Rac1 Activation Redistribution Assay

• Designed to assay compounds causing activation of Rac1 and thereby translocation of Pak1 to 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

Members of the Rho family of small GTPases (e.g. Rho, Rac, and Cdc42) are crucial regulators of biological responses in eukaryotic cells, including cytoskeletal dynamics, cell motility and cell cycle progression. Moreover, the Ras and Rho GTPases contribute to the initiation and progression of cancer by destabilizing regulation of specific intracellular signalling pathways. As a result, Ras and Rho play significant roles in the development of malignant phenotypes by promoting cell cycle progression, resistance to apoptotic stimuli, neo-vascularisation and tumour cell motility, invasiveness and metastasis, which makes these GTPases attractive targets for development of anti-cancer chemotherapeutic agents.

The p21-activated kinases (PAKs) 1-3 are serine/threonine protein kinases that function as downstream effectors of Rac and Cdc42; their activity is stimulated by the binding of active Rac and Cdc42 GTPases. GTP-bound Rac1 activates Pak by recruiting Pak to the plasma membrane. Rac1/Pak interaction is dependent on Rac1:GTP binding, and this has lead to the use of the Rac1-binding domain of Pak1 (Pak1RBD) as a capture agent for analyzing the activation state of Rac1. The Rac1 activation Redistribution assay utilizes Pak1RBD fused to EGFP as a read-out for Rac1 activity. Active Rac1 recruits Pak1RBD-EGFP to the plasma membrane whereas inactive Rac1 leaves Pak1RBD-EGFP free in the cytosol.

Figure 1. Translocation of Pak1RBD-EGFP. Cells were treated with 1 μM IGF-1 for 4 min (right panel) or untreated (DMSO control, left panel). Arrows indicate the membrane localization detected by the image analysis algorithm.

Figure 2. IGF-1 concentration response in the Rac1 Activation Redistribution assay. Concentration response was measured in 9 point half log dilution series (n=16). Cells were incubated with IGF-1 for 4 min. Cells were then fixed and the cytoplasm to membrane translocation was measured using the Cellomics ArrayScan V^TI Reader and the CytoCellMemTrans.V2 BioApplication. % activity was calculated relative to the positive (1 μM IGF-1) and negative control (0.25% DMSO). The EC₅₀ of IGF-1 is ~10nM.