Molecular and Cellular Biochemistry

, Volume 271, Issue 1–2, pp 215–223 | Cite as

A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo

  • Karène Antoine
  • Marie-Thérèse Prospéri
  • Didier Ferbus
  • Catherine Boule
  • Gérard Goubin


The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1–6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif. (Mol Cell Biochem 271: 215–223, 2005)

Key words

zinc finger protein SUMO-1 UBE2I yeast two-hybrid assay In vitro mutagenesis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Rosenberg UB, Schröder C, Preiss A, Kienlein A, Coté S, Riede I, Jackle H: Structural homology of the product of the Drosophila Krüppel gene with Xenopus transcription factor IIIA. Nature 319: 336–339, 1986Google Scholar
  2. 2.
    Chowdhury K, Deutsch U, Gruss P: A multigene family encoding several ”finger“ structures is present and differentially active in mammalian genomes. Cell 48: 771–778, 1987Google Scholar
  3. 3.
    Bellefroid EJ, Lecocq PJ, Benhida A, Poncelet DA, Belayew A, Martial JA: The human genome contains hundreds of genes coding for finger proteins of the Krueppel type. DNA 8: 377–387, 1989Google Scholar
  4. 4.
    Black AR, Black JD, Azizkhan-Clifford J: Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 188: 143–160, 2001Google Scholar
  5. 5.
    Le Chalony C, Prospéri M-T, Haluza R, Apiou F, Dutrillaux B, Goubin G: The OZF gene encodes a protein consisting essentially of zinc finger motifs. J Mol Biol 236: 399–404, 1994Google Scholar
  6. 6.
    Ferbus D, Le Chalony C, Prosperi M-T, Muleris M, Vincent-Salomon A, Goubin G: Identification, nuclear localization, and binding activities of OZF, a human protein solely composed of zinc-finger motifs. Eur J Biochem 236: 991–995, 1996Google Scholar
  7. 7.
    Ferbus D, Fléchon A, Muleris M, Yong L, Hanash S, Terris B, Hammel P, Pibouin L, Dutrillaux B, Goubin G: Amplification and over-expression of OZF, a gene encoding a zinc finger protein, in human pancreatic carcinomas. Int J Cancer 80: 369–372, 1999Google Scholar
  8. 8.
    Ferbus D, Bovin C, Validire P, Goubin G: The zinc finger protein OZF (ZNF146) is overexpressed in colorectal cancer. J Pathol 200: 177–182, 2003Google Scholar
  9. 9.
    Fernandez PC, Frank SR, Wang L, Schroeder M, Liu S, Greene J, Cocito A, Amati B: Genomic targets of the human c-Myc protein. Genes Dev 17: 1115–1129, 2003Google Scholar
  10. 10.
    Lee JS, Galvin KM, Shi Y: Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1. Proc Natl Acad Sci USA 90: 6145–6419, 1993Google Scholar
  11. 11.
    Perkins ND, Agranoff AB, Pascal E, Nabel GJ: An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation. Mol Cell Biol 14: 6570–6583, 1994Google Scholar
  12. 12.
    Merika M, Orkin SH: Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Kruppel family proteins Sp1 and EKLF. Mol Cell Biol 15: 2437–2447, 1995Google Scholar
  13. 13.
    Muller S, Hoege C, Pyrowolakis G, Jentsch S: SUMO, ubiquitin’s mysterious cousin. Nat Rev Mol Cell Biol 2: 202–210, 2001Google Scholar
  14. 14.
    Seeler JS, Dejean A: Nuclear and unclear functions of SUMO. Nat Rev Mol Cell Biol 4: 690–699, 2003CrossRefPubMedGoogle Scholar
  15. 15.
    Rodriguez MS, Dargemont C, Hay RT: SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J Biol Chem 276: 12654–12659, 2001Google Scholar
  16. 16.
    Sampson DA, Wang M, Matunis MJ: The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J Biol Chem 276: 21664–21669, 2001Google Scholar
  17. 17.
    Yamada K, Wang J-C, Osawa H, Scott DK, Granner DK: Efficient large-scale transformation of yeast. Biotechniques 24: 596–600, 1998Google Scholar
  18. 18.
    Ferbus D, Antoine K, Goubin G: Production and characterization of mouse monoclonal antibodies to human zinc finger OZF protein overexpressed in pancreatic carcinomas. Hybridoma 18: 431–436, 1999Google Scholar
  19. 19.
    Hahn SL, Criqui P, Wasylyk B: Modulation of ETS-1 transcriptional activity by huUBC9, a ubiquitin-conjugating enzyme. Oncogene 15: 1489–1495, 1997Google Scholar
  20. 20.
    Blottière L, Apiou F, Ferbus D, Guenzi C, Dutrillaux B, Prospéri M-T, Goubin G: Cloning, characterization, and chromosome assignment of Zfp146 the mouse ortholog of human ZNF146, a gene amplified and overexpressed in pancreatic cancer, and Zfp260 a closely related gene. Cytogenet Cell Genet 85: 297–300, 1999Google Scholar
  21. 21.
    Muller S, Berger M, Lehembre F, Seeler J-S, Haupt Y, Dejean A: c-Jun and p53 activity is modulated by SUMO-1 modification. J Biol Chem 275: 13321–13329, 2000Google Scholar
  22. 22.
    Duprez E, Saurin AJ, Desterro JM, Lallemand-Breitenbach V, Howe K, Boddy MN, Solomon E, de The H, Hay RT, Freemont PS: SUMO-1 modification of the acute promyelocytic leukaemia protein PML: Implications for nuclear localization. J Cell Sci 112: 381–393, 1999Google Scholar
  23. 23.
    Rangasamy D, Woytek K, Khan SA, Wilson VG: SUMO-1 modification of bovine papillomavirus E1 protein is required for intranuclear accumulation. J Biol Chem 275: 37999–38004, 2000Google Scholar
  24. 24.
    Lin D, Tatham MH, Yu B, Kim S, Hay RT, Chen Y: Identification of a substrate recognition site on Ubc9. J Biol Chem 277: 21740–21748, 2002Google Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Karène Antoine
    • 1
  • Marie-Thérèse Prospéri
    • 1
  • Didier Ferbus
    • 1
  • Catherine Boule
    • 1
  • Gérard Goubin
    • 1
    • 2
  1. 1.Laboratoire d’Oncogenèse, FRE 2584 CNRSInstitut CurieParis Cedex 05France
  2. 2.Biologie Oro-faciale et Pathologie, INSERM EMI 110Institut Biomédical des CordeliersParis CEDEX 06France

Personalised recommendations