Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence.
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. Boettger, A. Boettger, 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.
Mesh Terms:
Amino Acid Sequence, Biotinylation, DNA-Binding Proteins, Enzyme-Linked Immunosorbent Assay, Genes, Tumor Suppressor, Glutathione Transferase, Molecular Sequence Data, Nuclear Proteins, Organometallic Compounds, Phosphopeptides, Protein Binding, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-mdm2, Recombinant Fusion Proteins, Spectrometry, Fluorescence, Streptavidin, Tumor Suppressor Protein p53, Tumor Suppressor Proteins
Amino Acid Sequence, Biotinylation, DNA-Binding Proteins, Enzyme-Linked Immunosorbent Assay, Genes, Tumor Suppressor, Glutathione Transferase, Molecular Sequence Data, Nuclear Proteins, Organometallic Compounds, Phosphopeptides, Protein Binding, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-mdm2, Recombinant Fusion Proteins, Spectrometry, Fluorescence, Streptavidin, Tumor Suppressor Protein p53, Tumor Suppressor Proteins
Anal. Biochem.
Date: Feb. 01, 2000
PubMed ID: 10640350
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