MdmX Binding to ARF Affects Mdm2 Protein Stability and p53 Transactivation

Abstract

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 The plasmid was by of a p53 binding of a contains of a p53 binding of the The plasmid was used to In the p53 transactivation and expression were used. were In a plasmid was with each to of cells with For in and were by the of a plasmid (Promega) in each between MdmX and ARF Mdm2 and inhibits p53 of from U2OS cells was used to Mdm2, and MdmX protein levels. of MdmX with Mdm2 in the of Mdm2. However, expression of increasing of ARF in the presence of MdmX in a dose-dependent decrease in the of Mdm2 protein by MdmX. from U2OS cells were used in to and ARF proteins levels. The of Mdm2 following the coexpression of ARF is not by an of ARF and MdmX not protein U2OS cells were with human MdmX and increasing of or and increasing of human MdmX. cell were and was to ARF and MdmX protein levels. The protein stability levels of MdmX and ARF are not by was U2OS cells on were in with in and for with 10% goat in antibody was to a of and for The cells were and with the secondary antibody for were with for immunofluorescence was 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 and cell were following by cell for in containing a inhibitor was by of cell with of monoclonal V5 antibody in a of of containing and of protein was and an additional for each were in of and 10% by of proteins to a a was as described previously S.J. D. D. 1999; PubMed Scopus Google Scholar) primary antibody of and secondary of For p53 were p53 transactivation was by activity in from cell a activity was by protein in and for activity was by the into of activity of and were in of p53 transactivation the for the by the of studies have demonstrated that ARF is 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, Y. Xiong Y. Mol. Cell. 1999; 3: 579-591Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 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, not found in in the absence of can be to following either coexpression of ARF or 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). on the homology between Mdm2 and we examined MdmX was found in or could be with ARF. U2OS which not ARF 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 with an expression plasmid a human MdmX protein in the presence or absence of an expression plasmid an protein. In the absence of ARF in the presence of data not MdmX no nucleolar localization However, coexpression of MdmX to the nucleolus and MdmX and ARF signals were demonstrated and ARF localization to the nucleolus was in U2OS cells by that ARF with the nucleolus protein and the effect of ARF expression on MdmX of p53 U2OS cells were with the of the into cells wild-type p53 protein to a increase in p53 transactivation with of a p53 plasmid containing p53 binding of a expression vector to a decrease in the activity of the p53 increasing levels of expression vector, p53 transactivation levels to decrease in U2OS cells not Interestingly, the addition of a or expression to a dose-dependent increase in p53 levels of p53 transactivation in the absence of MdmX or ARF. The increase in p53 transactivation activity on the with the U2OS cells that the ARF protein levels both the MdmX and Mdm2, and the of p53 protein on the for a of basic amino or in the RING 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 MdmX contains a similar the NrLS of Mdm2 with the NrLS of both human and murine MdmX. human and murine MdmX sequences have a 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 M.A. Ashcroft M. Kubbutat M.H. Vousden K.H. Nat. Cell Biol. 2000; 2: 179-181Crossref PubMed Scopus (171) Google Scholar). the of the NrLS in a of MdmX the RING finger which contains the was and its ability to colocalize with ARF was The a similar in Mdm2, which demonstrated the for a NrLS in the Mdm2 RING finger for Mdm2·ARF 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). with Mdm2, which the MdmX RING was unable to colocalize to the nucleolus with ARF ARF was not from the nucleolus with MdmX-(1–446) the results with 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 expression of MdmX-(1–446) to a of ARF in the with coexpression with full-length MdmX and The of MdmX-(1–446) to ARF within the in ARF binding to MdmX and H1299 cells were as described in MdmX protein was and the interaction with ARF was by on results both full-length MdmX and MdmX-(1–446) were capable of interacting with ARF. the to a to be a decrease in the of ARF to MdmX-(1–446) to full-length MdmX. the interaction between ARF and MdmX is not as in the as it is in the nucleolus. The nucleolar localization of ARF in H1299 cells that ARF localization was with that in U2OS We also examined a more of amino acids which also to ARF is unable to colocalize to the nucleolus with ARF not Taken together, the data in and are with the similar for Mdm2, that MdmX must contain a functional RING finger in for ARF to it to the nucleolus not for association with ARF. ARF and MdmX have both been 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, 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 examined MdmX the stability of Mdm2 either or in the presence of increasing levels of ARF protein. Although studies expression have to Berberich S.J. Cell Biol. 1999; 18: PubMed Scopus Google Scholar, A. N. van T. P. M. van van van W. G. van Eb A.J. Jochemsen A.G. EMBO J. 1996; PubMed Scopus Google Scholar), has levels of in the of both human and A. M. P. Ramos Y.F. G. van van van W. van Eb A.J. Jochemsen A.G. PubMed Scopus Google Scholar) that MdmX protein in specific In with previously MdmX was to stabilize the Mdm2 protein In coexpression of MdmX and ARF a dose-dependent antagonistic effect on the ability of both proteins to stabilize Mdm2 protein Interestingly, was also a decrease in MdmX protein levels as ARF protein levels and Mdm2 protein levels is that the decrease in MdmX protein levels results from a decrease in complex be in with recent in which MdmX the RING finger protein stability to full-length MdmX S. S. K. K. A. M. 1999; PubMed Scopus Google Scholar). of MdmX-(1–446) protein was not to stabilize Mdm2 protein expressed in U2OS cells was it to the ability of ARF protein to stabilize Mdm2 protein The of MdmX-(1–446) to reverse the stability of Mdm2 that the association of MdmX and ARF not be to reverse the ARF stability of Mdm2 and that nucleolar localization of ARF and MdmX is required to Mdm2. The results shown in demonstrated that the stability of both MdmX and Mdm2 as ARF protein levels the decrease in MdmX stability was Mdm2 or MdmX was expressed in U2OS cells with increasing ARF in the absence of Mdm2. Under these conditions, increasing ARF protein levels not MdmX protein levels with the stability of MdmX in more on the presence of Mdm2 ARF levels. Finally, was no in ARF levels as in containing increasing levels of MdmX expression vector Taken together, the results shown in that the association between MdmX and ARF has no effect on the stability of either protein. Finally, to the effects of Mdm2, and ARF on p53 U2OS cells were with the and the of Mdm2 and MdmX expression together, to a decrease in p53 transactivation The inhibition of p53 transactivation by with and expression was in a dose-dependent by coexpression with ARF expression The increase in p53 transactivation in of increasing ARF in the presence of constant Mdm2 and MdmX 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 or the protein stability of Mdm2 and MdmX Finally, with the antagonist effects of ARF and MdmX through nucleolar a increase in p53 transactivation ARF was in U2OS cells was in a dose-dependent manner with of increasing levels of expression plasmid Taken with the results in these data demonstrate that MdmX of p53 transactivation is not to its association with p53 A. M. P. Ramos Y.F. G. van van van W. van Eb A.J. Jochemsen A.G. PubMed Scopus Google Scholar) or with Mdm2 (22Jackson M. Berberich S.J. Mol. Cell. Biol. 2000; 20: 1001-1007Crossref PubMed Scopus (183) Google Scholar) can also be through MdmX association with ARF. ARF and MdmX are capable of regulating p53 protein levels through binding to and Mdm2 However, MdmX also to p53, resulting in the 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 that in addition to Mdm2 to the ARF can also with and the nucleolar localization of MdmX Redistribution of MdmX requires the C-terminal RING of MdmX that contains a of basic amino acids recently as an NrLS in Mdm2 of MdmX from the to the MdmX from p53, resulting in a decrease in the ability of MdmX to with and inhibit p53 transactivation we that coexpression of MdmX with ARF and Mdm2 results in a decrease in Mdm2 that interaction of MdmX and ARF the interaction between Mdm2 and ARF and MdmX with Mdm2. Interestingly, the coexpression of Mdm2, and ARF results in Mdm2 protein levels to MdmX and Mdm2 it also in a of p53 transactivation to ARF data are with a in which the nucleolar sequestration of MdmX through Mdm2, to its for degradation the ability of MdmX to stabilize Mdm2 is coexpression of we that the of an between and the levels of both Mdm2 and p53 and a in effect is the of MdmX and Mdm2 protein. is that the MdmX protein has a it is that in these present studies of and the MdmX protein is that for Mdm2 protein. the various binding between the Mdm2, and ARF are the we that in as the and where levels of transcripts have been to other A. N. van T. P. M. van van van W. G. van Eb A.J. Jochemsen A.G. EMBO J. 1996; PubMed Scopus Google Scholar, A. M. P. Ramos Y.F. G. van van van W. van Eb A.J. Jochemsen A.G. PubMed Scopus Google Scholar), that a with in these Taken together, the here a MdmX is for maintaining the localization of Mdm2 and p53 in a in which p53 and Mdm2 MdmX be of because from and have shown that MdmX proteins are not following Berberich S.J. Cell Biol. 1999; 18: PubMed Scopus Google Scholar, A. N. van T. P. M. van van van W. G. van Eb A.J. Jochemsen A.G. EMBO J. 1996; PubMed Scopus Google Scholar). However, induction of ARF by as expression F. T. Sherr C.J. Roussel M.F. Genes Dev. 1998; PubMed Scopus Google Scholar), and its subsequent of Mdm2 be in levels of MdmX. The resulting antagonistic effect of MdmX on ARF the ability of ARF to Mdm2, resulting in more Mdm2 and p53 and an of p53 with a of human contain an p53 mutation or containing no of p53 or Mdm2 protein M.J. R. M. J. J. J.A. U. G. Cancer Res. 1999; Google Scholar). We are the that in these Although 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 here that MdmX with ARF act to more Mdm2 both are to levels of Mdm2 and p53 in is also that the interaction between MdmX and ARF pathways other to p53 and Mdm2. In expressed ARF can a cell cycle arrest in cells p53, and ARF J.D. G. Roussel M.F. Sherr C.J. Genes Dev. 2000; PubMed Scopus Google Scholar). Interestingly, the Mdm2 binding of amino acids is required for ARF to an arrest in a Although it is to that ARF with MdmX or similar proteins to reverse the inhibition of a protein, studies into the effects of in the absence of Mdm2 to be We Dr. for the and with the expression