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Journal of Biomolecular Screening
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Pharmacological Characterization of Purified Recombinant mTOR FRB-Kinase Domain Using Fluorescence-Based Assays

Laurie J. Reichling

Invitrogen Discovery Sciences, Madison, Wisconsin

Connie S. Lebakken

Invitrogen Discovery Sciences, Madison, Wisconsin

Steven M. Riddle

Invitrogen Discovery Sciences, Madison, Wisconsin

Kevin L. Vedvik

Invitrogen Discovery Sciences, Madison, Wisconsin

Matthew B. Robers

Invitrogen Discovery Sciences, Madison, Wisconsin

Leisha M. Kopp

Invitrogen Discovery Sciences, Madison, Wisconsin

Rica Bruinsma

Invitrogen Discovery Sciences, Madison, Wisconsin

Kurt W. Vogel

Invitrogen Discovery Sciences, Madison, Wisconsin, kurt.vogel{at}invitrogen.com

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in nutrient sensing and cell growth and is a validated target for oncology and immunosuppression. Two modes of direct small-molecule inhibition of mTOR activity are known: targeting of the kinase active site and a unique mode in which the small molecule rapamycin, in complex with FKBP12 (the 12-kDa FK506 binding protein), binds to the FRB (FKBP12/rapamycin binding) domain of mTOR and inhibits kinase activity through a poorly defined mechanism. To facilitate the study of these processes, the authors have expressed and purified a truncated version of mTOR that contains the FRB and kinase domains and have developed homogeneous fluorescence-based assays to study mTOR activity. They demonstrate the utility of these assays in studies of active site-directed and FRB domain-directed mTOR inhibition. The results suggest that these assays can replace traditional radiometric or Western blot—based assays. (Journal of Biomolecular Screening 2008:238-244)

Key Words: FRAP1 • mTOR • rapamycin • kinase • assay

Journal of Biomolecular Screening, Vol. 13, No. 3, 238-244 (2008)
DOI: 10.1177/1087057108314609
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