Characterization of the Rheb-mTOR signaling pathway in mammalian cells: constitutive active mutants of Rheb and mTOR.
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 for rapamycin treatment, which directly inhibits mTOR and can be used to investigate the mTOR signaling pathway in cell growth, cell size, etc.
Sato
Tatsuhiro
T
Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California, USA.
Umetsu
Akiko
A
Tamanoi
Fuyuhiko
F
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D018797:Cell Cycle Proteins; D006367:HeLa Cells; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D046912:Multiprotein Complexes; D009479:Neuropeptides / Q000235:genetics* / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics* / Q000502:physiology*; D011506:Proteins; D000076205:Ras Homolog Enriched in Brain Protein; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D046912:Multiprotein Complexes; D009479:Neuropeptides; D010750:Phosphoproteins; D011506:Proteins; C490211:RHEB protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D014157:Transcription Factors; D011494:Protein Kinases; C546842:MTOR protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins; D020123:Sirolimus