Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23

ABSTRACT The Arf tumor suppressor inhibits cell cycle progression through both p53-dependent and p53-independent mechanisms, including interference with rRNA processing. Using tandem-affinity-tagged p19Arf, we purified Arf-associated proteins from mouse NIH 3T3 fibroblasts undergoing cell cycle arrest. Tagged p19Arf associated with nucleolar and ribosomal proteins, including nucleophosmin/B23 (NPM), a protein thought to foster the maturation of preribosomal particles. NPM is an abundant protein, only a minor fraction of which binds to p19Arf; however, a significant proportion of p19Arf associates with NPM. The interaction between p19Arf and NPM requires amino acid sequences at the Arf amino terminus, which are also required for Mdm2 binding, as well as the central acidic domain of NPM and an adjacent segment that regulates NPM oligomerization. The interaction between p19Arf and NPM occurs in primary mouse embryonic fibroblasts, including those lacking both Mdm2 and p53. In an NIH 3T3 derivative cell line (MT-Arf) engineered to conditionally express an Arf transgene, induced p19Arf associates with NPM and colocalizes with it in high-molecular-weight complexes (2 to 5 MDa). An NPM mutant lacking its carboxyl-terminal nucleic acid-binding domain oligomerizes with endogenous NPM, inhibits p19Arf from entering into 2- to 5-MDa particles, and overrides the ability of p19Arf to retard rRNA processing.

[1]  W. Franke,et al.  Identification of a small, very acidic constitutive nucleolar protein (NO29) as a member of the nucleoplasmin family. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  F. Zindy,et al.  Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest , 1995, Cell.

[3]  D. Beach,et al.  p16INK4A and p19ARF act in overlapping pathways in cellular immortalization , 2000, Nature Cell Biology.

[4]  D. Eichler,et al.  Processing of eukaryotic ribosomal RNA. , 1994, Progress in nucleic acid research and molecular biology.

[5]  Mei-Ling Kuo,et al.  Nucleolar Arf tumor suppressor inhibits ribosomal RNA processing. , 2003, Molecular cell.

[6]  R. Reddel,et al.  Pex19p Dampens the p19ARF-p53-p21WAF1 Tumor Suppressor Pathway* , 2001, The Journal of Biological Chemistry.

[7]  M. Olson,et al.  Interaction of nucleolar phosphoprotein B23 with nucleic acids. , 1989, Biochemistry.

[8]  J. Shabanowitz,et al.  Composition and functional characterization of yeast 66S ribosome assembly intermediates. , 2001, Molecular cell.

[9]  A. Levine,et al.  P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Bouvet,et al.  Structure and functions of nucleolin. , 1999, Journal of cell science.

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

[12]  E. Brambilla,et al.  Human ARF binds E2F1 and inhibits its transcriptional activity , 2001, Oncogene.

[13]  Richard A. Ashmun,et al.  Tumor Suppression at the Mouse INK4a Locus Mediated by the Alternative Reading Frame Product p19 ARF , 1997, Cell.

[14]  F. Zindy,et al.  Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging , 1997, Oncogene.

[15]  H. F. Horn,et al.  Specific Phosphorylation of Nucleophosmin on Thr199 by Cyclin- dependent Kinase 2-Cyclin E and Its Role in Centrosome Duplication* , 2001, The Journal of Biological Chemistry.

[16]  C. Korgaonkar,et al.  Cyclin G1 has growth inhibitory activity linked to the ARF-Mdm2-p53 and pRb tumor suppressor pathways. , 2003, Molecular cancer research : MCR.

[17]  K. Nagata,et al.  The RNA binding activity of a ribosome biogenesis factor, nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype. , 2002, Molecular biology of the cell.

[18]  David Tollervey,et al.  60S pre‐ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm , 2002, The EMBO journal.

[19]  J L Cleveland,et al.  Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. , 1998, Genes & development.

[20]  U. Hoffmann‐Rohrer,et al.  Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes , 1998, The EMBO journal.

[21]  J. Steitz,et al.  U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus. , 1989, The EMBO journal.

[22]  M. Olson,et al.  Mapping the Functional Domains of Nucleolar Protein B23* , 2000, The Journal of Biological Chemistry.

[23]  Nobuko Katoku-Kikyo,et al.  Reversible disassembly of somatic nucleoli by the germ cell proteins FRGY2a and FRGY2b , 2003, Nature Cell Biology.

[24]  Jun Qin,et al.  Histone-like TAFs within the PCAF Histone Acetylase Complex , 1998, Cell.

[25]  M. Kubbutat,et al.  Identification of a cryptic nucleolar-localization signal in MDM2 , 2000, Nature Cell Biology.

[26]  Pier Giuseppe Pelicci,et al.  Nucleophosmin regulates the stability and transcriptional activity of p53 , 2002, Nature Cell Biology.

[27]  P. Bouvet,et al.  Nucleolin functions in the first step of ribosomal RNA processing , 1998, The EMBO journal.

[28]  T. Parisi,et al.  The Human Tumor Suppressor ARF Interacts with Spinophilin/Neurabin II, a Type 1 Protein-phosphatase-binding Protein* , 2001, The Journal of Biological Chemistry.

[29]  H. F. Horn,et al.  Nucleophosmin/B23 Is a Target of CDK2/Cyclin E in Centrosome Duplication , 2000, Cell.

[30]  David Tollervey,et al.  Making ribosomes. , 2002, Current opinion in cell biology.

[31]  S. Piñol-Roma.  Association of nonribosomal nucleolar proteins in ribonucleoprotein complexes during interphase and mitosis. , 1999, Molecular biology of the cell.

[32]  Charles J. Sherr,et al.  The INK4a/ARF network in tumour suppression , 2001, Nature Reviews Molecular Cell Biology.

[33]  Charles J. Sherr,et al.  Nucleolar Arf sequesters Mdm2 and activates p53 , 1999, Nature Cell Biology.

[34]  A. Szalay,et al.  The p14ARF Tumor Suppressor Protein Facilitates Nucleolar Sequestration of Hypoxia-inducible Factor-1α (HIF-1α) and Inhibits HIF-1-mediated Transcription* , 2001, The Journal of Biological Chemistry.

[35]  D. Quelle,et al.  Cancer-associated mutations at the INK4a locus cancel cell cycle arrest by p16INK4a but not by the alternative reading frame protein p19ARF. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[36]  R. Savkur,et al.  Preferential cleavage in pre-ribosomal RNA byprotein B23 endoribonuclease. , 1998, Nucleic acids research.

[37]  J. R. Warner,et al.  Nascent Ribosomes , 2001, Cell.

[38]  M. Roussel,et al.  p53-independent functions of the p19(ARF) tumor suppressor. , 2000, Genes & development.

[39]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[40]  F. Peter.  Strategies for protein purification and characterization , 1996 .

[41]  M. Roussel,et al.  Cooperative Signals Governing ARF-Mdm2 Interaction and Nucleolar Localization of the Complex , 2000, Molecular and Cellular Biology.

[42]  David M. Livingston,et al.  p19ARF targets certain E2F species for degradation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[43]  M. Dundr,et al.  Partially processed pre-rRNA is preserved in association with processing components in nucleolus-derived foci during mitosis. , 1998, Molecular biology of the cell.

[44]  H. Busch,et al.  Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis. , 1985, Biochimica et biophysica acta.

[45]  J. Steitz,et al.  Sno Storm in the Nucleolus: New Roles for Myriad Small RNPs , 1997, Cell.

[46]  P. Grandi,et al.  Identification of a 60S preribosomal particle that is closely linked to nuclear export. , 2001, Molecular cell.

[47]  C. Larsen,et al.  Human ARF protein interacts with Topoisomerase I and stimulates its activity , 2001, Oncogene.

[48]  B. Séraphin,et al.  The tandem affinity purification (TAP) method: a general procedure of protein complex purification. , 2001, Methods.

[49]  A. Görg,et al.  Improved silver staining protocols for high sensitivity protein identification using matrix‐assisted laser desorption/ionization‐time of flight analysis , 2001, Proteomics.

[50]  Deanna Naeve,et al.  Arf induces p53-dependent and -independent antiproliferative genes. , 2003, Cancer research.

[51]  R. Simard.  The nucleus: action of chemical and physical agents. , 1970, International review of cytology.

[52]  M. Olson,et al.  Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction. , 1986, Biochemistry.

[53]  P. Bouvet,et al.  Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA. , 1996, Journal of molecular biology.

[54]  N. Perkins,et al.  p53- and Mdm2-Independent Repression of NF-κB Transactivation by the ARF Tumor Suppressor , 2003 .

[55]  M. Olson,et al.  Nucleolar protein B23 has molecular chaperone activities , 2008, Protein science : a publication of the Protein Society.