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The involvement of protein kinases in most cellular processes, and their role in regulating cellular function through complex signaling pathways, is a fascinating and productive avenue of life sciences research. Protein kinases represent primary targets in drug discovery programs aimed at developing inhibitors of these enzymes as therapeutics. The study of kinase interactions with other cellular proteins offers promising avenues for therapeutic research, particularly as abnormalities in kinase activity are implicated in many disease pathologies.
A key challenge in kinase research, however, is the difficulty in measuring the activity of endogenous protein kinases in primary cells. While several assay technologies can be used for assessing modulation of receptor-triggered kinase activation in cells that over-express these kinase targets, it is often difficult to find an assay that is sensitive enough to detect phosphorylation of endogenous cellular proteins in untransfected primary cells.
AlphaScreen® Surefire® technology, a homogenous bead-based proximity assay, is a highly sensitive tool for measuring cellular protein phosphorylation. In the challenging realm of kinase research, AlphaScreen® SureFire® assays provide a cell-based environment for measuring basal phosphorylation and activation of endogenous proteins in primary cells, as well as studying key signal transduction pathways implicated in many disease states.
Sensitive enough to measure basal activation in primary cells
Sensitivity of the AlphaScreen® Surefire® assay platform was assessed by measuring the basal level of endogenous ERK phosphorylation in unstimulated primary human umbilical vein endothelial cells (HUVEC) using the AlphaScreen® Surefire® phospho-ERK assay kit. The basal ERK phosphorylation was measured as an indication of MAPK pathway activation in these cells. HUVEC cells were treated with increasing concentrations of either U0126, an inhibitor of MAPK/ERK kinase (MEK) 1 and 2 (Calbiochem), or LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K) (Calbiochem). Cell lysates were then analyzed for the expression levels of phosphorylated ERK. The mean of 3 independent experiments +/– standard deviation are plotted in Figure 1. The results suggest that U0126 inhibits MEK 1/2–induced ERK phosphorylation with an IC50 (concentration which induces 50% inhibition) of approximately 100 nM, which is consistent with the IC50 of 70 nM for MEK 1 and 60 nM for MEK 2 reported by Duncia et al. (1998). The absence of inhibition by LY294002 showed the specificity of the inhibition observed with U0126.
Figure 1. Inhibition of endogenous ERK phosphorylation by U0126 in unstimulated HUVEC cells. Confluent HUVEC cells were treated with various concentrations of either the MEK-1/2 inhibitor U0126 (●) or the PI3K inhibitor LY294002 (▲) for 2 hours. Phosphorylation of ERK was measured using AlphaScreen® SureFire®phospho-ERK and AlphaScreen Protein A detection reagents on the EnVision® multilabel plate reader with alpha.
The basal Akt/mTOR signaling was also measured in unstimulated HUVEC cells using the AlphaScreen® Surefire® phospho-S6 RP (Ser235/Ser236) assay kit. Cells were treated with increasing concentrations of either U0126 or LY294002. Cell lysates were analyzed for phosphorylated S6 ribosomal protein. The mean of 3 independent experiments +/– standard deviation are plotted in Figure 2. The results suggest that LY294002 inhibits PI3 kinase–induced S6 RP phosphorylation in a dose-dependent manner with an IC50 of approximately 3 µM, which is similar to the IC50 of 1.4 µM reported by Vlahos et al. (1994). The absence of inhibition by U0126 showed the specificity of the inhibition observed with LY294002.
Figure 2. Inhibition of endogenous Akt/mTOR signaling by LY294002 in unstimulated HUVEC cells. Confluent HUVEC cells were treated with various concentrations of either the MEK-1/2 inhibitor U0126 (●) or the PI3K inhibitor LY294002 (▲) for 2 hours. Phosphorylation of S6 ribosomal protein was measured using the AlphaScreen® SureFire®phospho-S6 RP (Ser235/Ser236) assay kit and AlphaScreen Protein A detection reagents on the EnVision miltilabel plate reader with alpha.
Detecting endogenous kinase activation in stimulated cells
AlphaScreen® Surefire® assay kits are optimized for directly measuring kinase activation following treatment of cells with activators of signaling pathways. To demonstrate the ability of the assay platform to directly measure the activation of endogenous kinases, HUVEC cells were stimulated either with increasing concentrations of vascular endothelial growth factor (VEGF) for 10 minutes (Figure 3A) or treated with a high dose of VEGF (2 µg/mL) for various time points (Figure 3B). Phosphorylation of VEGF receptor 2 (VEGF R2), a major receptor for VEGF-mediated signaling in endothelial cells, was detected using the AlphaScreen® SureFire® phospho-VEGF receptor 2 (Tyr1175) kit.
Upon ligand binding, VEGF R2 undergoes autophosphorylation to become activated. The dose response curve (Figure 3A) shows that the maximal response is induced at 10 minutes when approximately 100 ng/mL of VEGF was used, with higher concentrations of VEGF increasing the rate of receptor phosphorylation/dephosphorylation. The time course study using a high dose of VEGF demonstrates a very rapid maximal phosphorylation after stimulation, followed by dephosphorylation (Figure 3B).
Dose-Response (10 min)
Time Course (2µg/mL VEGF)
Figure 3. Detection of endogenous VEGF receptor phosphorylation in stimulated HUVEC cells. Confluent HUVEC cells were serum-starved overnight, and stimulated either with varying concentrations of VEGF for 10 minutes (A) or with 2 µg/mL of VEGF for varying time points. The stimulation medium was removed, and cells were lysed and analyzed for VEGF receptor phosphorylation levels using the AlphaScreen® SureFire® phospho-VEGF receptor 2 (Tyr1175) kit and AlphaScreen Protein A detection reagents on the EnVision multilabel plate reader with alpha.
Conclusion
The AlphaScreen® Surefire® technology is an extremely precise assay that is sensitive enough to measure basal activation in primary cells expressing endogenous levels of kinase or receptor target. The cell-based assay platform provides a reliable tool for direct measurement of endogenous receptor activation, and can also be used to assess responses to intracellular kinase inhibitors. The homogeneous nature of this technology allows high throughput analysis of kinase signaling pathways and can be used to identify drugs that modulate kinase activity.
References
Vlahos CJ, Matter WF, Hui KY and Brown RF. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) J. Biol. Chem. 269: 5241–5248.
Duncia JV, Santella JB 3rd, Higley CA, Pitts WJ, Wityak J, Frietze WE, Rankin FW, Sun JH, Earl RA, Tabaka AC, Teleha CA, Blom KF, Favata MF, Manos EJ, Daulerio AJ, Stradley DA, Horiuchi K, Copeland RA, Scherle PA, Trzaskos JM, Magolda RL, Trainor GL, Wexler RR, Hobbs FW, Olson RE.(1998). MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products. Bioorg Med Chem Lett. 8: 2839-44.
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