Akt1 Redistribution Assay

• Designed to assay compounds for their ability to modulate membrane translocation of the Akt1-domain
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The serine/threonine kinase Akt, also known as protein kinase B (PKB), is essential for cell signaling downstream of growth factors, cytokines, and other cellular stimuli. Akt thereby contributes to a variety of cellular functions such as cell survival, growth, proliferation, angiogenesis, metabolism, and migration. The Akt/PKB family consists of three highly homologous members known as Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ in mammalian cells. The Akt signaling pathway is activated by various growth factors (e.g. insulin, IGF-I, EGF) and upon activation Akt translocates from the cytoplasmic space to the inner surface of the plasma membrane, where it binds to specific phospholipids produced by phosphatidylinositol 3-kinase (PI3K) at the membrane. Deregulation of Akt such as aberrant loss or gain of Akt activation often results in a variety of complex diseases, including type-2 diabetes and cancer, making Akt a viable drug target for cancer therapy.

The Akt1 Redistribution assay monitors translocation of GFP-Akt1 fusion protein from the cytoplasm to the plasma membrane. Insulin-like growth factor-I (IGF-I) is used as reference agonist, and compounds are assayed for their ability to inhibit IGF-I-stimulated membrane translocation of Akt1. The PI3K inhibitor wortmannin is used as reference antagonist, having an EC₅₀ value of ~ 35 nM in the assay. Compounds interfering with membrane translocation of Akt1 can be analyzed for isoform selectivity using the reagents in the table of Related Products below.

Figure 1. Images illustrating IGF-I-treated cells in the absence (DMSO control, left panel) or presence of 300 nM wortmannin for 4 min (right panel). Arrows indicate IGF-I-mediated membrane translocation detected by the image analysis algorithm.

Figure 2. Effect of wortmannin in the Akt1 Redistribution assay. Concentration-response curves of wortmmanin in the Akt1 Redistribution assay with 4 min compound incubation in medium containing 0.05 % FBS. The EC₅₀ value of wort-mannin is approximately 35 nM, n=16. Cells were pre-incubated with 100 nM IGF-1 for 60 min. and treated with wortmannin for 4 min. Cells were then fixed and membrane translocation was measured using the Cellomics ArrayScan V^TI Reader and the CytoCellMemTrans.V2 BioApplication. % activity was calculated relative to the positive (500 nM wortmannin) and negative control (0.25% DMSO).