for the (HdmxR) alone (800 ng) or a combination of both wild-type proteins as analysis. Hdmx does not prevent Hdm2NLS-induced p53(K305N) deg- outlined in the transfection scheme. Luciferase activity is shown as -fold radation. Dapi, 4,6-diamidino-2-phenylindole. induction compared with no transfection of the reporter with the S.D. of triplicates. pressed Hdmx is mainly localized to the cytoplasm. However, in all reported experiments but one (12), the effect of Hdmx was HdmxR, which is partly localized in the nucleus (see Fig. 1, f investigated by transient transfection using various p53-defi- and h), slightly, yet reproducibly, reduced the p53 transcrip- cient cell lines (Hep3B, H1299, Calu, and p53/ murine em- tional ability. Strikingly, simultaneous expression of Hdm2 bryonic fibroblasts), overexpressed p53, and various reporter and Hdmx resulted in an almost complete repression of p53 constructs (14, 15). In these experimental conditions, overex- pG13Luc transcription. These findings demonstrate that regu- pressed p53 might trigger recruitment of Hdmx into the nu- lation of Hdmx by Hdm2 is an absolute requirement for the cleus indirectly through its effect on the endogenous Hdm2 efficient regulation of p53 activity by Hdmx and suggest that promoter. To test this possibility, we have transfected U2OS only the nuclear fraction of Hdmx regulates p53-dependent cells, which express endogenous p53, with the reporter con- transcription. struct pG13Luc containing 13 copies of the p53 consensus se- DISCUSSION quence upstream of a minimal promoter driving transcription of the luciferase gene. As expected, endogenous p53 is able to We have shown here that exogenous Hdmx, when expressed sustain a significant basal level of expression of the reporter alone, is mainly cytoplasmic, while, in the presence of Hdm2, it construct (Fig. 6). Overexpression of Hdm2, but not of Hdmx, becomes mainly nuclear. Nuclear relocalization of Hdmx by significantly inhibited pG13Luc transcription. Expression of overexpressed Hdm2 depends on their reciprocal interaction as Regulation of p53 Stability and Activity by Nuclear Hdmx 7323 suggested by the finding that this phenomenon requires the Hdmx, however, remains to be elucidated. Recent reports, in- integrity of the RING domain of both proteins. Surprisingly, cluding observations presented here, indicate that stabilized however, RING mutants of Hdmx (R and R-NLS) were found p53 protein is not able to activate transcription, and it was in both the nucleus and cytoplasm even when expressed alone proposed that Hdmx secures the presence of a pool of inactive suggesting that Hdmx binds other cellular factor(s) that might p53 that can be instantly activated when needed. Overex- sequester Hdmx in the cytoplasm in the absence of Hdm2. pressed Hdmx, however, is an artificial situation, and it is not Since Hdm2 binds Hdmx in the same RING-finger region, it known whether or not it corresponds to any physiological sit- might compete for binding and displace this putative interac- uations. Indeed, Hdmx protein levels seem to be very low in all tion. Alternatively, formation of Hdmx oligomers might occur primary cells tested. On the other hand, increased Hdmx levels in the cytoplasm and inhibit the nuclear import of Hdmx by, for were found in a significant fraction of tumor cell lines when instance, masking its putative NLS, which is located in the compare with normal cells (28). In general Hdmx expression in RING-finger motif of the protein. In this context, Hdm2, which these tumor cell lines correlates with the presence of wild-type forms stable heterodimers with Hdmx (14), might disrupt the p53, suggesting that deregulated expression of Hdmx plays a formation of Hdmx oligomers. role in carcinogenesis as an alternative way to inactivate p53. We then investigated the mechanisms by which nuclear High levels of Hdmx may also explain the efficient response to Hdmx inhibits Hdm2-induced p53 degradation. Since this ef- radiation and chemotherapy displayed by a subset of tumors, fect was previously reported to be mediated by the Hdmx including germ-cell tumors, which express high levels of wild- RING-finger and to involve hetero-oligomerization with the type p53 and Hdm2 (29, 30). The now available mouse models Hdm2 RING-finger, we hypothesized that Hdmx inhibits the further support the notion that, at least during embryogenesis, ubiquitin ligase activity of Hdm2. 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