Characterization of the Rheb-mTOR signaling pathway in mammalian cells: constitutive active mutants of Rheb and mTOR.

Methods in Enzymology
Tatsuhiro SatoFuyuhiko Tamanoi

Abstract

Rheb (Ras homolog enriched in brain) is a GTPase conserved from yeast to human and belongs to a unique family within the Ras superfamily of GTPases. Rheb plays critical roles in the activation of mTOR, a serine/threonine kinase that is involved in the activation of protein synthesis and growth. mTOR forms two distinct complexes, mTORC1 and mTORC2. While mTORC1 is implicated in the regulation of cell growth, proliferation, and cell size in response to amino acids and growth factors, mTORC2 is involved in actin organization. However, the mechanism of activation is not fully understood. Therefore, studies to elucidate the Rheb-mTOR signaling pathway are of great importance. Here we describe methods to characterize this pathway and to evaluate constitutive active mutants of Rheb and mTOR that we recently identified. Constitutive activity of the mutants can be demonstrated by the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eIF4E-binding protein 1 (4E-BP1) both in vivo and in vitro after starving cells for amino acids and growth factors. In addition, formation and activity of mTORC1 and mTORC2 can be measured by immunoprecipitating these complexes and carrying out in vitro kinase assays. We also describe a protocol fo...Continue Reading

Citations

Oct 20, 2011·Molecular Therapy : the Journal of the American Society of Gene Therapy·Sang Ryong KimRobert E Burke
Mar 21, 2009·The Journal of Biological Chemistry·Tatsuhiro SatoFuyuhiko Tamanoi
Jan 30, 2013·Molecular and Cellular Biology·Andreas M MärzFelix Hausch
Feb 10, 2017·Bioscience Trends·Lawrence E Goldfinger, James V Michael
Jun 23, 2018·Physiological Reports·Brandee Decker, Kevin Pumiglia
Sep 15, 2020·The International Journal of Neuroscience·Shivani R SainaniShivani Desai
Jun 10, 2011·Molecular Biology Reports·Ashley Bonneau, Nitika Parmar
Aug 10, 2017·Oncology Letters·Zixuan Zhang, Mengzhao Wang
Mar 6, 2019·Scientific Reports·Abdullah R AhmedStanley W Botchway
Apr 10, 2016·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Tatsuhiro SatoSeisuke Hattori
Sep 26, 2015·Genes, Brain, and Behavior·T M KazdobaJ N Crawley
Oct 15, 2019·Frontiers in Neuroscience·Minori Kadoya, Noriaki Sasai
Mar 26, 2011·Annals of Neurology·Sang Ryong KimRobert E Burke
Mar 7, 2021·International Journal of Molecular Sciences·Aleksandra BabichevaJason X-J Yuan

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