Isolation of hyperactive mutants of mammalian target of rapamycin.
The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that plays essential roles in the regulation of a wide array of growth-related processes such as protein synthesis, cell sizing, and autophagy. mTOR forms two functionally distinct complexes, termed the mTOR complex 1 (mTORC1) and 2 (mTORC2); only the former of which is inhibited by rapamycin. Based on the similarity between the cellular responses caused by rapamycin treatment and by nutrient starvation, it has been widely accepted that modulation in the mTORC1 activity in response to nutrient status directs these cellular responses, although direct evidence has been scarce. Here we report isolation of hyperactive mutants of mTOR. The isolated mTOR mutants exhibited enhanced kinase activity in vitro and rendered cells refractory to the dephosphorylation of the mTORC1 substrates upon amino acid starvation. Cells expressing the hyperactive mTOR mutant displayed larger cell size in a normal growing condition and were resistant to cell size reduction and autophagy induction in an amino acid-starved condition. These results indicate that the activity of mTORC1 actually directs these cellular processes in response to nutrient status and confirm the biological functions of mTORC1, which had been proposed solely from loss-of-function analyses using rapamycin and (molecular)genetic techniques. Additionally, the hyperactive mTOR mutant did not induce cellular transformation of NIH/3T3 cells, suggesting that concomitant activation of additional pathways is required for tumorigenesis. This hyperactive mTOR mutant will be a valuable tool for establishing physiological consequences of mTOR activation in cells as well as in organisms.
Ohne
Yoichiro
Y
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-0032, Japan.
Takahara
Terunao
T
Hatakeyama
Riko
R
Matsuzaki
Tomoko
T
Noda
Makoto
M
Mizushima
Noboru
N
Maeda
Tatsuya
T
D000818:Animals; D001343:Autophagy; D002460:Cell Line; D048429:Cell Size; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D046912:Multiprotein Complexes; D009154:Mutation / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000378:metabolism
D046912:Multiprotein Complexes; D011506:Proteins; D014157:Transcription Factors; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D000076222:Mechanistic Target of Rapamycin Complex 1; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases