TR-FRET biochemical assays for detecting posttranslational modifications of p53.
The p53 tumor suppressor protein plays a pivotal role in suppressing oncogenesis by regulating a range of cellular functions, including DNA repair, cell growth, cell cycle progression, and cellular death. A network of different pathways converge upon p53, ultimately regulating the response of the tumor suppressor protein by posttranslational modifications. The authors have developed a time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput compatible assay to analyze the critical posttranslational modifications of p53, including phosphorylation, acetylation, and ubiquitination. By using full-length p53 protein fused with GFP (GFP-p53) as the substrate, they were able to measure all 3 different posttranslational modifications with a single substrate. In addition, with a few additional steps, the GFP-p53 substrate can also be used to assay deacetylation to aid in the discovery of inhibitors for sirtuins or other deacetylase enzymes. The flexibility of the assay to measure a diverse range of posttranslational modifications allows one to further dissect the complex regulating mechanisms of p53 and enable the discovery of specific inhibitors for these processes.
Dudek
Jeanne M
JM
Invitrogen, Part of Life Technologies, Madison, WI 53719, USA.
Horton
Robert A
RA
D000107:Acetylation; D001681:Biological Assay / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D037761:Sirtuins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D037761:Sirtuins