Cancer-Associated Mutations in the MDM2 Zinc Finger Domain Disrupt Ribosomal Protein Interaction and Attenuate MDM2-Induced p53 Degradation

ABSTRACT The p53-inhibitory function of the oncoprotein MDM2 is regulated by a number of MDM2-binding proteins, including ARF and ribosomal proteins L5, L11, and L23, which bind the central acidic domain of MDM2 and inhibit its E3 ubiquitin ligase activity. Various human cancer-associated MDM2 alterations targeting the central acidic domain have been reported, yet the functional significance of these mutations in tumor development has remained unclear. Here, we show that cancer-associated missense mutations targeting MDM2's central zinc finger disrupt the interaction of MDM2 with L5 and L11. We found that the zinc finger mutant MDM2 is impaired in undergoing nuclear export and proteasomal degradation as well as in promoting p53 degradation, yet retains the function of suppressing p53 transcriptional activity. Unlike the wild-type MDM2, whose p53-suppressive activity can be inhibited by L11, the MDM2 zinc finger mutant escapes L11 inhibition. Hence, the MDM2 central zinc finger plays a critical role in mediating MDM2's interaction with ribosomal proteins and its ability to degrade p53, and these roles are disrupted by human cancer-associated MDM2 mutations.

[1]  Petra de Graaf,et al.  Critical Role for a Central Part of Mdm2 in the Ubiquitylation of p53 , 2003, Molecular and Cellular Biology.

[2]  A. Levine,et al.  Nuclear Export Is Required for Degradation of Endogenous p53 by MDM2 and Human Papillomavirus E6 , 1998, Molecular and Cellular Biology.

[3]  F. Rilke,et al.  DISTINCT mdm2/p53 EXPRESSION PATTERNS IN LIPOSARCOMA SUBGROUPS: IMPLICATIONS FOR DIFFERENT PATHOGENETIC MECHANISMS , 1997, The Journal of pathology.

[4]  Muyang Li,et al.  Mono- Versus Polyubiquitination: Differential Control of p53 Fate by Mdm2 , 2003, Science.

[5]  A. Weissman,et al.  RING Finger Proteins Mediators of Ubiquitin Ligase Activity , 2000, Cell.

[6]  E. Yeh,et al.  Nucleocytoplasmic Shuttling Modulates Activity and Ubiquitination-Dependent Turnover of SUMO-Specific Protease 2 , 2006, Molecular and Cellular Biology.

[7]  Lawrence A. Donehower,et al.  Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53 , 1995, Nature.

[8]  T. Hupp,et al.  Dual-site regulation of MDM2 E3-ubiquitin ligase activity. , 2006, Molecular cell.

[9]  Yue Xiong,et al.  ARF Promotes MDM2 Degradation and Stabilizes p53: ARF-INK4a Locus Deletion Impairs Both the Rb and p53 Tumor Suppression Pathways , 1998, Cell.

[10]  M. Schwab,et al.  Cytogenetic evolution of MYCN and MDM2 amplification in the neuroblastoma LS tumour and its cell line. , 1995, European journal of cancer.

[11]  H. Taubert,et al.  Alternative and aberrant splicing of MDM2 mRNA in human cancer. , 2002, Cancer cell.

[12]  Y. Xiong,et al.  A p53 Amino-Terminal Nuclear Export Signal Inhibited by DNA Damage-Induced Phosphorylation , 2001, Science.

[13]  W. Sellers,et al.  Interaction between the retinoblastoma protein and the oncoprotein MDM2 , 1995, Nature.

[14]  Bert Vogelstein,et al.  Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53 , 1993, Nature.

[15]  K. Shirouzu,et al.  MDM2 interacts with MDMX through their RING finger domains , 1999, FEBS letters.

[16]  A. Levine,et al.  The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes , 1994, Molecular and cellular biology.

[17]  M. Kubbutat,et al.  Regulation of HDM2 activity by the ribosomal protein L11. , 2003, Cancer cell.

[18]  L. Donehower,et al.  Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  I. Weissman,et al.  LYAR, a novel nucleolar protein with zinc finger DNA-binding motifs, is involved in cell growth regulation. , 1993, Genes & development.

[20]  T. Allio,et al.  Ribosomal Protein L11 Negatively Regulates Oncoprotein MDM2 and Mediates a p53-Dependent Ribosomal-Stress Checkpoint Pathway , 2003, Molecular and Cellular Biology.

[21]  Tony Kouzarides,et al.  Stimulation of E2F1/DP1 transcriptional activity by MDM2 oncoprotein , 1995, Nature.

[22]  G. Reifenberger,et al.  Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. , 1993, Cancer research.

[23]  R. Honda,et al.  Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase , 2000, Oncogene.

[24]  M. Dai,et al.  Balance of Yin and Yang: ubiquitylation-mediated regulation of p53 and c-Myc. , 2006, Neoplasia.

[25]  J. Trent,et al.  WAF1, a potential mediator of p53 tumor suppression , 1993, Cell.

[26]  M. Dai,et al.  Ribosomal Protein L23 Activates p53 by Inhibiting MDM2 Function in Response to Ribosomal Perturbation but Not to Translation Inhibition , 2004, Molecular and Cellular Biology.

[27]  A. Levine,et al.  Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[28]  C. Cordon-Cardo,et al.  Impact of alterations affecting the p53 pathway in bladder cancer on clinical outcome, assessed by conventional and array-based methods. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[29]  A. Levine,et al.  Molecular abnormalities of mdm2 and p53 genes in adult soft tissue sarcomas. , 1994, Cancer research.

[30]  Shengyun Fang,et al.  Mdm2 Is a RING Finger-dependent Ubiquitin Protein Ligase for Itself and p53* , 2000, The Journal of Biological Chemistry.

[31]  F. Zindy,et al.  Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[32]  C. Maki,et al.  The MDM2 RING-finger domain is required to promote p53 nuclear export , 2000, Nature Cell Biology.

[33]  Hirofumi Tanaka,et al.  Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53 , 1997, FEBS letters.

[34]  G. Warren,et al.  Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1–Npl4 , 2002, The EMBO journal.

[35]  Stephen N. Jones,et al.  Regulation of p53 stability by Mdm2 , 1997, Nature.

[36]  Y. Xiong,et al.  Control of p53 ubiquitination and nuclear export by MDM2 and ARF. , 2001, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[37]  K. Bhat,et al.  Essential role of ribosomal protein L11 in mediating growth inhibition‐induced p53 activation , 2004, The EMBO journal.

[38]  M. Dai,et al.  Inhibition of MDM2-mediated p53 Ubiquitination and Degradation by Ribosomal Protein L5* , 2004, Journal of Biological Chemistry.

[39]  G. Wahl,et al.  A leucine‐rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking , 1999, The EMBO journal.

[40]  M. Oren,et al.  Mdm2 promotes the rapid degradation of p53 , 1997, Nature.

[41]  David Hawke,et al.  Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation. , 2003, Molecular cell.

[42]  P. Meltzer,et al.  Amplification of a gene encoding a p53-associated protein in human sarcomas , 1992, Nature.

[43]  D. Lane,et al.  Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53. , 2001, Experimental cell research.

[44]  M. Pierotti,et al.  Analysis of the molecular species generated by mdm2 gene amplification in liposarcomas , 2001, International journal of cancer.

[45]  A. Levine,et al.  Molecular Abnormalities of mdm 2 and p 53 Genes in Adult Soft Tissue Sarcomas ' , 2022 .

[46]  R. Copeland,et al.  A second p53 binding site in the central domain of Mdm2 is essential for p53 ubiquitination. , 2006, Biochemistry.

[47]  Kevin Ryan,et al.  The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2 , 1998, The EMBO journal.

[48]  R. Tjian,et al.  Repression of p53-mediated transcription by MDM2: a dual mechanism. , 1997, Genes & development.

[49]  A. Fersht,et al.  Solution structure of the C4 zinc finger domain of HDM2 , 2006, Protein science : a publication of the Protein Society.

[50]  K. Tsai,et al.  An intact HDM2 RING-finger domain is required for nuclear exclusion of p53 , 2000, Nature Cell Biology.

[51]  H. Ke,et al.  Essential Role of the B23/NPM Core Domain in Regulating ARF Binding and B23 Stability* , 2006, Journal of Biological Chemistry.

[52]  K. Itahana,et al.  Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23 , 2004, Molecular and Cellular Biology.

[53]  Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. , 1998, The EMBO journal.

[54]  Ken Chen,et al.  The Ink4a Tumor Suppressor Gene Product, p19Arf, Interacts with MDM2 and Neutralizes MDM2's Inhibition of p53 , 1998, Cell.

[55]  Guillermina Lozano,et al.  MDM2, an introduction. , 2003, Molecular cancer research : MCR.

[56]  T. Schlott,et al.  POINT MUTATIONS AND NUCLEOTIDE INSERTIONS IN THE MDM2 ZINC FINGER STRUCTURE OF HUMAN TUMOURS , 1997, The Journal of pathology.

[57]  Guillermina Lozano,et al.  Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53 , 1995, Nature.

[58]  Yanping Zhang,et al.  Nucleocytoplasmic Shuttling of p53 Is Essential for MDM2-Mediated Cytoplasmic Degradation but Not Ubiquitination , 2003, Molecular and Cellular Biology.

[59]  A. Levine,et al.  The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation , 1992, Cell.

[60]  D. George,et al.  Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. , 1991, The EMBO journal.

[61]  Scott D Emr,et al.  Ubiquitin interactions of NZF zinc fingers , 2004, The EMBO journal.

[62]  B. Wasylyk,et al.  The contribution of the acidic domain of MDM2 to p53 and MDM2 stability , 2001, Oncogene.

[63]  T. Pieler,et al.  Protein-mediated nuclear export of RNA: 5S rRNA containing small RNPs in xenopus oocytes , 1990, Cell.