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Homogeneous time-resolved fluorescence-based assay to monitor extracellular signal-regulated kinase signaling in a high-throughput format.

Ayoub MA, Trebaux J, Vallaghe J, Charrier-Savournin F, Al-Hosaini K, Gonzalez Moya A, Pin JP, Pfleger KD, Trinquet E - Front Endocrinol (Lausanne) (2014)

Bottom Line: This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs).The validation was performed for agonists, antagonists, and inhibitors in dose-response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced.Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia , Nedlands, WA , Australia.

ABSTRACT
The extracellular signal-regulated kinases (ERKs) are key components of multiple important cell signaling pathways regulating diverse biological responses. This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We report the development of a new cell-based Phospho-ERK1/2 assay (designated Phospho-ERK), which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently, or stably expressing the different receptors. The validation was performed for agonists, antagonists, and inhibitors in dose-response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z'-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signaling. Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling.

No MeSH data available.


Comparison of the Phospho-ERK and western blot assays, and effect of cell density. A431 cells were used for the detection of the phosphorylation of ERK1/2 upon cell stimulation with 100 nM of EGF for 5 min (A) as well as the corresponding total ERK levels using the one-plate protocol (B). S/N represents the signal-to-noise ratio through the different cell densities. For this, serial dilutions of whole cells or cell lysate were dispensed as indicated and analyzed side-by-side using the HTRF assay [top of (A,B)] and western blot [bottom of (A,B)] as described in Section “Materials and Methods.” (C) The detection limit of the total ERK levels using the two methods was represented by plotting the total HTRF signals (HTRF) with the enhanced chemiluminescence (ECL) signal obtained by densitometry from the western blot (WB) normalized to the signal obtained with 60,000 cells/well as 100%. The data are mean ± SEM of three independent experiments performed in duplicate.
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Figure 3: Comparison of the Phospho-ERK and western blot assays, and effect of cell density. A431 cells were used for the detection of the phosphorylation of ERK1/2 upon cell stimulation with 100 nM of EGF for 5 min (A) as well as the corresponding total ERK levels using the one-plate protocol (B). S/N represents the signal-to-noise ratio through the different cell densities. For this, serial dilutions of whole cells or cell lysate were dispensed as indicated and analyzed side-by-side using the HTRF assay [top of (A,B)] and western blot [bottom of (A,B)] as described in Section “Materials and Methods.” (C) The detection limit of the total ERK levels using the two methods was represented by plotting the total HTRF signals (HTRF) with the enhanced chemiluminescence (ECL) signal obtained by densitometry from the western blot (WB) normalized to the signal obtained with 60,000 cells/well as 100%. The data are mean ± SEM of three independent experiments performed in duplicate.

Mentions: As mentioned above, the assessment of ERK1/2 activation by western blot using an antibody recognizing the phosphorylated forms of ERK1/2 represents the widely used method. First, we performed a comparative study on A431 cells. The phosphorylation of ERK1/2 upon stimulation with 100 nM of EGF for 5 min was measured on the same number of cells comparing the western blot and HTRF®-based assay (Phospho-ERK assay) with the one-plate protocol. As shown in Figure 3A, the HTRF®-based assay used to detect the phosphorylated from of ERK1/2 presents a very good signal-to-noise ratio (S/N = 18). The Phospho-ERK assay was substantially more sensitive than the western blot assay, since ~400 cells were enough to start detecting specific EGF-induced ERK1/2 phosphorylation using the HTRF®-based assay while ~7500–15,000 cells were needed for the western blot method (Figure 3A). Such a significant difference in the sensitivity between the Phospho-ERK assay and western blot method was also confirmed when the total ERK1/2 levels were quantified in the different cell samples (Figure 3B). Indeed, the normalized quantification of the intensities of western blot bands of the total ERK1/2 clearly indicated that proteins were effectively detected at 1875–3750 cells while 234–468 cells per well were sufficient for the specific HTRF signal reflecting the total ERK1/2 (Figure 3C) showing a sensitivity factor of about 10-fold between the two methods.


Homogeneous time-resolved fluorescence-based assay to monitor extracellular signal-regulated kinase signaling in a high-throughput format.

Ayoub MA, Trebaux J, Vallaghe J, Charrier-Savournin F, Al-Hosaini K, Gonzalez Moya A, Pin JP, Pfleger KD, Trinquet E - Front Endocrinol (Lausanne) (2014)

Comparison of the Phospho-ERK and western blot assays, and effect of cell density. A431 cells were used for the detection of the phosphorylation of ERK1/2 upon cell stimulation with 100 nM of EGF for 5 min (A) as well as the corresponding total ERK levels using the one-plate protocol (B). S/N represents the signal-to-noise ratio through the different cell densities. For this, serial dilutions of whole cells or cell lysate were dispensed as indicated and analyzed side-by-side using the HTRF assay [top of (A,B)] and western blot [bottom of (A,B)] as described in Section “Materials and Methods.” (C) The detection limit of the total ERK levels using the two methods was represented by plotting the total HTRF signals (HTRF) with the enhanced chemiluminescence (ECL) signal obtained by densitometry from the western blot (WB) normalized to the signal obtained with 60,000 cells/well as 100%. The data are mean ± SEM of three independent experiments performed in duplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4066300&req=5

Figure 3: Comparison of the Phospho-ERK and western blot assays, and effect of cell density. A431 cells were used for the detection of the phosphorylation of ERK1/2 upon cell stimulation with 100 nM of EGF for 5 min (A) as well as the corresponding total ERK levels using the one-plate protocol (B). S/N represents the signal-to-noise ratio through the different cell densities. For this, serial dilutions of whole cells or cell lysate were dispensed as indicated and analyzed side-by-side using the HTRF assay [top of (A,B)] and western blot [bottom of (A,B)] as described in Section “Materials and Methods.” (C) The detection limit of the total ERK levels using the two methods was represented by plotting the total HTRF signals (HTRF) with the enhanced chemiluminescence (ECL) signal obtained by densitometry from the western blot (WB) normalized to the signal obtained with 60,000 cells/well as 100%. The data are mean ± SEM of three independent experiments performed in duplicate.
Mentions: As mentioned above, the assessment of ERK1/2 activation by western blot using an antibody recognizing the phosphorylated forms of ERK1/2 represents the widely used method. First, we performed a comparative study on A431 cells. The phosphorylation of ERK1/2 upon stimulation with 100 nM of EGF for 5 min was measured on the same number of cells comparing the western blot and HTRF®-based assay (Phospho-ERK assay) with the one-plate protocol. As shown in Figure 3A, the HTRF®-based assay used to detect the phosphorylated from of ERK1/2 presents a very good signal-to-noise ratio (S/N = 18). The Phospho-ERK assay was substantially more sensitive than the western blot assay, since ~400 cells were enough to start detecting specific EGF-induced ERK1/2 phosphorylation using the HTRF®-based assay while ~7500–15,000 cells were needed for the western blot method (Figure 3A). Such a significant difference in the sensitivity between the Phospho-ERK assay and western blot method was also confirmed when the total ERK1/2 levels were quantified in the different cell samples (Figure 3B). Indeed, the normalized quantification of the intensities of western blot bands of the total ERK1/2 clearly indicated that proteins were effectively detected at 1875–3750 cells while 234–468 cells per well were sufficient for the specific HTRF signal reflecting the total ERK1/2 (Figure 3C) showing a sensitivity factor of about 10-fold between the two methods.

Bottom Line: This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs).The validation was performed for agonists, antagonists, and inhibitors in dose-response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced.Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia , Nedlands, WA , Australia.

ABSTRACT
The extracellular signal-regulated kinases (ERKs) are key components of multiple important cell signaling pathways regulating diverse biological responses. This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We report the development of a new cell-based Phospho-ERK1/2 assay (designated Phospho-ERK), which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently, or stably expressing the different receptors. The validation was performed for agonists, antagonists, and inhibitors in dose-response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z'-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signaling. Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling.

No MeSH data available.