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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

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.
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Figure 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.

Mentions: To validate the AlphaScreen assay for the discovery of 14-3-3 inhibitors, we tested inhibition of the interaction of 14-3-3 with the R18 by two untagged antagonist peptides: an R18 peptide and an ASK1 peptide, pS967-ASK1. The untagged R18 and pS967-ASK1 peptides are expected to compete with GST-R18 for the binding of 14-3-3 proteins. Indeed, incubation of increasing concentrations of untagged R18 peptide with biotin-14-3-3γ and GST-R18 led to a dose-dependent decrease in Alpha signal with an IC50 value of 28 ± 1.1 nM (Fig. 4A). On the other hand, untagged R18Lys, a R18 peptide with a mutated 14-3-3 binding residue, failed to displace GST-R18 from the 14-3-3 protein, supporting a specific effect of the R18 competitor. Many 14-3-3 client proteins, including ASK1, require phosphorylation to mediate the binding to 14-3-3 proteins. The pS967-ASK1 peptide is a 14-3-3 binding peptide that contains phosphorylated Ser-967 and its surrounding sequence within the ASK1 protein [21, 22]. As shown in Fig. (4B), the free pS967-ASK1 peptide competed with the binding of GST-R18 to 14-3-3γ, resulting in a dose-dependent decrease in Alpha signal with an IC50 value of 549.4 ± 1.1 nM. The same ASK1 peptide without phosphorylation, however, had no effect on the Alpha signal generated from the 14-3-3 binding to R18 (Fig. 4). These results are consistent with reported interactions of 14-3-3 with phosphorylated client peptides [6, 7]. Thus, small molecules that compete for the binding of R18 to 14-3-3 proteins can be revealed in the AlphaScreen-based assay platform.


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)

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

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

Figure 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.
Mentions: To validate the AlphaScreen assay for the discovery of 14-3-3 inhibitors, we tested inhibition of the interaction of 14-3-3 with the R18 by two untagged antagonist peptides: an R18 peptide and an ASK1 peptide, pS967-ASK1. The untagged R18 and pS967-ASK1 peptides are expected to compete with GST-R18 for the binding of 14-3-3 proteins. Indeed, incubation of increasing concentrations of untagged R18 peptide with biotin-14-3-3γ and GST-R18 led to a dose-dependent decrease in Alpha signal with an IC50 value of 28 ± 1.1 nM (Fig. 4A). On the other hand, untagged R18Lys, a R18 peptide with a mutated 14-3-3 binding residue, failed to displace GST-R18 from the 14-3-3 protein, supporting a specific effect of the R18 competitor. Many 14-3-3 client proteins, including ASK1, require phosphorylation to mediate the binding to 14-3-3 proteins. The pS967-ASK1 peptide is a 14-3-3 binding peptide that contains phosphorylated Ser-967 and its surrounding sequence within the ASK1 protein [21, 22]. As shown in Fig. (4B), the free pS967-ASK1 peptide competed with the binding of GST-R18 to 14-3-3γ, resulting in a dose-dependent decrease in Alpha signal with an IC50 value of 549.4 ± 1.1 nM. The same ASK1 peptide without phosphorylation, however, had no effect on the Alpha signal generated from the 14-3-3 binding to R18 (Fig. 4). These results are consistent with reported interactions of 14-3-3 with phosphorylated client peptides [6, 7]. Thus, small molecules that compete for the binding of R18 to 14-3-3 proteins can be revealed in the AlphaScreen-based assay platform.

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