Pharmacological characterization of purified recombinant mTOR FRB-kinase domain using fluorescence-based assays.
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.
Reichling
Laurie J
LJ
Invitrogen Discovery Sciences, Madison, Wisconsin 53719, USA.
Lebakken
Connie S
CS
Riddle
Steven M
SM
Vedvik
Kevin L
KL
Robers
Matthew B
MB
Kopp
Leisha M
LM
Bruinsma
Rica
R
Vogel
Kurt W
KW
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002384:Catalysis / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis / Q000187:drug effects; D020128:Inhibitory Concentration 50; D007313:Insecta; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000737:chemistry* / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000494:pharmacology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus