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Journal of Biomolecular Screening
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Development of LanthaScreenTM Cellular Assays for Key Components within the PI3K/AKT/mTOR Pathway

Coby B. Carlson

Invitrogen Discovery Sciences, Madison, Wisconsin, coby.carlson{at}invitrogen.com

Matthew B. Robers

Invitrogen Discovery Sciences, Madison, Wisconsin

Kurt W. Vogel

Invitrogen Discovery Sciences, Madison, Wisconsin

Thomas Machleidt

Invitrogen Discovery Sciences, Madison, Wisconsin

The PI3K/AKT/mTOR pathway is central to cell growth and survival, cell cycle regulation, and programmed cell death. Aberrant activation of this signaling cascade is linked to several disease states, and thus many components of the pathway are attractive targets for therapeutic intervention. However, the considerable degree of complexity, crosstalk, and feedback regulation that exists within the pathway (especially with respect to the regulation of mTOR and its complexes) underscores the need for a comprehensive set of cell-based assays to properly identify and characterize small-molecule modulators. Here, the development and application of time-resolved Förster resonance energy transfer (TR-FRET)—based assays to enable the phosphoprotein analysis of key pathway components in a cellular format are reported. The LanthaScreenTM cellular assay platform uses FRET between a terbium-labeled phosphorylation site-specific antibody and an expressed green fluorescent protein fusion of particular kinase substrate and provides an assay readout that is ratiometric, robust, and amenable to high-throughput screening applications. Assays specific for 5 different targets within the pathway are highlighted: Ser183 and Thr246 on the proline-rich AKT substrate 40 kDa (PRAS40), Ser457 on programmed cell death protein 4 (PDCD4), and Thr308 and Ser473 on AKT. Each assay was evaluated under various experimental conditions and individually optimized for performance. Known pathway agonists and a small panel of commercially available compounds were also used to complete the assay validation. Taken together, these data demonstrate the utility of a related set of cell-based assays to interrogate PI3K/AKT/mTOR signaling and provide a template for the development of similar assays for other targets. (Journal of Biomolecular Screening 2009:121-132)

Key Words: PI3K • AKT • mTOR • TR-FRET • GFP

This version was published on February 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 2, 121-132 (2009)
DOI: 10.1177/1087057108328132
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