TORC2 Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of TORC2
• 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 TORC proteins (transducers of regulated CREB activity, TORC1, TORC2, TORC3) are regulators of the gluconeogenic program in response to hormonal and intracellular signals. TORCs are regulators of CREB activity and translocate to the nucleus after an increase in the level of intracellular cAMP or calcium. In fact, TORC proteins can be viewed as integrators of cAMP and calcium signals. TORC2 shuttles between the nucleus and the cytoplasm by means of an NLS in aa 56-144, and two NES’s between aa 145-320. TORC2 is dephosphorylated at Ser171 and presumable Ser369 under conditions of high levels of cAMP, leading to nuclear translocation. Nuclear TORC2 interacts with the DNA binding domain of CREB and thereby enhances gene expression of CREB target genes such as gluconeogenic genes. High levels of cAMP can be induced by glucagon, fasting treatment, or forskolin.

The effect of glucagon and/or fasting on TORC2 can be attenuated by expression of the salt-induced kinases (SIK1 or SIK2) or by activation of the AMPK pathway, e.g. by exposure to the AMP analog AICAR. Compounds that induce or inhibit nuclear translocation of TORC2 are of interest as neuroprotective/memory enhancing agents or for treatment of type II diabetes, respectively.

Figure 2. Concentration response curves in the TORC2 assay: A) Forskolin concentration response. The EC₅₀ is approximately 160 nM. Concentration response was measured in 9 point half log dilution series (n=8). Cells were treated with forskolin for 1 hr. Cells were then fixed and nuclear translocation was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (10 μM forskolin) and negative control (0.25% DMSO). B) Glucagon concentration response in the TORC2 assay cell line that was stably transfected with the glucagon receptor. Concentration response was measured in 9 point half log dilution series (n=8). The EC₅₀ of glucagon is approximately 2 pM. Cells analyzed as described in A).