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Title: 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

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 (K {sub m}) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 {mu}M, 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.

Authors:
 [1];  [1];  [2];  [2];  [1];  [1]
  1. Department of Discovery Oncology, Wyeth Research, Pearl River, NY 10965 (United States)
  2. Department of Chemical and Screening Sciences, Wyeth Research, Pearl River, NY 10965 (United States)
Publication Date:
OSTI Identifier:
20710826
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 332; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2005.04.117; PII: S0006-291X(05)00877-6; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; FLUORESCENCE; IMMUNOASSAY; IN VITRO; INHIBITION; NEOPLASMS; PHOSPHOTRANSFERASES; RARE EARTHS; SERINE; SUBSTRATES; THREONINE

Citation Formats

Toral-Barza, Lourdes, Weiguo, Zhang, Lamison, Craig, LaRocque, James, Gibbons, James, and Yu, Ker. 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. United States: N. p., 2005. Web. doi:10.1016/j.bbrc.2005.04.117.
Toral-Barza, Lourdes, Weiguo, Zhang, Lamison, Craig, LaRocque, James, Gibbons, James, & Yu, Ker. 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. United States. https://doi.org/10.1016/j.bbrc.2005.04.117
Toral-Barza, Lourdes, Weiguo, Zhang, Lamison, Craig, LaRocque, James, Gibbons, James, and Yu, Ker. Fri . "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". United States. https://doi.org/10.1016/j.bbrc.2005.04.117.
@article{osti_20710826,
title = {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},
author = {Toral-Barza, Lourdes and Weiguo, Zhang and Lamison, Craig and LaRocque, James and Gibbons, James and Yu, Ker},
abstractNote = {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 (K {sub m}) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 {mu}M, 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.},
doi = {10.1016/j.bbrc.2005.04.117},
url = {https://www.osti.gov/biblio/20710826}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 1,
volume = 332,
place = {United States},
year = {2005},
month = {6}
}