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.
Mesh Terms:
Adenosine Triphosphate, Cloning, Molecular, Enzyme Activation, Enzyme Inhibitors, Fluorescence Polarization Immunoassay, Kinetics, Protein Kinases, Recombinant Proteins
Adenosine Triphosphate, Cloning, Molecular, Enzyme Activation, Enzyme Inhibitors, Fluorescence Polarization Immunoassay, Kinetics, Protein Kinases, Recombinant Proteins
Biochem. Biophys. Res. Commun.
Date: Jun. 24, 2005
PubMed ID: 15896331
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