Related Experiment Video

Updated: Aug 30, 2026

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method
08:04

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Published on: October 23, 2018

Modulation of the protein kinase activity of mTOR

J C Lawrence1, T A Lin, L P McMahon

  • 1Department of Pharmacology, University of Virginia School of Medicine, 1300 Jefferson Park Avenue, Charlottesville, VA 22908-0735, USA. JCL3p@virginia.edu

Current Topics in Microbiology and Immunology
|October 17, 2003
PubMed

Insights

Mammalian target of rapamycin (mTOR) activity is regulated by phosphorylation, and rapamycin inhibits mTOR by disrupting substrate recognition, not direct inhibition. This reveals a novel regulatory mechanism for mTOR signaling.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The mechanistic target of rapamycin (mTOR) is a protein kinase involved in cellular signaling pathways.
  • mTOR plays a role in insulin signaling, adipocyte differentiation, and translational regulation.
  • Insulin stimulates mTOR phosphorylation at Ser2448, a site also targeted by protein kinase B (PKB).

Purpose of the Study:

  • To investigate the regulatory mechanisms of mTOR activity.
  • To elucidate the mechanism by which rapamycin inhibits mTOR.
  • To identify key regulatory sites and domains within mTOR.

Main Methods:

  • Site-directed mutagenesis of mTOR, including a mutant lacking the Ser2448-containing stretch.
  • In vitro kinase assays using wild-type and mutant mTOR.
  • Treatment with rapamycin-FKBP12 complex and antibody mTAb-1.
  • Analysis of mTOR phosphorylation of substrates p70S6K and PHAS-I.

Main Results:

  • A mutant mTOR lacking the Ser2448-containing region showed increased activity.
  • Binding of mTAb-1 to this region also increased mTOR activity.
  • Rapamycin-FKBP12 inhibited mTOR phosphorylation of p70S6K and PHAS-I.
  • A mutation in the FKBP12-rapamycin binding domain conferred rapamycin resistance but decreased substrate phosphorylation.
  • These findings suggest rapamycin disrupts substrate recognition.

Conclusions:

  • mTOR activity is controlled by phosphorylation of an inhibitory regulatory domain.
  • Rapamycin inhibits mTOR by disrupting substrate recognition rather than directly inhibiting its enzymatic activity.
  • The Ser2448 region is critical for mTOR regulation and is targeted by an inhibitory phosphorylation event.

More Related Videos

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells08:47

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Published on: May 1, 2020

Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants07:38

Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants

Published on: June 6, 2025

Related Concept Videos

PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
mTOR Signaling and Cancer Progression03:03

mTOR Signaling and Cancer Progression

The mammalian target of rapamycin or mTOR protein was discovered in 1994 due to its direct interaction with rapamycin. The protein gets its name from a yeast homolog called TOR. The mTOR protein complex in mammalian cells plays a major role in balancing anabolic processes such as the synthesis of proteins, lipids, and nucleotides and catabolic processes, such as autophagy in response to environmental cues, such as availability of nutrients and growth factors.
The mTOR pathway or the...
mTOR Signaling and Cancer Progression03:03

mTOR Signaling and Cancer Progression

The mammalian target of rapamycin or mTOR protein was discovered in 1994 due to its direct interaction with rapamycin. The protein gets its name from a yeast homolog called TOR. The mTOR protein complex in mammalian cells plays a major role in balancing anabolic processes such as the synthesis of proteins, lipids, and nucleotides and catabolic processes, such as autophagy in response to environmental cues, such as availability of nutrients and growth factors.
The mTOR pathway or the...
The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
Calmodulin-dependent Signaling01:16

Calmodulin-dependent Signaling

Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...