Characterization of the cloned full-length and a truncated human target of rapamycin: activity, specificity, and enzyme inhibition as studied by a high capacity assay.
The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (Km) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 microM, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors.
Toral-Barza
Lourdes
L
Department of Discovery Oncology, Wyeth Research, Pearl River, NY 10965, USA.
Zhang
Wei-Guo
WG
Lamison
Craig
C
Larocque
James
J
Gibbons
James
J
Yu
Ker
K
D000255:Adenosine Triphosphate / Q000737:chemistry*; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D007700:Kinetics; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D058570:TOR Serine-Threonine Kinases
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases