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| Biochem. Biophys. Res. Commun. Jun (2005); 332(1):304-10 | |
| 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. | |
| Toral-Barza L, Zhang WG, Lamison C, Larocque J, Gibbons J, Yu K | |
| Department of Discovery Oncology, Wyeth Research, Pearl River, NY 10965, USA. | |
| Abstract: 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. | |
| [PUBMED: 15896331] |   |
| Copyright © 2009, Tyers Lab, The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, All Rights Reserved. |
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| BioGRID: A General Repository for Interaction Datasets. Chris Stark, Bobby-Joe Breitkreutz, Teresa Reguly, Lorrie Boucher, Ashton Breitkreutz, Mike Tyers. Nucleic Acids Res. Jan 1;34:D535-9. |