Btk-PH Redistribution Assay

• Designed to assay compounds for their ability to modulate formation of PIP3 in the plasma membrane
• 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

Bruton’s tyrosine kinase (Btk) is a non-receptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases. Btk mediates B lymphocyte proliferation, differentiation and signaling. Btk translocates to the plasma membrane via binding of its pleckstrin homology (PH) domain to phosphatidylinositol-3,4,5-triphosphate (PIP3) following growth factor stimulation and cross linking of the B cell receptor. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA). This disorder is characterized by lack of mature B cells resulting in primary immunodeficiency. The Btk-PH Redistribution assay monitors the translocation of the Btk-PH-EGFP fusion protein from the cytoplasm to the plasma membrane. Insulin-like growth factor-I (IGF-I) is used as the reference agonist.

Test compounds are assayed for their ability to inhibit IGF-I-stimulated membrane translocation of the Btk-PH-EGFP fusion protein. Wortmannin inhibits the phosphatidylinositol 3-kinase (PI3K) and hereby decreases the production of PIP3. Thus, wortmannin is used in the assay as reference antagonist. Test compounds inhibiting IGF-I-induced cytoplasm to membrane translocation of Btk may interfere directly with Btk-PH translocation or may act upstream of Btk. The Btk Redistribution assay can be used as a selectivity assay for the Akt Redistribution assays to deselect or identify compounds working upstream of Akt at the level of PI3K.

Figure 1. Membrane translocation of Btk-PH-EGFP. Cells were treated with 10 nM IGF-1 without (DMSO control, left panel) and with (right panel) addition of 500 nM wortmannin. Arrows indicate IGF-1 induced membrane translocation of EGFP-PDK1 detected by the image analysis algorithm.

Figure 2. Concentration response curve in the Btk-PK antagonist assay. Wortmannin concentration response curve in the Btk-PH antagonist Redistribution assay stimulated by 100 nM IGF-1 (n=8). The EC₅₀ of wortmannin is 45 nM. Concentration response was measured in 9 point half log dilution series. Cells were incubated with 100 nM IGF-1 and 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).