Open AccessPharmacological Characterization of Purified Recombinant mTOR FRB-Kinase Domain Using Fluorescence-Based Assays
Author(s) -
Laurie J. Reichling,
Connie S. Lebakken,
Steven M. Riddle,
Kevin L. Vedvik,
Matthew B. Robers,
Leisha Kopp,
Rica Bruinsma,
Kurt W. Vogel
Publication year - 2008
Publication title -
slas discovery
Language(s) - English
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/1087057108314609
Subject(s) - pi3k/akt/mtor pathway , protein kinase domain , kinase , fkbp , small molecule , serine , biochemistry , biology , rptor , chemistry , microbiology and biotechnology , phosphorylation , signal transduction , gene , mutant
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.