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

Du Y, Khuri FR, Fu H - Curr Chem Genomics (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: With the establishment of a general platform for monitoring 14-3-3 interactions, we extended our study to examine the utility of the AlphaScreen technology for the binding of 14-3-3 to a phosphorylated peptide derived from Raf-1, a mitogen-activated protein kinase regulator. It has been well-established that phosphorylation of Raf-1 at Ser-259 induces its association with the 14-3-3 proteins, which plays a critical role in Raf-1-mediated cell signaling [6, 8, 23-25]. To test the 14-3-3/Raf-1 interaction in the AlphaScreen system, we generated a biotinylated Raf peptide, pS259-Raf-1, for coupling to streptavadin-coated donor beads. 14-3-3γ was produced and purified as a GST-tagged fusion protein for coupling to the GST-antibody coated acceptor beads. The interaction of biotin-pS259-Raf-1 peptide with GST-14-3-3γ would bring donor and acceptor beads together and induce the production of Alpha signal (Fig. 2). Indeed, mixing increasing concentrations of GST-14-3-3γ and biotin-pS259-Raf-1 with both beads resulted in a dose-dependent increase in Alpha signal (Fig. 5A). When incubated with 30 or 100 nM of biotin-pS259-Raf-1, the S/B ratio significantly increased with increasing concentration of 14-3-3 proteins (Fig. 5B). The calculated Z’ factors were above 0.6 when 100 nM of biotin-pS259-Raf-1 was incubated with increasing concentrations of GST-14-3-3 protein (Fig. 5C). Z’ factors reached 0.9 when 30 or 100 nM of 14-3-3 was used, indicating a robust assay performance.


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

Du Y, Khuri FR, Fu H - Curr Chem Genomics (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803432&req=5

Figure 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.
Mentions: With the establishment of a general platform for monitoring 14-3-3 interactions, we extended our study to examine the utility of the AlphaScreen technology for the binding of 14-3-3 to a phosphorylated peptide derived from Raf-1, a mitogen-activated protein kinase regulator. It has been well-established that phosphorylation of Raf-1 at Ser-259 induces its association with the 14-3-3 proteins, which plays a critical role in Raf-1-mediated cell signaling [6, 8, 23-25]. To test the 14-3-3/Raf-1 interaction in the AlphaScreen system, we generated a biotinylated Raf peptide, pS259-Raf-1, for coupling to streptavadin-coated donor beads. 14-3-3γ was produced and purified as a GST-tagged fusion protein for coupling to the GST-antibody coated acceptor beads. The interaction of biotin-pS259-Raf-1 peptide with GST-14-3-3γ would bring donor and acceptor beads together and induce the production of Alpha signal (Fig. 2). Indeed, mixing increasing concentrations of GST-14-3-3γ and biotin-pS259-Raf-1 with both beads resulted in a dose-dependent increase in Alpha signal (Fig. 5A). When incubated with 30 or 100 nM of biotin-pS259-Raf-1, the S/B ratio significantly increased with increasing concentration of 14-3-3 proteins (Fig. 5B). The calculated Z’ factors were above 0.6 when 100 nM of biotin-pS259-Raf-1 was incubated with increasing concentrations of GST-14-3-3 protein (Fig. 5C). Z’ factors reached 0.9 when 30 or 100 nM of 14-3-3 was used, indicating a robust assay performance.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

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.

No MeSH data available.


Related in: MedlinePlus