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Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin

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  • Nucleolar Proteins
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Abstract

The nucleolar protein gar2 of fission yeast is structurally related to the multifunctional nucleolar protein nucleolin from vertebrates and has been shown to be implicated in production of 18S rRNA. gar2 contains several potential casein kinase 2 (CK2) phosphorylation sites and a single putative p34cdc2 phosphorylation site in the consensus S50PKK. Here, we show that, like nucleolin, gar2 is phosphorylated in vitro by both highly purified CK2 from CHO cells and p34cdc2 from starfish oocytes. Moreover, the substitution of alanine for the N-terminal serine 50 abolishes phosphorylation by p34cdc2 in vitro. We also provide evidence that gar2 is phosphorylated in vitro by a p13suc1-Sepharose-bound kinase fromSchizosaccharomyces pombe extracts that displays cell cycle-regulated activity similar to that of the p34cdc2 kinase. In vivo32P labeling of cells indicates that gar2 is a phosphoprotein and that incorporation of phosphate on residue 50 occurs specifically at mitosis. Taken together, these results lead us to propose that gar2 is likely to be an in vivo substrate for the mitotic p34cdc2 kinase. However, this posttranslational modification of the gar2 protein does not appear to be essential for normal production of 18S rRNA.

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References

  • Alderuccio F, Chan EKL, Tan EM (1991) Molecular characterization of an autoantigen PM-Scl in the polymyositis/schleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75 kD acidic protein of the nucleolar complex. J Exp Med 173:941–952

    Article  PubMed  CAS  Google Scholar 

  • Azum-Gélade MC, Noaillac-Depeyre J, Caizergues-Ferrer M, Gas N (1994) Cell cycle redistribution of U3 snRNA and fibrillarin. J Cell Sci 107:463–475

    PubMed  Google Scholar 

  • Basi G, Schmid E, Maundrell K (1993) Tata box mutations in theSchizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123:131–136

    Article  PubMed  CAS  Google Scholar 

  • Belenguer P, Caizergues-Ferrer M, Labbé JC, Dorée M, Amalric F (1990) Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase. Mol Cell Biol 10:3607–3618

    PubMed  CAS  Google Scholar 

  • Booher RN, Alfa CE, Hyams JS, Beach DH (1989). The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization. Cell 58:485–497

    Article  PubMed  CAS  Google Scholar 

  • Brizuela L, Draetta G, Beach D (1987) p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J 6:3507–3514

    PubMed  CAS  Google Scholar 

  • Caizergues-Ferrer M, Belenguer P, Lapeyre B, Amalric F, Wallace MO, Olson MOJ (1987). Phosphorylation of nucleolin by a nucleolar Type NII protein kinase. Biochemistry 26:7876–7883

    Article  PubMed  CAS  Google Scholar 

  • Ducommun B, Brambilla P, Draetta G (1991). Mutation at sites involved in suc1 binding inactivate cdc2. Mol Cell Biol 11:6177–6184

    PubMed  CAS  Google Scholar 

  • Fantes P (1979) Epistatic gene interactions in the control of division in fission yeast. Nature 279:428–430

    Article  PubMed  CAS  Google Scholar 

  • Gallagher IM, Alfa CE, Hyams JS (1993) p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus. Mol Biol Cell 4:1087–1096

    PubMed  CAS  Google Scholar 

  • Gulli MP, Girard JP, Zabetakis D, Lapeyre B, Mélèse T, Caizergues-Ferrer M (1995). gar 2 is a nucleolar protein fromSchizosacchharonyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation. Nucleic Acids Res 23:1912–1918

    PubMed  CAS  Google Scholar 

  • Heald R, Mc Keon F (1990) Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Cell 61:579–589

    Article  PubMed  CAS  Google Scholar 

  • Hiraoka Y, Toda T, Yanagida M (1984) The nda3 gene of fission yeast encodes β-tubulin: a cold sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39:349–358

    Article  PubMed  CAS  Google Scholar 

  • Kharrat A, Derancourt J, Dorée M, Amalric F, Erard M. (1991) Synergistic effects of histone H1 and nucleolin on chromatin condensation in mitosis: role of phosphorylated heteromer. Biochemistry 30:10329–10336

    Article  PubMed  CAS  Google Scholar 

  • Kunkel TA, Roberts JD, Zakour RA (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–403

    PubMed  CAS  Google Scholar 

  • Labbé PC, Picard A, Peaucellier G, Cavadore JC, Nurse P, Dorée M (1989) Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation. Cell 57:253–263

    Article  PubMed  Google Scholar 

  • Labbé JC, Cavadore JC, Dorée M (1991) M. phase-specific cdc2 kinase: preparation from starfish oocytes and properties. Methods Enzymol 200:291–301

    PubMed  Google Scholar 

  • Lapeyre B, Bourbon HM, Amalric F (1987) Nucleolin, the major nucleolar protein structure revealed by nucleotide sequence. Proc Natl Acad Sci USA. 84:1472–1476

    Article  PubMed  CAS  Google Scholar 

  • Lee WC, Xue Z, Mélèse T (1991) The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs. J Cell Biol 113:1–12

    Article  PubMed  CAS  Google Scholar 

  • Lischwe MA, Richards RL, Busch RK, Busch H (1981) Localization of phosphoprotein C23 to nucleolar structures and to the nucleolus organizer regions. Exp Cell Res 136:101–109

    Article  PubMed  CAS  Google Scholar 

  • Mc Cully EK, Robinow CF (1971) Mitosis in the fission yeastSchizosaccharomyces pombe: a comparative study with light and electron microscopy. J Cell Sci 9:475–507

    CAS  Google Scholar 

  • Moll T, Tebb G, Surana U, Robitsch H, Nasmyth K (1991) The role of phosphorylation and the cdc28 protein kinase in cell cycle-regulated nuclear import of theS. cerevisiae transcription factor SWI 5. Cell 66:1–20

    Article  Google Scholar 

  • Moreno S, Klar A, Nurse P (1991) Guide to yeast genetics and molecular biology. Molecular genetic analysis of fission yeastSchizosaccharomyces pombe. Methods Enzymol 194:795–823

    PubMed  CAS  Google Scholar 

  • Morgan DO (1995) Principles of CDK regulation. Nature 374:131–134

    Article  PubMed  CAS  Google Scholar 

  • Nigg EA (1995) Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays 17:471–480

    Article  PubMed  CAS  Google Scholar 

  • Norbury C, Nurse P (1992) Animal cell cycles and their control. Annu Rev Biochem 61:441–470

    Article  PubMed  CAS  Google Scholar 

  • Ookata K, Hisanaga SI, Bulinski JC, Murofushi H, Aizawa H, Itoh TJ, Hotani H, Okumura E, Tachibana K, Kishimoto T (1995) Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 to microtubules and is a potential regulator of M-phase microtubule dynamics. J Cell Biol 128:849–862

    Article  PubMed  CAS  Google Scholar 

  • Peter M, Nakagawa J, Dorée M, Labbé JC, Nigg EA (1990a). In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell 61:591–602

    Article  PubMed  CAS  Google Scholar 

  • Peter M, Nakagawa J, Dorée M, Labbé JC, Nigg EA (1990b) Identification of major nuclear proteins as candidate mitotic substrates of cdc2 kinase. Cell 60:791–801

    Article  PubMed  CAS  Google Scholar 

  • Pinna L (1990) Casein kinase II: an “éminence grise” in cellular regulation? Biochim Biophys Acta 1054:267–284

    Article  PubMed  CAS  Google Scholar 

  • Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:203–211

    Google Scholar 

  • Roussel P, Hernandez-Verdun D (1994) Identification of Ag-NOR proteins, markers of proliferation related to ribosomal gene activity. Exp Cell Res 214:465–472

    Article  PubMed  CAS  Google Scholar 

  • Russell P, Nurse P (1986) cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell 45:145–153

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fristsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Shägger H, Von Jagow G (1987) Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    Article  Google Scholar 

  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89

    Article  PubMed  CAS  Google Scholar 

  • Tyers M, Tokiwa G, Nash R, Futcher B (1992) The Cln3-Cdc28 kinase complex ofS. cerevisiae is regulated by proteolysis and phosphorylation. EMBO J 11:1773–1784

    PubMed  CAS  Google Scholar 

  • Weisenberger D, Scheer U (1995) A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis. J Cell Biol 129:561–575

    Article  PubMed  CAS  Google Scholar 

  • Xue Z, Mélèse T (1994) Nucleolar proteins that bind NLSs: a role in nucleolar import or ribosome biogenesis? Trends Cell Biol 4:414–417

    Article  PubMed  CAS  Google Scholar 

  • Yanagida M (1990) Higher-order chromosome structure in yeast. J Cell Sci 96:1–3

    PubMed  Google Scholar 

Download references

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Author notes

  1. Marie-Pierre Gulli

    Present address: ISREC, Chemin des Boveresses 155, CH-1066, Epalinges, Switzerland

Authors and Affiliations

  1. Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 Route de Narbonne, F-31062, Toulouse Cedex, France

    Marie-Pierre Gulli, Marlène Faubladier, Hélène Sicard & Michèle Caizergues-Ferrer

Authors
  1. Marie-Pierre Gulli
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  2. Marlène Faubladier
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  3. Hélène Sicard
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  4. Michèle Caizergues-Ferrer
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Correspondence to Michèle Caizergues-Ferrer.

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Edited by: S.A. Gerbi

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Gulli, MP., Faubladier, M., Sicard, H. et al. Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin. Chromosoma 105, 532–541 (1997). https://doi.org/10.1007/BF02510490

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  • DOI: https://doi.org/10.1007/BF02510490

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