Monitoring 14–3–3 protein interactions with a homogeneous fluorescence polarization assay

Department of Pharmacology, Emory University School of Medicine and Emory Chemistry-Biology Discovery Center, Emory University, Atlanta, GA 30322, USA.
Journal of Biomolecular Screening (Impact Factor: 2.42). 05/2006; 11(3):269-76. DOI: 10.1177/1087057105284862
Source: PubMed

ABSTRACT The 14-3-3 proteins mediate phosphorylation-dependent protein-protein interactions. Through binding to numerous client proteins, 14-3-3 controls a wide range of physiological processes and has been implicated in a variety of diseases, including cancer and neurodegenerative disorders. To better understand the structure and function of 14-3-3 proteins and to develop small-molecule modulators of 14-3-3 proteins for physiological studies and potential therapeutic interventions, the authors have designed and optimized a highly sensitive fluorescence polarization (FP)-based 14-3-3 assay. Using the interaction of 14-3-3 with a fluorescently labeled phosphopeptide from Raf-1 as a model system, they have achieved a simple 1-step "mix-and-measure" method for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used to monitor the binding of 14-3-3 with a variety of client proteins. The 14-3-3 FP assay is highly stable and has achieved a robust performance in a 384-well format with a demonstrated signal-to-noise ratio greater than 10 and a Z' factor greater than 0.7. Because of its simplicity and high sensitivity, this assay is generally applicable to studying 14-3-3/client-protein interactions and especially valuable for high-throughput screening of 14-3-3 modulators.

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    • "Regulation of PACS-2 by Akt and 14-3-3 Molecular Cell 34, 497–509, May 29, 2009 ª2009 Elsevier Inc. 503 ranging from 1.34 to 7.84 mM (Figure 5D), similar to other 14-3-3 client proteins (Du et al., 2006). "
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    ABSTRACT: TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.
    Molecular cell 06/2009; 34(4):497-509. DOI:10.1016/j.molcel.2009.04.011 · 14.02 Impact Factor
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    • "(A) Biotinylation has no effect on the ligand binding capability of His-14-3-3γ proteins. Binding of biotin-14-3-3γ to pS259-Raf-1 peptide was determined by FP assay [11]. Biotin-14-3-3 proteins were incubated with a TMR-pS259-Raf peptide (1 nM) at RT for 30 min and the FP signal [expressed in millipolarization units (mP)] was read with an Analyst HT reader (Molecular Devices). "
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    ABSTRACT: 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.
    Current Chemical Genomics 11/2008; 2:40-7. DOI:10.2174/1875397300802010040
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    ABSTRACT: First Page of the Article
    Solid-State Device Research Conference, 1998. Proceeding of the 28th European; 10/1998
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