Hdmx and Mdm2 can repress transcription activation by p53 ... - Nature

ã

Oncogene (2001) 20, 4576 ± 4580 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Hdmx and Mdm2 can repress transcription activation by p53 but not by p63 Natalie A Little1 and Aart G Jochemsen*,1 1

Department of Molecular and Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Centre, PO Box 9503, 2300 RA Leiden, The Netherlands

The p53 protein is involved in cell cycle arrest and apoptosis. To ensure that cells under non-stressed conditions are able to grow, p53 sets up a negative feedback loop by inducing Mdm2. Mdm2 is able to both inhibit the transcriptional regulation by p53 and to degrade it, thus maintaining p53 inactive until it is required. The Mdm2 related protein, Hdmx, has also been shown to inhibit the transcriptional activation of p53 but is unable to degrade it. A few years ago, the p53 family member, p63 was identi®ed. Like p53, p63 is able to induce p53 target genes and it was shown to be able to cause cell cycle arrest and apoptosis. In this study we report that, despite the similarities between p53 and p63, neither Hdmx nor Mdm2 are able to interact with p63, to repress p63-induced transcription or to aect its halflife. Oncogene (2001) 20, 4576 ± 4580.

ubiquitin ligase for p53 (Fang et al., 2000; Honda et al., 1997). The Mdmx protein, structurally related to Mdm2, also inhibits p53-induced transcription by binding to its N-terminus (Shvarts et al., 1996). However, unlike Mdm2, Mdmx and its human

Keywords: p53; p63; Mdmx; Mdm2; transcriptional activation

The tumour suppressor gene p53 is mutated in over 50% of human tumours (Hollstein et al., 1994). Its role is to generate a response to cellular stresses such as DNA damage, hypoxia or uncontrolled mitogenic stimulation to prevent accumulation of genomic instability. When cells are subjected to stress, the p53 protein is stabilized and activated, leading to either cell cycle arrest or apoptosis. These events are mainly brought about by the induction of p53 target genes, and the number of those being identi®ed is expanding (Yu et al., 1999). Classic examples of p53 target genes are p21 (El-Deiry et al., 1993), which is involved in cell cycle arrest, and bax (Miyashita and Reed, 1995), which is involved in apoptosis. In addition, p53 also activates the mdm2 gene. The main function of the Mdm2 protein is to downregulate the activity of p53, thus setting up a negative feedback loop (Wu et al., 1993). Mdm2 can bind to p53 at the N-terminal transcriptional activation domain thereby inhibiting p53's transcriptional activity (Chen et al., 1993). Furthermore, upon binding, Mdm2 also targets p53 for proteosomal degradation, as Mdm2 is the E3

*Correspondence: AG Jochemsen, E-mail: [email protected] Received 12 March 2001; revised 24 April 2001; accepted 9 May 2001

Figure 1 Reporter assays showing the eects of Hdmx and Mdm2 on p53- and p63-induced transcription activation. Bax-luc; p21-luc and Mdm2-luc were the reporters used and the activity was determined as follows: 26105 SAOS-2 cells were seeded on six-well plates and maintained in Dulbecco's Modi®ed Eagle's Medium supplemented with 10% fetal bovine serum and antibiotics. Eight hours later, cells were transfected using the calcium phosphate method (van der Eb and Graham, 1980). To achieve approximate equal levels of expressed protein, 100 ng of HA-p53; 400 ng HA-p63 TAa; and 800 ng HA-p63TAg were transfected. Two mg Hdmx or Mdm2; 1 mg lacZ and 2.5 mg reporter plasmid were co-transfected. CMV vector was used to correct for total DNA input. Thirty-six hours after transfection, luciferase assays were performed as described previously (Stad et al., 2000). The co-transfected lacZ was used to correct for transfection eciency, as determined by a b-galactosidase assay. These experiments were done at least twice, each one in triplicate, and the average values taken, with the standard deviation between the samples shown

Hdmx and Mdm2 can interact with p53 but not p63 NA Little and AG Jochemsen

4577

Figure 2 The eect of Hdmx and Mdm2 on the induction of endogenous p21 and Mdm2 proteins by p53 and p63 and their eect on the half-life of p53 and p63. 46105 SAOS-2 cells were grown on 5 cm plates and transfected as for the luciferase assays. 36 h after transfection cells were lysed in IPB 0.7 (20 mM triethanolamine pH 7.4, 0.7 M NaCl, 0.5% Nonidet P-40, 0.2% sodium deoxycholic acid, 1 mM PMSF, 3.5 mM trypsin inhibitor, 1 mM leupeptin) for 30 min on ice, and cell debris were removed by centrifugation. Twenty mg of cell lysate was separated on SDS ± PAGE, and a Western blot carried out to detect the proteins of interest. The antibodies used were: Anti-lacZ mouse monoclonal, clone D19-2F3-2 (Roche Molecular Diagnostics). Anti-Mdm2 mouse monoclonal 4B2 (Chen et al., 1993). Anti-HA monoclonal antibody, HA11 (Babco). Anti-p21 rabbit polyclonal C-19 (Santa Cruz Biotechnology)

homologue Hdmx cannot target p53 for degradation, and in fact are able to inhibit the degradation of p53 by Mdm2 (Jackson and Berberich, 2000; Sharp et al., 1999; Stad et al., 2000). Recently a new p53 family member was identi®ed, and called p63/p51/p73L/CUSP/KET (Lee et al., 1999; Osada et al., 1998; Senoo et al., 1998; Trink et al., 1998; Yang et al., 1998). The p63 gene is expressed into several protein isoforms. There are two groups of isoforms ± those that contain the transcriptional activation domain (TA isoforms) and those that lack it (DN isoforms). The TA isoforms are able to induce p53 target genes and cause cell cycle arrest and apoptosis (Osada et al., 1998; Shimada et al., 1999; Yang et al., 1998). The DN isoforms, which are unable to induce p53 target genes, may act in a dominant negative manner. The p63 isoforms also dier in the length of their C-terminus, with the alpha isoforms having the longest and the gamma isoforms containing the shortest C-terminus. A regulatory domain in the Cterminus of p63a represses the transcription activation domain, rendering it less active than the gamma (Yang et al., 1998). Unlike p53, which is ubiquitously expressed and acts as a tumour suppressor (Donehower et al., 1992), p63 is expressed in a more con®ned manner and plays an important role in development (Mills et al., 1999; Yang et al., 1999). Since the transcriptional activation domains of p53 and p63 are partly homologous, we investigated whether Hdmx and Mdm2 would have a similar repressive eect on p63 function. Three dierent reporter constructs known to be activated by p53 were used: Bax-luc (Ryan and Vousden, 1998); p21-luc (ElDeiry et al., 1993) and Mdm2-luc (Zauberman et al., 1995). These reporter constructs, together with either p53 or p63, were transfected into SAOS-2 cells and a

clear activation was seen (Figure 1). As expected, when Hdmx or Mdm2 were co-transfected, the induction by p53 was repressed, with Mdm2 having the strongest eect. However, neither Hdmx nor Mdm2 signi®cantly repressed the induction caused by the p63 isoforms on any of the three dierent reporters used. These results indicate that, despite the similarity between p53 and p63 in their transcriptional activation properties, the regulation of this activity by Hdmx and Mdm2 is dierent. To validate the results of the reporter assays, the eect on the endogenous promoters was also investigated. SAOS-2 cells were transfected as described for the reporter assays and the induction of the endogenous p21 and Mdm2 proteins was assessed. The eect on endogenous Bax could not be determined, as the basal levels were too high to be able to see a further increase after induction. When p53 was transfected, the endogenous levels of p21 and Mdm2 were induced compared to the basal levels seen in the mocktransfected cells (Figure 2, compare lanes 1 and 4). Upon co-transfection of Hdmx or Mdm2, the levels of endogenous p21 decreased, and as expected Mdm2 caused a greater decrease than Hdmx (compare lanes 5 and 6). The transfected Mdm2 also repressed the induction of endogenous Mdm2 by p53. For Hdmx the situation is more complex because although it is expected to repress the induction of the endogenous mdm2 gene by p53, this is not seen. This is probably because Hdmx stabilizes the Mdm2 protein (Sharp et al., 1999; Stad et al., 2000) and this eect is greater than the repression of p53 by Hdmx, which leads to elevated Mdm2 levels. When p63TAa and p63TAg were transfected, the endogenous levels of p21 and Mdm2 were also induced (compare lanes 7 and 10, respectively, with lane 1). Neither Hdmx nor Mdm2 Oncogene


4578

Figure 3 Hdmx and Mdm2 can bind to p53 but not to p63. (a) GST-pull downs. In vitro translated (IVT) p53 or p63 proteins were prepared in a T7-driven rabbit reticulocyte-coupled transcription and translation system (Promega). 0.5 mg of bacterially expressed GST-Hdmx or GST-Mdm2 were mixed with 35S-methionine-labeled IVT proteins in 150 ml of HSAB (500 mM NaCl, 100 mM Tris at pH 8, 0.1% NP40) at 48C for 2 h. Complexes were precipitated with glutathione beads, washed three times in HSAB, resolved by SDS ± PAGE and detected by ¯uorography. (b) Co-immunoprecipitations. 16106 SAOS-2 cells were grown on 9 cm plates and transfected with 4 mg of either Hdmx or Mdm2 together with 600 ng HA-p53; 2.4 mg HA-p63TAa; 4.8 mg HA-p63DNa; 4.8 mg HAp63TAg; or 2.4 mg HA-p63DNg. Thirty-six hours after transfection cells were lysed in Giordano buer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 0.1% Triton X-100, 5 mM EDTA, 1 mM PMSF, 3.5 mM trypsin inhibitor, 1 mM leupeptin) for 30 min on ice, followed by removal of cell debris by centrifugation. The cell lysate was incubated with either anti-Hdmx (6B1A; Ramos et al. in preparation) or anti-MDM2 (2A10; Chen et al., 1993), coupled to protein G-Sepharose. The immunoprecipitation was carried out overnight at 48C with tumbling. After washing three times in Giordano buer, the samples were resolved by SDS ± PAGE, and a Western blot was carried out with the use of anti-HA antibody to detect the dierent p53 family members. Panel 1 is 5% of the total cell lysate and panel 2 is the results of the co-immunoprecipitation

repressed this induction by p63TAa or p63TAg (compare lanes 8 and 9 with lane 7, and lanes 11 and 12 with lane 10, respectively). Therefore, both the reporter assays and the assessment of endogenous genes show that Hdmx and Mdm2 can repress transcription activation by p53 but not by p63. To determine whether Mdm2, and/or Hdmx, can in¯uence the half-life of p63, the eect on expression levels of p63 was investigated. As previously observed and shown in Figure 2, Mdm2 clearly degraded p53 (compare lanes 4 and 6), whereas Hdmx did not (compare lanes 4 and 5). Hdmx and Mdm2 appear to have no eect on p63 stability (compare lanes 8 and 9 with lane 7 for p63TAa, and lanes 11 and 12 with lane 10 for p63TAg). The levels of p53 and p63 were also investigated by immuno¯uorescence. The levels of p53 were reduced when Mdm2 was co-transfected, whereas Oncogene

the levels of p63 remained unchanged (data not shown), again indicating that Mdm2 does not degrade p63. Hdmx was unable to degrade either p53 or p63. The immuno¯uorescence also showed that the subcellular localization of p53 and p63 remained unchanged in the presence of Hdmx or Mdm2. The experiments indicate that Hdmx and Mdm2 are not able to repress p63 or aect its half-life. It was considered that this may be the result of an inability to interact with p63. To investigate this, a GST-pull down assay was carried out. A strong interaction between p53 and both Hdmx and Mdm2 was detected (Figure 3a). However, only a weak interaction was seen with p63 (p63TAa and p63TAg) but it was also observed for the DN isoforms (p63DNa and p63DNg), which lack the N-terminal domain. These results suggest that the observed binding is either non-speci®c, or not via the


N-terminal domain of p63. To con®rm these ®ndings in vivo, co-immunoprecipitation assays were carried out on SAOS-2 cells, which had been transfected with p53 or p63 together with Hdmx or Mdm2 (Figure 3b, panel 1). Immunoprecipitations were carried out on the cell lysates with antibodies against Hdmx or Mdm2. None of the p63 isoforms were detected in the Hdmx and Mdm2 immunoprecipitations whereas p53 was present (Figure 3b, panel 2). These data demonstrate that, unlike p53, p63 is unable to bind to Hdmx or Mdm2. Despite the fact that p63 has a transcriptional activation domain which is partly conserved with that of p53, divergences between them cause a dierent behaviour towards Hdmx and Mdm2. It is believed that the minimal region of human p53 to which Mdm2 and Hdmx bind is 16QPTFSDYWKLLP27. Within this peptide, the crucial amino acids required for binding are F19, W23 and L26 (BoÈttger et al., 1999). These crucial amino acids are conserved in p63, although the entire peptide sequence is only 33% conserved. This dierence in the entire peptide sequence may be enough to abolish the binding between p63 and Hdmx and Mdm2, although C-terminal sequences may also play a part (Kubbutat et al., 1998; Maki, 1999). Since Mdm2 is not able to interact with p63, it is not surprising that it cannot degrade p63. It has been shown previously that Mdm2 is able to bind to p73. Reporter assays have shown that Mdm2 can repress the transcriptional activation by p73, but it does not

target p73 for degradation (BaÂlint et al., 1999; Dobbelstein et al., 1999; Zeng et al., 1999). Amino acids 92 ± 112 of p53 are required for p53 degradation, and that sequence is not conserved in p73 (Gu et al., 2000). Like p73, p63 does not contain this motif and so, even if Mdm2 were able to bind to p63, it is unlikely that it would be targeted for degradation. This suggests that Mdm2 acts as a speci®c E3 ligase for p53 and not for the other family members. Recently, interactions between p53, p63 and p73 have been described, suggesting that they are all able to in¯uence one another's function (Gaiddon et al., 2001; Ratovitski et al., 2001; Strano et al., 2000). It would be interesting to investigate whether Mdm2 is able to degrade p53 when it is complexed to the other p53 family members. The results presented here are supported by two recent reports that appeared after submission of this manuscript (Kojima et al., 2001; Wang et al., 2001).

4579

Acknowledgments We would like to thank H van Bokhoven for the kind gifts of the p63 expression plasmids; M Oren for the Mdm2-luc, Bax-luc and p21-luc constructs; A Levine for the antiMdm2 antibodies; and R Stad for critically reading the manuscript. NA Little is supported by the Association for International Cancer Research (AICR).

References BaÂlint E, Bates S and Vousden KH. (1999). Oncogene, 18, 3923 ± 3929. BoÈttger V, BoÈttger A, Garcia-Echeverria C, Ramos YFM, van der Eb AJ, Jochemsen AG and Lane DP. (1999). Oncogene., 18, 189 ± 199. Chen J, Marechal V and Levine AJ. (1993). Mol. Cell. Biol., 13, 4107 ± 4114. Dobbelstein M, Wienzek S, KoÈnig C and Roth J. (1999). Oncogene, 18, 2101 ± 2106. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA, Butel JS and Bradley A. (1992). Nature, 356, 215 ± 221. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer E, Kinzler KW and Vogelstein B. (1993). Cell, 75, 817 ± 825. Fang S, Jensen JP, Ludwig RL, Vousden KH and Weissman AM. (2000). J. Biol. Chem., 275, 8945 ± 8951. Gaiddon C, Lokshin M, Ahn J, Zhang T and Prives C. (2001). Mol. Cell. Biol., 21, 1874 ± 1887. Gu J, Chen D, Rosenblum J, Rubin R and Yuan Z-M. (2000). Mol. Cell. Biol., 20, 1243 ± 1253. Hollstein M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sorlie T, Hovig E, Smith-Sorensen B, Montesano R and Harris CC. (1994). Nucleic Acids Res., 22, 3551 ± 3555. Honda R, Tanaka H and Yasuda H. (1997). FEBS. Lett., 420, 25 ± 27. Jackson MW and Berberich SJ. (2000). Mol. Cell. Biol., 20, 1001 ± 1007. Kojima T, Ikawa Y and Katoh I. (2001). Biochem. Biophys. Res. Commun., 281, 1170 ± 1175.

Kubbutat MH, Ludwig RL, Ashcroft M and Vousden KH. (1998). Mol. Cell. Biol., 18, 5690 ± 5698. Lee LA, Walsh P, Prater CA, Su LJ, Marchbank A, Egbert TB, Dellavalle RP, Targo IN, Kaufman KM, Chorzelski TP and Jablonska S. (1999). J. Invest. Dermatol., 113, 146 ± 151. Maki CG. (1999). J. Biol. Chem., 274, 16531 ± 16535. Mills AA, Zheng B, Wang X-J, Vogel H, Roop DR and Bradley A. (1999). Nature, 398, 708 ± 713. Miyashita T and Reed JC. (1995). Cell, 80, 293 ± 299. Osada M, Ohba M, Kawahara C, Ishioka C, Kanamaru R, Katoh I, Ikawa Y, Nimura Y, Nakagawara A, Obinata M and Ikawa S. (1998). Nat. Med., 4, 839 ± 843. Ratovitski EA, Patturajan M, Hibi K, Trink B, Yamaguchi K and Sidransky D. (2001). Proc. Natl. Acad. Sci. USA, 98, 1817 ± 1822. Ryan KM and Vousden KH. (1998). Mol. Cell. Biol., 18, 3692 ± 3698. Senoo M, Seki N, Ohira M, Sugano S, Watanabe M, Inuzuka S, Okamoto T, Tachibana M, Tanaka T, Shinkai Y and Kato H. (1998). Biochem. Biophys. Res. Commun., 248, 603 ± 607. Sharp DA, Kratowicz SA, Sank MJ and George DL. (1999). J. Biol. Chem., 274, 38189 ± 38196. Shimada A, Kato S, Enjo K, Osada M, Ikawa Y, Kohno K, Obinata M, Kanamaru R, Ikawa S and Ishioka C. (1999). Cancer Res., 59, 2781 ± 2786.

Oncogene


4580

Oncogene

Shvarts A, Steegenga WT, Riteco N, van Laar T, Dekker P, Bazuine M, van Ham RCA, van der Houven van Oordt W, Hateboer G, van der Eb AJ and Jochemsen AG. (1996). EMBO J., 15, 5349 ± 5357. Stad R, Ramos YFM, Little N, Grivell S, Attema J, van der Eb AJ and Jochemsen AG. (2000). J. Biol. Chem., 275, 28039 ± 28044. Strano S, Munarriz E, Rossi M, Cristofanelli B, Shaul Y, Castagnoli L, Levine AJ, Sacchi A, Cesareni G, Oren M and Blandino G. (2000). J. Biol. Chem., 275, 29503 ± 29512. Trink B, Okami K, Wu L, Sriuranpong V, Jen J and Sidransky D. (1998). Nat. Med., 4, 747 ± 748. van der Eb AJ and Graham FL. (1980). Methods Enzymol., 65, 826 ± 839. Wang X, Arooz T, Siu WY, Chiu CH, Lau A, Yamashita K and Poon RY. (2001). FEBS Lett., 490, 202 ± 208.

Wu X, Bayle JH, Olson D and Levine AJ. (1993). Genes Dev., 7, 1126 ± 1132. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, DoÈtsch V, Andrews NC, Caput D and McKeon F. (1998). Mol. Cell., 2, 305 ± 316. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C and McKeon F. (1999). Nature, 398, 714 ± 718. Yu L, Zhang L, Hwang PM, Rago C, Kinzler K and Vogelstein B. (1999). Proc. Natl. Sci. USA, 96, 14517 ± 14522. Zauberman A, Flusberg D, Haupt Y, Barak Y and Oren M. (1995). Nucleic Acid Res., 23, 2584 ± 2592. Zeng X, Chen L, Jost CA, Maya R, Keller D, Wang X, Kaelin Jr WG, Oren M, Chen J and Lu H. (1999). Mol. Cell. Biol., 19, 3257 ± 3266.