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. Böttger, A. Böttger, 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.
Kane
S A
SA
Department of Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. stefanie_kane@merck.com
Fleener
C A
CA
Zhang
Y S
YS
Davis
L J
LJ
Musselman
A L
AL
Huang
P S
PS
D000595:Amino Acid Sequence; D019921:Biotinylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D016147:Genes, Tumor Suppressor; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009942:Organometallic Compounds; D010748:Phosphopeptides / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019809:Streptavidin; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009942:Organometallic Compounds; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C070734:europium(III) trisbipyridine cryptate; D019809:Streptavidin; D051736:Proto-Oncogene Proteins c-mdm2; D005982:Glutathione Transferase