A Homogenous Luminescent Proximity Assay for 14-3-3 Interactions with Both Phosphorylated and Nonphosphorylated Client Peptides

Yuhong Du1, 3, Fadlo R Khuri2, Haian Fu*, 1, 2, 3
1 Department of Pharmacology Emory University School of Medicine
2 Department of Hematology and Medical Oncology, Emory University School of Medicine
3 Emory Chemical Biology Discovery Center, Atlanta, GA 30322, USA

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© Du et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the Department of Pharmacology, Emory University School of Medicine and Emory Chemical Biology Discovery Center, Atlanta, GA 30322, USA; Tel: 404-727-0368; Fax: 404-727-0365; E-mail:


The 14-3-3 proteins are a family of dimeric eukaryotic proteins that mediate both phosphorylation-dependent and -independent protein-protein interactions. Through these interactions, 14-3-3 proteins participate in the regulation of a wide range of cellular processes, including cell proliferation, cell cycle progression, and apoptosis. Because of their fundamental importance, 14-3-3 proteins have also been implicated in a variety of diseases, including cancer and neurodegenerative disorders. In order to monitor 14-3-3/client protein interactions for the discovery of small molecule 14-3-3 modulators, we have designed and optimized 14-3-3 protein binding assays based on the amplified luminescent proximity homogeneous assay (AlphaScreen) technology. Using the interaction of 14-3-3 with a phosphorylated Raf-1 peptide and a nonphosphorylated R18 peptide as model systems, we have established homogenous “add-and-measure” high-throughput screening assays. Both assays achieved robust performance with S/B ratios above 7 and Z’ factors above 0.7. Application of the known antagonistic peptides in our studies further validated the assay for screening of chemical compound libraries to identify small molecules that can modulate 14-3-3 protein-protein interactions.

Keywords: 14-3-3, AlphaScreen, protein-protein interaction, HTS.