Post-translational Modification of p53 by Ubiquitin

Part of the Methods in Molecular Biology book series (MIMB, volume 647)


Post-translational modification of p53 by ubiquitin resides in the center of a fine-tuned regulatory network that activates the tumor suppressor in response to genotoxic stress. Inhibition of p53 ubiquitination by DNA damage not only prevents p53 from degradation but also promotes its nuclear accumulation leading to transactivation of a number of downstream genes that are essential for the control of cell cycle progression, cell survival, and cellular senescence. Therefore, there are growing interests in studying p53 ubiquitination under physiological/pathological conditions. We describe herein a cell-based method and an in vitro reconstituted assay that are convenient, reproducible, and adaptable for various experimental conditions for detection of p53 ubiquitination. Wide application of these methods in studying mechanisms underlying regulation of p53 ubiquitination shall assist us in better understanding of the function of the tumor suppressor.

Key words

p53 Ubiquitination Ubiquitin Post-translational modification MDM2 Reconstituted assay system Transfection 


  1. 1.
    Levine A (1997) p53, the cellular gatekeeper for growth and division. Cell 88:323–331PubMedCrossRefGoogle Scholar
  2. 2.
    Toledo F, Wahl G (2006) Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat Rev Cancer 6:909–923PubMedCrossRefGoogle Scholar
  3. 3.
    Bode A, Dong Z (2008) Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer 4:793–805CrossRefGoogle Scholar
  4. 4.
    Geyer R, Yu Z, Maki C (2000) The MDM2 RING-finger domain is required to promote p53 nuclear export. Nat Cell Biol 2:569–573PubMedCrossRefGoogle Scholar
  5. 5.
    Lohrum M, Woods D, Ludwig R, Balint E, Vousden K (2001) C-terminal ubiquitination of p53 contributes to nuclear export. Mol Cell Biol 21:8521–8532PubMedCrossRefGoogle Scholar
  6. 6.
    Vogelstein B, Lane D, Levine A (2000) Surfing the p53 network. Nature 408:307–310PubMedCrossRefGoogle Scholar
  7. 7.
    Vousden K (2002) Activation of the p53 tumor suppressor protein. Biochim Biophys Acta 1602:47–59PubMedGoogle Scholar
  8. 8.
    Appella E, Anderson C (2001) Post-translational modifications and activation of p 53 by genotoxic stresses. Eur J Biochem 268:2764–2772PubMedCrossRefGoogle Scholar
  9. 9.
    Yan C, Lu D, Hai T, Boyd D (2005) Activating transcription factor 3, a stress sensor, activates p53 by blocking its ubiquitination. EMBO J 24:2425–2435PubMedCrossRefGoogle Scholar
  10. 10.
    Hershko A, Ciechanover A (1998) The ubiqtuitin system. Annu Rev Biochem 67:425–479PubMedCrossRefGoogle Scholar
  11. 11.
    Fang S, Jensen J, Ludwig R, Vousden K, Weissman A (2000) Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem 275:8945–8951PubMedCrossRefGoogle Scholar
  12. 12.
    Walerych D, Kudla G, Gutkowska M, Wawrzynow B, Muller L, King FW, Helwak A, Boros J, Zylicz A, Zylicz M (2004) Hsp90 chaperones wild-type p53 tumor suppressor protein. J Biol Chem 279:48836–48845PubMedCrossRefGoogle Scholar
  13. 13.
    Nie L, Sasaki M, Maki C (2007) Regulation of p53 nuclear export through sequential changes in conformation and ubiquitination. J Biol Chem 282:14616–14625PubMedCrossRefGoogle Scholar
  14. 14.
    Sasaki M, Nie L, Maki C (2007) MDM2 binding induces a conformation change in p53 that is opposed by heat-shock protein 90 and precedes p53 proteasomal degradation. J Biol Chem 282:14626–14634PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Center for Cell Biology and Cancer ResearchAlbany Medical CollegeAlbanyUSA

Personalised recommendations