Molecular Biology Reports

, Volume 36, Issue 4, pp 717–723

Ran GTPase guanine nucleotide exchange factor RCC1 is phosphorylated on serine 11 by cdc2 kinase in vitro

  • Yukiko Horiike
  • Hideki Kobayashi
  • Takeshi Sekiguchi
Article

Abstract

RCC1, a guanine nucleotide exchange factor for Ran GTPase, plays essential roles in the growth and viability of mammalian cells. Here, we examined the phosphorylation of specific serine and threonine residues of RCC1 in vivo and showed that RCC1 is indeed phosphorylated. Analysis by two-dimensional (2D) gel electrophoresis suggested that serine 11 (S11) of hamster RCC1 is phosphorylated in vivo. A point mutation of S11 of hamster RCC1 resulted in a decrease in the number of 2D gel spots, indicating a lack of phosphorylation at the mutant residue. S11 phosphorylation in vitro depended on cyclin B-cdc2 kinase. An RCC1 mutant in which all N-terminal serine and threonine residues were substituted with glutamate residues to mimic phosphorylation at these residues showed decreased binding to the karyopherin, KPNA4, compared with wild type RCC1. We conclude that RCC1 undergoes post-translational phosphorylation.

Keywords

CDK/cyclin Karyopherin Phosphorylation RCC1 tsBN2 

References

  1. 1.
    Bischoff FR, Maier G, Tilz G et al (1990) A 47-kDa human nuclear protein recognized by antikinetochore autoimmune sera is homologous with the protein encoded by RCC1, a gene implicated in onset of chromosome condensation. Proc Natl Acad Sci USA 87:8617–8621PubMedCrossRefGoogle Scholar
  2. 2.
    Kai R, Ohtsubo M, Sekiguchi M et al (1986) Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation. Mol Cell Biol 6:2027–2032PubMedGoogle Scholar
  3. 3.
    Nishitani H, Kobayashi H, Ohtsubo M et al (1990) Cloning of Xenopus RCC1 cDNA, a homolog of the human RCC1 gene: complementation of tsBN2 mutation and identification of the product. J Biochem (Tokyo) 107:228–235Google Scholar
  4. 4.
    Frasch M (1991) The maternally expressed Drosophila gene encoding the chromatin-binding protein BJ1 is a homolog of the vertebrate gene regulator of chromatin condensation, RCC1. EMBO J 10:1225–1236PubMedGoogle Scholar
  5. 5.
    Clark KL, Sprague GF Jr (1989) Yeast pheromone response pathway: characterization of a suppressor that restores mating to receptorless mutants. Mol Cell Biol 9:2682–2694PubMedGoogle Scholar
  6. 6.
    Aebi M, Clark MW, Vijayraghavan U et al (1990) A yeast mutant, PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is defective in a gene homologous to the human gene RCC1 which is involved in the control of chromosome condensation Mol Gen Genet 224:72–80PubMedCrossRefGoogle Scholar
  7. 7.
    Kadowaki T, Goldfarb D, Spitz LM et al (1993) Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily EMBO J 12:2929–2937PubMedGoogle Scholar
  8. 8.
    Matsumoto T, Beach D (1991) The spil GTPase interacts with RCCl in cell cycle dependency. Cold Spring Harb Symp Quant Biol 56:385–398PubMedGoogle Scholar
  9. 9.
    Dasso M (1993) RCC1 in the cell cycle: the regulator of chromosome condensation takes on new roles. Trends Biochem Sci 18:96–101PubMedCrossRefGoogle Scholar
  10. 10.
    Izaurralde E, Mattaj IW (1995) RNA export. Cell 81:153–159PubMedCrossRefGoogle Scholar
  11. 11.
    Moore MS, Blobel G (1993) The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365:661–663PubMedCrossRefGoogle Scholar
  12. 12.
    Ohba T, Nakamura M, Nishitani H et al (1999) Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran. Science 284:1356–1358PubMedCrossRefGoogle Scholar
  13. 13.
    Gorlich D, Kutay U (1999) Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol 15:607–660PubMedCrossRefGoogle Scholar
  14. 14.
    Hetzer M, Bilbao-Cortes D, Walther TC et al (2000) GTP hydrolysis by Ran is required for nuclear envelope assembly. Mol Cell 5:1013–1024PubMedCrossRefGoogle Scholar
  15. 15.
    Seino H, Hisamoto N, Uzawa S et al (1992) DNA-binding domain of RCC1 protein is not essential for coupling mitosis with DNA replication. J Cell Sci:393–400Google Scholar
  16. 16.
    Nemergut ME, Mizzen CA, Stukenberg T et al (2001) Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2B. Science 292:1540–1543PubMedCrossRefGoogle Scholar
  17. 17.
    Zang WQ, Benedict YT (1999) Distinct export pathway utilized by the hepatitis B virus posttranscriptional regulatory element. Virology 259:299–304PubMedCrossRefGoogle Scholar
  18. 18.
    Bilbao-Cortes D, Hetzer M, Langst G et al (2002) Ran binds to chromatin by two distinct mechanisms. Curr Biol 12:1151–1156PubMedCrossRefGoogle Scholar
  19. 19.
    Nishimoto T, Eilen E, Basilico C (1978) Premature of chromosome condensation in a ts DNA-mutant of BHK cells. Cell 15:475–483PubMedCrossRefGoogle Scholar
  20. 20.
    Arnaoutov A, Dasso M (2003) The Ran GTPase regulates kinetochore function. Dev Cell 5:99–111PubMedCrossRefGoogle Scholar
  21. 21.
    Uchida S, Sekiguchi T, Nishitani H et al (1990) Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene. Mol Cell Biol 10:577–584PubMedGoogle Scholar
  22. 22.
    Fukumura J, Noguchi E, Sekiguchi T et al (2003) A temperature-sensitive mutant of the mammalian RNA helicase, DEAD-BOX X isoform, DBX, defective in the transition from G1 to S phase. J Biochem (Tokyo) 134:71–82Google Scholar
  23. 23.
    Makarova O, Kamberov E, Margolis B (2000) Generation of deletion and point mutations with one primer in a single cloning step. Biotechniques 29:970–972PubMedGoogle Scholar
  24. 24.
    Miyamoto Y, Imamoto N, Sekimoto T et al (1997) Differential modes of nuclear localization signal (NLS) recognition by three distinct classes of NLS receptors. J Biol Chem 272:26375–26381PubMedCrossRefGoogle Scholar
  25. 25.
    O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021PubMedGoogle Scholar
  26. 26.
    Azuma Y, Seino H, Seki T et al (1996) Conserved histidine residues of RCC1 are essential for nucleotide exchange on Ran. J Biochem (Tokyo) 120:82–91Google Scholar
  27. 27.
    Sekiguchi T, Noguchi E, Hayashida T et al (1996) D-type cyclin expression is decreased and p21 and p27 CDK inhibitor expression is increased when tsBN462 CCG1/TAFII250 mutant cells arrest in G1 at the restrictive temperature. Genes Cells 1:687–705PubMedCrossRefGoogle Scholar
  28. 28.
    Morgan DO (1995) Principles of CDK regulation. Nature 374:131–134PubMedCrossRefGoogle Scholar
  29. 29.
    Hutchins JR, Moore WJ, Hood FE et al (2004) Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis. Curr Biol 14:1099–1104PubMedCrossRefGoogle Scholar
  30. 30.
    Li HY, Zheng Y (2004) Phosphorylation of RCC1 in mitosis is essential for producing a high RanGTP concentration on chromosomes and for spindle assembly in mammalian cells. Genes Dev 18:512–527PubMedCrossRefGoogle Scholar
  31. 31.
    Kohler M, Speck C, Christiansen M et al (1999) Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import. Mol Cell Biol 19:7782–7791PubMedGoogle Scholar
  32. 32.
    Renault L, Nassar N, Wittinghofer A et al (1999) Crystallization and preliminary X-ray analysis of human RCC1, the regulator of chromosome condensation. Acta Crystallogr D Biol Crystallogr 55:272–275PubMedCrossRefGoogle Scholar
  33. 33.
    Hanson DA, Ziegler SF (2004) Fusion of green fluorescent protein to the C-terminus of granulysin alters its intracellular localization in comparison to the native molecule. J Negat Results Biomed 3:2PubMedCrossRefGoogle Scholar
  34. 34.
    Lenassi Zupan A, Trobec S, Gaberc-Porekar V et al (2004) High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles. J Biotechnol 109:115–122PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Yukiko Horiike
    • 1
  • Hideki Kobayashi
    • 1
    • 2
    • 3
  • Takeshi Sekiguchi
    • 1
    • 2
  1. 1.Department of Molecular BiologyGraduate School of Medical Science, Kyushu UniversityFukuokaJapan
  2. 2.CREST, Japanese Science and Technology AgencyKawaguchiJapan
  3. 3.Center for Faculty DevelopmentOkayama UniversityOkayamaJapan

Personalised recommendations