Development of a Binding Assay for p53/HDM2 by Using Homogeneous Time-Resolved Fluorescence

Abstract The p53 tumor suppressor protein is activated and stabilized in response to DNA damage, resulting in cell cycle arrest or apoptosis. HMD2 is a negative regulator of p53. Binding of p53 by HDM2 traffics p53 from the nucleus to the cytoplasm where it is recognized and targeted for ubiquitin-mediated degradation (D. A. Freedman, L. Wu, and A. J. Levine, 1999, Cell. Mol. Life Sci. 55, 96–107). Several reports have suggested that disruption of this complex in normal cells results in p53 signaling (V. Bottger, A. Bottger, A. Sparks, W.-L. Liu, S. F. Howard, and D. P. Lane, 1997, Curr. Biol. 7, 860–869; C. Wasylyk, R. Salvi, M. Argentini, C. Dureuil, I. Delumeau, J. Abecassis, L. Debussche, and B. Wasylyk, 1999, Oncogene 18, 1921–1934). A homogeneous time-resolved fluorescence (HTRF) assay has been developed to monitor p53/HDM2 binding. This assay employs a site-specific biotinylated p53 protein, a GST-fused HDM2 protein, and two fluorophore-conjugated detection reagents, streptavidin-XL665 and europium cryptate-labeled anti-GST antibody [Eu(K)-anti-GST]. Binding of p53 to HDM2 brings the fluorophores into close proximity, allowing fluorescence resonance energy transfer to occur. Development of this assay and comparison to a traditional ELISA are described in this report. The HTRF assay was then utilized to assess the effect of serine phosphorylation within the p53 N-terminus on HDM2 binding, and to determine the relative affinity of a p73 peptide for HDM2.