Related Experiment Video

Updated: May 27, 2026

Identification of Kinase-substrate Pairs Using High Throughput Screening
11:13

Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

An in vitro assay for the kinase activity of mTOR complex 2

Jingxiang Huang1

  • 1Department of Pathology, National University Hospital of Singapore, Singapore, Singapore. jingxiang_huang@nuhs.edu.sg

Methods in Molecular Biology (Clifton, N.J.)
|November 30, 2011
PubMed

Insights

The mechanistic target of rapamycin complex 2 (mTORC2) regulates key cell functions. A common kinase assay measures mTORC2 activity by tracking substrate phosphorylation, aiding in small molecule studies.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • The mechanistic target of rapamycin complex 2 (mTORC2) is a crucial protein kinase complex.
  • mTORC2 regulates vital physiological processes by phosphorylating substrates like Akt, SGK1, and PKC.
  • DEPTOR inhibits mTORC2, while the TSC1-TSC2 complex activates it.

Purpose of the Study:

  • To describe a common method for studying mTORC2 regulation.
  • To detail an in vitro kinase assay for measuring mTORC2 activity.
  • To highlight the assay's utility in assessing small molecule effects on mTORC2.

Main Methods:

  • In vivo examination of substrate phosphorylation.
  • In vitro kinase assay using immunoprecipitated mTORC2.
  • Phosphorylation of exogenously supplied Akt as a measure of mTORC2 activity.

Main Results:

  • The described kinase assay effectively measures mTORC2 activity.
  • The assay allows for the study of regulatory interactions, including those with DEPTOR and TSC1-TSC2.
  • The method can be adapted to investigate the impact of small molecules on mTORC2 function.

Conclusions:

  • Kinase assays are valuable for studying mTORC2 regulation.
  • The described in vitro assay provides a reliable method for assessing mTORC2 activity.
  • This assay is adaptable for screening small molecules that modulate mTORC2.

More Related Videos

Assaying the Kinase Activity of LRRK2 in vitro06:09

Assaying the Kinase Activity of LRRK2 in vitro

Published on: January 18, 2012

Assaying Protein Kinase Activity with Radiolabeled ATP08:05

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

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...