A homogenous luminescent proximity assay for 14-3-3 interactions with both phosphorylated and nonphosphorylated client peptides

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.

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

Fig. (1)

14-3-3 binds phosphorylated Ser/Thr motifs with RSxpS/TxP as a prototype. It plays an important role in multiple signaling pathways.

Fig. (2)

Design of AlphaScreen-based 14-3-3 binding assays. (A) Biotin-14-3-3 proteins are coupled to streptavidin-coated donor beads (D) while GST-R18 is attached to anti-GST antibody-coated acceptor beads (A). (B) Biotinylated pS259-Raf-1 peptides are coupled to streptavidin-coated donor beads while GST-14-3-3 proteins are attached to acceptor beads with coated anti-GST antibodies. Interaction of 14-3-3 with R18 or pS259-Raf-1 leads to generation of luminescence signals at the indicated wavelength.

Fig. (3)

AlphaScreen assay for measuring the interaction of 14-3-3γ with R18. (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). Specific binding (mP) was obtained by subtracting free peptide tracer values from values recorded in the presence of 14-3-3 proteins. (B) Interaction of biotin-14-3-3 with GST-R18 generates an Alpha luminescence signal. Increasing concentrations of GST-R18 protein were incubated with biotin-14-3-3 protein at RT for 30 min. Donor and acceptor beads were added with an interval of 30 min. The plate was assayed using an EnVision multilabel plate reader after 1.5 hr incubation at room temperature. Data are expressed as mean ± SD from triplicate samples. (C) S/B ratios of the 14-3-3γ/R18 AlphaScreen assay. (D) Z’ values of the assay.

Fig. (4)

Competition with untagged peptide antagonists, R18 and pS967-ASK1, in the 14-3-3γ/R18 AlphaScreen assay. Biotin-14-3-3γ (5 nM) and GST-R18 (15 nM) were incubated with increasing concentrations of untagged antagonists at RT for 1 hr. After incubation for 1.5 hr upon addition of donor and acceptor beads, Alpha signals were recorded. (A) Competition with the R18 peptide or the mutated control (R18Lys) peptide. (B) Competition with pS967-ASK1 or the non-phosphorylated control (ASK1) peptide. The y-axis of the graph represents the percentage of the control. The control is defined by maximal Alpha signal obtained in the absence of any inhibitor with background counts subtracted. Data are expressed as means ± SD from triplicate determinations.

Fig. (5)

AlphaScreen assay for measuring the interaction of 14-3-3γ with pS259-Raf-1. Experiments were performed as described in the legend to Fig. (4) using 100 nM of GST-14-3-3γ and 100 nM of biotin-pS259-Raf-1 peptide. For (A), (D), and (E), data are expressed as means ± SD from triplicate determinations. (A) Increasing concentrations of GST-14-3-3γ protein were incubated with biotin-pS-259-Raf peptide at RT for 30 min. Donor and acceptor beads were added with an interval of 30 min. Results were recorded after 3 hr of incubation at RT. (B) The S/B ratios of the assay. (C) The Z’ factor of the assay. (D) Competition with the R18 peptide or its mutant peptide, R18Lys, in the 14-3-3γ/pS259-Raf-1 AlphaScreen assay. (E) Competition assay using the pS967-ASK1 or its non-phosphorylated ASK1 control peptide.

Fig. (6)

Evaluation of the DMSO tolerance and stability of the 14-3-3 AlphaScreen assay. (A) Effect of DMSO on Alpha signal. The biotin-14-3-3γ (5 nM) and GST-R18 (15 nM) were incubated with increasing amounts of DMSO, and reactions were carried out as described in the legend to Fig. (4). Results were recorded after incubation with donor and acceptor beads for 1.5 hr. (B) Stability of the 14-3-3 AlphaScreen assay as evaluated in R18 competition assay. Experiments were carried out as described in Fig. (4) except that results were recorded after incubation for 1.5 hr or 16 hr. Dose-response data with the untagged R18 peptide antagonist were expressed as the percentage of maximal Alpha signal control that was obtained from samples in the absence of inhibitor. Data are expressed as means ± SD from triplicate determinations.

Fig. (7)

Evaluation of the 14-3-3 AlphaScreening assay for HTS. The binding assay was performed essentially as described in Fig. (4), using biotin-14-3-3γ (5 nM) and GST-R18 (15 nM) and 10 assay plates (384-well). (A) Z’ factor for the 14-3-3 AlphaScreen assay. The Z’ values across the 10 plates were consistently higher than 0.7. (B) The S/B values, calculated for each of the 10 plates, were all above 7. (C) Day to day variation. Day to day precision was assessed by comparison of 2 representative dose-response competition curves of R18 peptide which were performed on 2 different days. Results are shown as a percentage of control Alpha signal (obtained in the absence of inhibitor) after subtracting the background (biotin-14-3-3 only). Data are expressed as means ± SD from triplicate determinations.