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MdmX Binding to ARF Affects Mdm2 Protein Stability and p53 Transactivation

2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês

10.1074/jbc.m010685200

1083-351X

Mark W. Jackson, Mikael S. Lindström, Steven J. Berberich,

Folate and B Vitamins Research

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity. Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity. retinoblastoma protein functional nucleolar localization signal green fluorescent protein Cellular division is controlled predominantly by two distinct tumor suppressors, p53 and Rb.1 The p53 protein regulates cell cycle arrest and apoptosis because of its ability to transcriptionally activate specific target genes following various forms of genotoxic stress (1Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2292) Google Scholar). The Rb protein controls entry into S-phase through its interaction with various members of the E2F family of transcription factors (2Kaelin Jr., W.G. Bioessays. 1999; 21: 950-958Crossref PubMed Scopus (172) Google Scholar). Signaling between these two pathways involves an ever increasing number of proteins including p21WAF1, a target of p53-dependent transactivation and inhibitor of cyclin-dependent kinase activity (3Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5239) Google Scholar, 4el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar), and the Mdm2 protein, an antagonist of both p53 and Rb growth suppressive functions (5Oliner J.D. Pietenpol J.A. Thiagalingam S. Gyuris J. Kinzler K.W. Vogelstein B. Nature. 1993; 362: 857-860Crossref PubMed Scopus (1307) Google Scholar, 6Oliner J.D. Kinzler K.W. Meltzer P.S. George D.L. Vogelstein B. Nature. 1992; 358: 80-83Crossref PubMed Scopus (1802) Google Scholar, 7Xiao Z.X. Chen J. Levine A.J. Modjtahedi N. Xing J. Sellers W.R. Livingston D.M. Nature. 1995; 375: 694-698Crossref PubMed Scopus (573) Google Scholar, 8Finlay C.A. Mol. Cell. Biol. 1993; 13: 301-306Crossref PubMed Scopus (310) Google Scholar). Additional overlap between p53 and Rb is provided by theINK4A locus, which encodes both p16INK4a and p19ARF proteins (9Quelle D.E. Zindy F. Ashmun R.A. Sherr C.J. Cell. 1995; 83: 993-1000Abstract Full Text PDF PubMed Scopus (1317) Google Scholar, 10Stott F.J. Bates S. James M.C. McConnell B.B. Starborg M. Brookes S. Palmero I. Ryan K. Hara E. Vousden K.H. Peters G. EMBO J. 1998; 17: 5001-5014Crossref PubMed Scopus (1012) Google Scholar). Although the coding transcripts of these proteins share common exons, the primary amino acid sequences of both proteins are unique. Overexpression of p16INK4A or p19ARF results in a cell cycle arrest through distinct pathways involving either Rb or p53, respectively (9Quelle D.E. Zindy F. Ashmun R.A. Sherr C.J. Cell. 1995; 83: 993-1000Abstract Full Text PDF PubMed Scopus (1317) Google Scholar,11Liggett Jr., W.H. Sewell D.A. Rocco J. Ahrendt S.A. Koch W. Sidransky D. Cancer Res. 1996; 56: 4119-4123PubMed Google Scholar, 12Quelle D.E. Ashmun R.A. Hannon G.J. Rehberger P.A. Trono D. Richter K.H. Walker C. Beach D. Sherr C.J. Serrano M. Oncogene. 1995; 11: 635-645PubMed Google Scholar, 13Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3375) Google Scholar). p16INK4A inhibits cyclin D-dependent kinases, thus preventing the phosphorylation of Rb and the subsequent release of E2F proteins (12Quelle D.E. Ashmun R.A. Hannon G.J. Rehberger P.A. Trono D. Richter K.H. Walker C. Beach D. Sherr C.J. Serrano M. Oncogene. 1995; 11: 635-645PubMed Google Scholar, 13Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3375) Google Scholar). p19ARF activates the p53 protein by binding to Mdm2 and sequestering it to the nucleolus (14Pomerantz J. Schreiber-Agus N. Liegeois N.J. Silverman A. Alland L. Chin L. Potes J. Chen K. Orlow I. Lee H.W. Cordon-Cardo C. DePinho R.A. Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, 15Weber J.D. Taylor L.J. Roussel M.F. Sherr C.J. Bar-Sagi D. Nat. Cell Biol. 1999; 1: 20-26Crossref PubMed Scopus (803) Google Scholar). The resulting Mdm2·ARF complex is unable to mediate nucleocytoplasmic shuttling of p53 (16Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6937-6941Crossref PubMed Scopus (499) Google Scholar) or act as an E3 ligase to ubiquitinate p53 (17Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (614) Google Scholar). In addition to interacting with Mdm2, ARF must also contain two functional nucleolar localization signals (NrLS) (18Zhang Y. Xiong Y. Mol. Cell. 1999; 3: 579-591Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 19Weber J.D. Kuo M.L. Bothner B. DiGiammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar, 20Rizos H. Darmanian A.P. Mann G.J. Kefford R.F. Oncogene. 2000; 19: 2978-2985Crossref PubMed Scopus (91) Google Scholar). However, recent evidence indicates that an additional NrLS within the C-terminal RING finger of Mdm2 is also required for nucleolar localization of the ARF·Mdm2 complex. Deletion of the NrLS or a more subtle mutation of the basic residues of the NrLS to uncharged amino acids results in failure of the two proteins to colocalize in the nucleolus (19Weber J.D. Kuo M.L. Bothner B. DiGiammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar, 21Lohrum M.A. Ashcroft M. Kubbutat M.H. Vousden K.H. Nat. Cell Biol. 2000; 2: 179-181Crossref PubMed Scopus (171) Google Scholar). Recently, we demonstrated that the MdmX protein, a p53-binding protein with homology to Mdm2, could protect p53 from Mdm2-mediated degradation while maintaining suppression of p53-dependent transactivation (22Jackson M. Berberich S.J. Mol. Cell. Biol. 2000; 20: 1001-1007Crossref PubMed Scopus (183) Google Scholar). Moreover, additional studies have demonstrated that Mdm2 is also stabilized through association with MdmX (23Stad R. Ramos Y.F. Little N.A. Grivell S. Attema J. van De Eb A.J. Jochemsen A.G. J. Biol. Chem. 2000; 275: 28039-28044Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Sharp D.A. Kratowicz S.A. Sank M.J. George D.L. J. Biol. Chem. 1999; 274: 38189-38196Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Given the homology between MdmX and Mdm2, we examined the localization of MdmX in the presence or absence of ARF. Our data indicate that coexpression of ARF with MdmX results in the mobilization of full-length MdmX to the nucleoli, where it is not found in the absence of ARF. Redistribution of MdmX by ARF reverses the MdmX-mediated inhibition of p53 transactivation. Interestingly, MdmX and ARF have an antagonistic effect on each other with respect to their ability to stabilize Mdm2, whereas MdmX and Mdm2 work synergistically to reverse ARF induction of p53 transactivation. U2OS cells are an osteosarcoma cell line containing wild-type p53 and having no detectable ARF protein (10Stott F.J. Bates S. James M.C. McConnell B.B. Starborg M. Brookes S. Palmero I. Ryan K. Hara E. Vousden K.H. Peters G. EMBO J. 1998; 17: 5001-5014Crossref PubMed Scopus (1012) Google Scholar). H1299 cells are a non-small cell lung carcinoma devoid of p53. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Antibodies include monoclonal FLAG M2 (Sigma), monoclonal V5 (Invitrogen), monoclonal GFP (Zymed Laboratories Inc.), and polyclonal ARF Ab-1 (Neomarkers). For immunofluorescence analysis, a Texas Red-conjugated goat anti-mouse antibody (Jackson ImmunoResearch) was used. An horseradish peroxidase-conjugated secondary antibody (Promega) was used for chemiluminescent detection of proteins. The p14ARF-GFP expression plasmid has been described previously (25Lindstrom M.S. Klangby U. Inoue R. Pisa P. Wiman K.G. Asker C.E. Exp. Cell Res. 2000; 256: 400-410Crossref PubMed Scopus (74) Google Scholar). The p19ARF-Myc/His plasmid contains the murine ARF cDNA with a C-terminal Myc/His epitope tag. Mdm2-FLAG is in the pFLAG-CMV-2 vector (Sigma), which encodes a FLAG epitope tag onto the amino terminus of the Mdm2 protein. Human MdmX and MdmX-(1–446) are in the pcDNA3.1/V5-His vector, which encodes a V5 epitope tag and a six-histidine tag onto the C terminus of the expressed protein. The human MdmX cDNA used as a template for amplification was kindly provided by Dr. Aart Jochemsen. The PG13-luc plasmid was constructed by cloning 13 copies of a synthetic p53 DNA binding site upstream of a SV40 promoter-luciferase gene. MG15-luc contains 15 copies of a mutated p53 DNA binding site cloned upstream of the SV40 promoter-luciferase reporter gene. The pQE-β-gal plasmid was used to normalize transfection efficiency. In the p53 transactivation studies, ARF, mdm2, and mdmX expression plasmids were used. Transfections were performed using LipofectAMINE (Life Technologies, Inc.). In Fig. 3, a pHOOK plasmid (Invitrogen) was included with each transfection to allow immunoselection of transfected cells with Capture-Tec beads (Invitrogen). For Western analysis in Figs. 4 and 5, transfection efficiencies were normalized by the inclusion of a pGL3-luciferase reporter plasmid (Promega) in each transfection.Figure 4Interaction between MdmX and ARF destabilizes Mdm2 and inhibits p53 transactivation.A, Western blot analysis of cellular extracts from transfected U2OS cells was used to determine Mdm2, ARF, and MdmX protein levels. Coexpression of MdmX with Mdm2 resulted in the stabilization of Mdm2. However, expression of increasing amounts of ARF in the presence of MdmX resulted in a dose-dependent decrease in the stabilization of Mdm2 protein by MdmX. B, extracts from transfected U2OS cells were used in Western blot analysis to monitor MdmX-(1–466) and ARF proteins levels. The stabilization of Mdm2 observed following the coexpression of ARF is not affected by an NrLS-deficient MdmX.View Large Image Figure ViewerDownload (PPT)Figure 5Expression of ARF and MdmX does not alter protein stability. U2OS cells were transfected with cDNAs encoding: A, V5-tagged human MdmX and increasing amounts of ARF·GFP; or B, ARF·GFP and increasing amounts of V5-tagged human MdmX. After 24 h, cell extracts were made and Western analysis was performed to determine ARF and MdmX protein levels. The protein stability levels of MdmX and ARF are not altered by one another.View Large Image Figure ViewerDownload (PPT) Immunofluorescence analysis was performed 24 h after transfection. Transfected U2OS cells on chamber slides (LabTech) were fixed in 3% paraformaldehyde, permeabilized with 1% Triton X-100 in phosphate-buffered saline, and blocked for 2 h with 10% goat serum, 0.01% Tween 20 in phosphate-buffered saline. Primary antibody was added to a 1/10 dilution of blocking solution and incubated for 1 h. The cells were washed five times and incubated with the secondary antibody for 1 h. Nuclei were stained with 25 μg/ml Hoechst Dye (Sigma) for 2 min. B23 immunofluorescence was performed as described previously (25Lindstrom M.S. Klangby U. Inoue R. Pisa P. Wiman K.G. Asker C.E. Exp. Cell Res. 2000; 256: 400-410Crossref PubMed Scopus (74) Google Scholar). For immunoprecipitation and Western blot experiments, whole cell extracts were made 24 h following transfection by incubating cell pellets for 30 min in lysis buffer (50 mmTris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40) containing a protease inhibitor mixture (Sigma). Immunoprecipitation analysis was performed by adding 100 μg of pHOOK cell extracts with 1 μg of monoclonal V5 antibody in a total volume of 300 μl of phosphate-buffered saline containing 5 mm EDTA and 0.5% Triton X-100. Following overnight incubation, 25 μl of protein G-agarose was added and incubated an additional 1 h before washing three times for 30 min each time. Pellets were resuspended in 25 μl of 2× SDS loading buffer and resolved using 10% SDS-polyacrylamide gel electrophoresis followed by transfer of proteins to a polyvinylidene difluoride membrane (Millipore) using a Transblot system (Bio-Rad). Immunoblotting was performed as described previously (26Berberich S.J. Litteral V. Mayo L.D. Tabesh D. Morris D. Differentiation. 1999; 64: 205-212Crossref PubMed Scopus (22) Google Scholar) using primary antibody dilutions of 1:1,000–1:2,500 and secondary dilutions of 1:5,000–1:10,000. For p53 reporter assays, extracts were made 24 h after transfection. p53 transactivation was determined by quantifying luciferase activity in aliquots from whole cell extracts using a luciferase assay system (Promega). β-Galactosidase activity was determined by incubating protein extracts in 100 mmsodium phosphate, pH 7.3, 1 mm MgCl2, 50 mm β-mercaptoethanol, and 650 μ g/mlO-nitrophenyl-β-d-galactopyranoside at 37 °C for 0.5–2 h. β-Galactosidase activity was calculated by converting the absorbance read at 420 nm into milliunits of β-galactosidase activity per microliter of extract. All luciferase and β-galactosidase assays were performed in duplicate. Error bars of relative p53 transactivation represent the average deviation for the relative luciferase units divided by the milliunits of β-galactosidase. Previous studies have demonstrated that ARF is localized to the nucleolus (10Stott F.J. Bates S. James M.C. McConnell B.B. Starborg M. Brookes S. Palmero I. Ryan K. Hara E. Vousden K.H. Peters G. EMBO J. 1998; 17: 5001-5014Crossref PubMed Scopus (1012) Google Scholar, 15Weber J.D. Taylor L.J. Roussel M.F. Sherr C.J. Bar-Sagi D. Nat. Cell Biol. 1999; 1: 20-26Crossref PubMed Scopus (803) Google Scholar, 18Zhang Y. Xiong Y. Mol. Cell. 1999; 3: 579-591Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 25Lindstrom M.S. Klangby U. Inoue R. Pisa P. Wiman K.G. Asker C.E. Exp. Cell Res. 2000; 256: 400-410Crossref PubMed Scopus (74) Google Scholar). The Mdm2 protein, although not found in nucleoli in the absence of ARF, can be mobilized to nucleoli following either coexpression of ARF or induced ARF expression (14Pomerantz J. Schreiber-Agus N. Liegeois N.J. Silverman A. Alland L. Chin L. Potes J. Chen K. Orlow I. Lee H.W. Cordon-Cardo C. DePinho R.A. Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, 15Weber J.D. Taylor L.J. Roussel M.F. Sherr C.J. Bar-Sagi D. Nat. Cell Biol. 1999; 1: 20-26Crossref PubMed Scopus (803) Google Scholar). Based on the homology between Mdm2 and MdmX, we first examined whether MdmX was found in nucleoli or could be colocalized with ARF. U2OS cells, which do not express ARF yet do possess wild-type p53 protein (10Stott F.J. Bates S. James M.C. McConnell B.B. Starborg M. Brookes S. Palmero I. Ryan K. Hara E. Vousden K.H. Peters G. EMBO J. 1998; 17: 5001-5014Crossref PubMed Scopus (1012) Google Scholar), were transiently transfected with an expression plasmid encoding a V5-epitope tagged human MdmX protein in the presence or absence of an expression plasmid encoding an ARF·GFP fusion protein. In the absence of ARF (or in the presence of GFP, data not shown), MdmX showed no nucleolar localization (Fig.1A, panel l). However, upon coexpression of ARF, MdmX became localized to the nucleolus (Fig.1A, panels j and k). When MdmX and ARF signals were overlaid they demonstrated clear colocalization (Fig.1A, panels m and n). ARF localization to the nucleolus (Fig. 1A, panel e) was confirmed in transfected U2OS cells by demonstrating that ARF colocalized with the nucleolus protein B23 (Fig. 1A, panels p and q). To determine the effect of ARF expression on MdmX regulation of p53 transactivation, U2OS cells were transfected with the p53-responsive promoter PG13-luc. As expected, transfection of the p53-responsive promoter (PG13-luc) into cells possessing wild-type p53 protein led to a 6-fold increase in p53 transactivation when compared with transfection of a p53 reporter plasmid containing mutant p53 DNA binding sites (MG15-luc; Fig.1B). Coexpression of a mdmX expression vector led to a modest 20% decrease in the activity of the p53 responsive promoter (Fig. 1B). With increasing levels of mdmX expression vector, p53 transactivation levels do continue to decrease in U2OS cells (data not shown). Interestingly, the addition of a 1:1 or 1:2 ratio ofmdmX:ARF expression vectors led to a dose-dependent increase in p53 transactivation, surpassing levels of p53 transactivation seen in the absence of exogenous MdmX or ARF. The 2.7-fold increase in p53 transactivation reporter activity seen on comparing the 1:2 mdmX:ARF transfections with the PG13-luc only transfected U2OS cells most likely means that the elevated ARF protein levels effectively sequestered both the exogenous MdmX and endogenous Mdm2, thereby stabilizing and maximizing the nuclear pools of free p53 protein (Fig.1B). Based on the requirement for a conserved stretch of basic amino acids, or NrLS, in the RING domain of Mdm2 (19Weber J.D. Kuo M.L. Bothner B. DiGiammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar, 21Lohrum M.A. Ashcroft M. Kubbutat M.H. Vousden K.H. Nat. Cell Biol. 2000; 2: 179-181Crossref PubMed Scopus (171) Google Scholar), we examined whether MdmX contains a similar NrLS. Fig.2A compares the NrLS of Mdm2 with the putative NrLS of both human and murine MdmX. Both human and murine MdmX sequences have a conserved (R/K)(R/K)X(R/K) motif previously shown to be required for the nucleolar localization of the ARF·Mdm2 complex (19Weber J.D. Kuo M.L. Bothner B. DiGiammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar,21Lohrum M.A. Ashcroft M. Kubbutat M.H. Vousden K.H. Nat. Cell Biol. 2000; 2: 179-181Crossref PubMed Scopus (171) Google Scholar). To examine the importance of the putative NrLS in MdmX, a deletion mutant of MdmX lacking the RING finger domain, which contains the NrLS, was constructed, and its ability to colocalize with ARF was tested. The deletion matched a similar deletion made in Mdm2, which demonstrated the requirement for a NrLS in the Mdm2 RING finger for proper Mdm2·ARF nucleoli localization (19Weber J.D. Kuo M.L. Bothner B. DiGiammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar, 21Lohrum M.A. Ashcroft M. Kubbutat M.H. Vousden K.H. Nat. Cell Biol. 2000; 2: 179-181Crossref PubMed Scopus (171) Google Scholar). As seen with Mdm2, MdmX-(1–446), which lacked the MdmX RING domain, was unable to colocalize to the nucleolus with ARF (Fig. 2B, panel h). Surprisingly, ARF was not completely prohibited from entering the nucleolus when coexpressed with MdmX-(1–446) (Fig. 2B, panel e), contrasting the results reported with NrLS-deficient Mdm2 proteins (19Weber J.D. Kuo M.L. Bothner B. DiGiammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar, 21Lohrum M.A. Ashcroft M. Kubbutat M.H. Vousden K.H. Nat. Cell Biol. 2000; 2: 179-181Crossref PubMed Scopus (171) Google Scholar). In any event, expression of MdmX-(1–446) did lead to a greater accumulation of ARF in the nucleoplasm when compared with coexpression with full-length MdmX (Fig. 2B, comparepanels d and e). The inability of MdmX-(1–446) to maintain ARF within the nucleoplasm may simply represent differences in ARF binding to MdmX and MdmX-(1–446). To test this possibility, H1299 cells were transfected as described in Fig. 2B, MdmX protein was immunoprecipitated, and the interaction with ARF was confirmed by Western analysis. Based on immunoprecipitation results (Fig.3A), both full-length MdmX and MdmX-(1–446) were capable of interacting with ARF. Comparing the immunoprecipiations to a parallel immunoblot (Fig. 3A, lower panel), there appears to be a slight but reproducible decrease in the amount of ARF bound to MdmX-(1–446) relative to full-length MdmX. Perhaps the interaction between ARF and MdmX is not as stable in the nucleoplasm as it is in the nucleolus. The nucleolar localization of ARF in transfected H1299 cells (Fig. 3B) confirmed that ARF localization was comparable with that seen in U2OS cells. We also examined a more truncated version of MdmX, amino acids 1–363, which also binds to ARF but is unable to colocalize to the nucleolus with ARF (data not shown). Taken together, the data in Figs. 2 and 3 are consistent with the similar interpretation made for Mdm2, namely that MdmX must contain a functional RING finger domain in order for ARF to mobilize it to the nucleolus but not for association with ARF. Because ARF and MdmX have both been reported to individually stabilize the Mdm2 protein (10Stott F.J. Bates S. James M.C. McConnell B.B. Starborg M. Brookes S. Palmero I. Ryan K. Hara E. Vousden K.H. Peters G. EMBO J. 1998; 17: 5001-5014Crossref PubMed Scopus (1012) Google Scholar, 23Stad R. Ramos Y.F. Little N.A. Grivell S. Attema J. van De Eb A.J. Jochemsen A.G. J. Biol. Chem. 2000; 275: 28039-28044Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Sharp D.A. Kratowicz S.A. Sank M.J. George D.L. J. Biol. Chem. 1999; 274: 38189-38196Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), we next examined how MdmX affects the stability of Mdm2 either alone or in the presence of increasing levels of ARF protein. Although studies examining mdmX gene expression have failed to uncover any significant modulation (27Jackson M.W. Berberich S.J. DNA Cell Biol. 1999; 18: 693-700Crossref PubMed Scopus (27) Google Scholar, 28Shvarts A. Steegenga W.T. Riteco N. van Laar T. Dekker P. Bazuine M. van Ham R.C. van der Houven van Oordt W. Hateboer G. van der Eb A.J. Jochemsen A.G. EMBO J. 1996; 15: 5349-5357Crossref PubMed Scopus (519) Google Scholar), one group has reported substantially higher levels of mdmX mRNA in the thymus of both human and mouse (29Shvarts A. Bazuine M. Dekker P. Ramos Y.F. Steegenga W.T. Merckx G. van Ham R.C. van der Houven van Oordt W. van der Eb A.J. Jochemsen A.G. Genomics. 1997; 43: 34-42Crossref PubMed Scopus (124) Google Scholar) suggesting that elevated MdmX protein may occur in specific tissues. In agreement with previously reported data, MdmX was able to stabilize the Mdm2 protein (Fig.4A, lane 3). In contrast, coexpression of MdmX and ARF together had a dose-dependent antagonistic effect on the ability of both proteins to stabilize Mdm2 protein (Fig. 4A, lanes 4–6). Interestingly, there was also a slight, but reproducible, decrease in MdmX protein levels as ARF protein levels increased and Mdm2 protein levels decreased (Fig. 4A, lanes 3–6). It is possible that the decrease in MdmX protein levels results from a decrease in MdmX·Mdm2 complex formation. This would be in agreement with one recent study in which MdmX mutants lacking the RING finger domain showed decreased protein stability relative to full-length MdmX (30Tanimura S. Ohtsuka S. Mitsui K. Shirouzu K. Yoshimura A. Ohtsubo M. FEBS Lett. 1999; 447: 5-9Crossref PubMed Scopus (269) Google Scholar). As expected, overexpression of MdmX-(1–446) protein was not able to stabilize Mdm2 protein when expressed in U2OS cells (Fig.4B, lane 5), nor was it able to antagonize the ability of ARF protein to stabilize Mdm2 protein (Fig. 4B,lane 4). The inability of MdmX-(1–446) to reverse the ARF-induced stability of Mdm2 implies that the association of MdmX and ARF may not be sufficient to reverse the ARF stability of Mdm2 and that nucleolar localization of ARF and MdmX is required to destabilize Mdm2. The results shown in Fig. 4A demonstrated that the stability of both MdmX and Mdm2 decreased as ARF protein levels increased. To determine whether the decrease in MdmX stability was dependent upon Mdm2 or ARF, MdmX was expressed in U2OS cells with increasing ARF in the absence of Mdm2. Under these transfection conditions, increasing ARF protein levels did not affect MdmX protein levels (Fig.5A), consistent with the stability of MdmX in Fig. 4A being more dependent on the presence of elevated Mdm2 than ARF levels. Finally, there was no alteration in ARF levels as in transfections containing increasing levels of MdmX expression vector (Fig. 5B). Taken together, the results shown in Fig. 5 suggest that the association between MdmX and ARF has no dramatic effect on the stability of either protein. Finally, to examine the effects of coexpressing Mdm2, MdmX, and ARF on endogenous p53 transactivation, U2OS cells were transfected with the indicated plasmids and the p53-responsive promoter, PG13-luc (Fig. 6). As expected, transfection of Mdm2 and MdmX expression vectors together, led to a >90% decrease in p53 transactivation (Fig. 6A). The inhibition of p53 transactivation by transfection with mdm2 and mdmX expression vectors was reversed, in a dose-dependent manner, by coexpression with ARF expression vectors (Fig.6A). The increase in p53 transactivation seen in transfections of increasing ARF in the presence of constant Mdm2 and MdmX most likely results from with ARF nucleolar sequestration of Mdm2 (14Pomerantz J. Schreiber-Agus N. Liegeois N.J. Silverman A. Alland L. Chin L. Potes J. Chen K. Orlow I. Lee H.W. Cordon-Cardo C. DePinho R.A. Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, 15Weber J.D. Taylor L.J. Roussel M.F. Sherr C.J. Bar-Sagi D. Nat. Cell Biol. 1999; 1: 20-26Crossref PubMed Scopus (803) Google Scholar) and MdmX (Fig. 1) or the decreased protein stability of Mdm2 and MdmX (Fig. 4A). Finally, consistent with the antagonist effects of ARF and MdmX functioning through nucleolar sequestration, a 5-fold increase in p53 transactivation detected when ARF was overexpressed in U2OS cells was inhibited in a dose-dependent manner with transfection of increasing levels of mdmX expression plasmid (Fig.6B). Taken together with the results in Fig. 1B, these data demonstrate that MdmX regulation of p53 transactivation is not limited to its direct association with p53 (29Shvarts A. Bazuine M. Dekker P. Ramos Y.F. Steegenga W.T. Merckx G. van Ham R.C. van der Houven van Oordt W. van der Eb A.J. Jochemsen A.G. Genomics. 1997; 43: 34-42Crossref PubMed Scopus (124) Google Scholar) or with Mdm2 (22Jackson M. Berberich S.J. Mol. Cell. Biol. 2000; 20: 1001-1007Crossref PubMed Scopus (183) Google Scholar) but can also be elicited through MdmX association with ARF. Both ARF and MdmX are capable of regulating p53 protein levels through binding to and inhibiting Mdm2 function. However, MdmX also binds directly to p53, resulting in the nucleoplasmic sequestration of p53 protein and inhibition of p53 transactivation (22Jackson M. Berberich S.J. Mol. Cell. Biol. 2000; 20: 1001-1007Crossref PubMed Scopus (183) Google Scholar). We demonstrate in this report that in addition to mobilizing Mdm2 to the nucleolus, ARF can also interact with and direct the nucleolar localization of MdmX (Fig. 1A). Redistribution of MdmX requires the C-terminal RING domain of MdmX that contains a conserved stretch of basic amino acids recently identified as an NrLS in Mdm2 (Fig. 2). Mobilization of MdmX from the nucleoplasm to the nucleoli segregates MdmX from p53, resulting in a decrease in the ability of MdmX to interact with and inhibit p53 transactivation (Fig. 1B). Furthermore, we show that coexpression of MdmX with ARF and Mdm2 results in a decrease in Mdm2 stability, indicating that interaction of MdmX and ARF prohibits the stabilizing interaction between Mdm2 and ARF and likely blocks MdmX interactions with Mdm2. Interestingly, although the coexpression of MdmX, Mdm2, and ARF results in decreased Mdm2 protein levels relative to MdmX and Mdm2 alone, it also resulted in a lower level of p53 transactivation relative to ARF alone (Fig.6A). These data are consistent with a model in which the nucleolar sequestration of MdmX through MdmX·ARF complexes releases Mdm2, leading to its eventual for degradation (Fig.7). Because the ability of MdmX to stabilize Mdm2 (Fig. 7A) is lost upon coexpression of ARF, we predict that the existence of an equilibrium between MdmX·Mdm2, Mdm2·ARF, and MdmX·ARF complexes may ultimately balance the levels of both Mdm2 and p53 (Fig. 7,B and C). Clearly a key factor in this effect is the ratio of MdmX and Mdm2 protein. It is worth noting that the MdmX protein has a much greater half-life than Mdm2; therefore, it is likely that in these present studies when cotransfecting equal molar amounts of mdm2 and mdmX plasmids, the MdmX protein concentration is significantly higher than that for Mdm2 protein. Studies comparing the various binding affinities between the Mdm2, MdmX, and ARF heterodimeric complexes are currently ongoing. At the cellular level we speculate that in tissues such as the thymus, brain, and testis, where higher levels of mdmX transcripts have been reported relative to other tissues (28Shvarts A. Steegenga W.T. Riteco N. van Laar T. Dekker P. Bazuine M. van Ham R.C. van der Houven van Oordt W. Hateboer G. van der Eb A.J. Jochemsen A.G. EMBO J. 1996; 15: 5349-5357Crossref PubMed Scopus (519) Google Scholar, 29Shvarts A. Bazuine M. Dekker P. Ramos Y.F. Steegenga W.T. Merckx G. van Ham R.C. van der Houven van Oordt W. van der Eb A.J. Jochemsen A.G. Genomics. 1997; 43: 34-42Crossref PubMed Scopus (124) Google Scholar), that ARF-MdmX interactions (Fig. 7C) may play a role consistent with those observed in these overexpression studies. Taken together, the observations detailed here support a model whereby MdmX is responsible for maintaining the nucleoplasmic localization of Mdm2 and p53 in actively dividing cells. During a DNA-damaging event in which p53 and Mdm2 would become induced, MdmX would likely be of little consequence because reports from our laboratory and others have shown that MdmX proteins are not induced following DNA damage (27Jackson M.W. Berberich S.J. DNA Cell Biol. 1999; 18: 693-700Crossref PubMed Scopus (27) Google Scholar, 28Shvarts A. Steegenga W.T. Riteco N. van Laar T. Dekker P. Bazuine M. van Ham R.C. van der Houven van Oordt W. Hateboer G. van der Eb A.J. Jochemsen A.G. EMBO J. 1996; 15: 5349-5357Crossref PubMed Scopus (519) Google Scholar). However, induction of ARF by oncogenic stimuli, such as deregulated Myc expression (31Zindy F. Eischen C.M. Randle D.H. Kamijo T. Cleveland J.L. Sherr C.J. Roussel M.F. Genes Dev. 1998; 12: 2424-2433Crossref PubMed Scopus (1060) Google Scholar), and its subsequent regulation of Mdm2 may be affected differentially in tissues expressing high levels of MdmX. The resulting antagonistic effect of MdmX on ARF may weaken the ability of ARF to sequester Mdm2, resulting in more rapid Mdm2 and p53 turnover and an inactivation of p53 function. Consistent with this hypothesis, a small percentage of human gliomas contain an amplified mdmX gene without p53 mutation or mdm2 gene amplification. Furthermore, gliomas containing amplified mdmX showed no accumulation of p53 or Mdm2 protein (32Riemenschneider M.J. Buschges R. Wolter M. Reifenberger J. Bostrom J. Kraus J.A. Schlegel U. Reifenberger G. Cancer Res. 1999; 59: 6091-6096PubMed Google Scholar). We are presently exploring the possibility that ARF-MdmX interactions in these tumors produce this malignant phenotype. Although our original model outlined MdmX as an inhibitor of Mdm2-mediated degradation (22Jackson M. Berberich S.J. Mol. Cell. Biol. 2000; 20: 1001-1007Crossref PubMed Scopus (183) Google Scholar), the data presented here argue that MdmX together with ARF may act antagonistically to allow more rapid Mdm2 turnover (Fig. 7). Most likely, both mechanisms are utilized to maintain steady state levels of Mdm2 and p53 in normal cells. It is also possible that the interaction between MdmX and ARF affects pathways other than those relating to p53 and Mdm2. In fact, exogenously expressed ARF can induce a cell cycle arrest in triple knock-out cells lacking p53, mdm2, and ARF (33Weber J.D. Jeffers J.R. Rehg J.E. Randle D.H. Lozano G. Roussel M.F. Sherr C.J. Zambetti G.P. Genes Dev. 2000; 14: 2358-2365Crossref PubMed Scopus (333) Google Scholar). Interestingly, the Mdm2 binding domain of ARF, amino acids 1–14, is required for ARF to induce an arrest in a p53/Mdm2 null background. Although it is tempting to speculate that ARF interacts with MdmX or similar Mdm2-like proteins to reverse the inhibition of a yet unidentified protein, further studies into the effects of ARF-MdmX interactions in the absence of Mdm2 remain to be completed. We thank Dr. Madhavi Kadakia for reviewing the manuscript and providing us with the V5-MdmX expression vector.