Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells.
Rheb is a unique member of the Ras superfamily GTP-binding proteins. We as well as others previously have shown that Rheb is a critical component of the TSC/TOR signaling pathway. In fission yeast, Rheb is encoded by the rhb1 gene. Rhb1p is essential for growth and directly interacts with Tor2p. In this article, we report identification of 22 single amino acid changes in the Tor2 protein that enable growth in the absence of Rhb1p. These mutants also exhibit decreased mating efficiency. Interestingly, the mutations are located in the C-terminal half of the Tor2 protein, clustering mainly within the FAT and kinase domains. We noted some differences in the effect of a mutation in the FAT domain (L1310P) and in the kinase domain (E2221K) on growth and mating. Although the Tor2p mutations bypass Rhb1p's requirement for growth, they are incapable of suppressing Rhb1p's requirement for resistance to stress and toxic amino acids, pointing to multiple functions of Rhb1p. In mammalian systems, we find that mammalian target of rapamycin (mTOR) carrying analogous mutations (L1460P or E2419K), although sensitive to rapamycin, exhibits constitutive activation even when the cells are starved for nutrients. These mutations do not show significant difference in their ability to form complexes with Raptor, Rictor, or mLST8. Furthermore, we present evidence that mutant mTOR can complex with wild-type mTOR and that this heterodimer is active in nutrient-starved cells.
Urano
Jun
J
Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
Sato
Tatsuhiro
T
Matsuo
Tomohiko
T
Otsubo
Yoko
Y
Yamamoto
Masayuki
M
Tamanoi
Fuyuhiko
F
D000595:Amino Acid Sequence; D019281:Dimerization; D020558:GTP Phosphohydrolases / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D013045:Species Specificity; D058570:TOR Serine-Threonine Kinases
D020558:GTP Phosphohydrolases; D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D029702:Schizosaccharomyces pombe Proteins; D058570:TOR Serine-Threonine Kinases; C546842:MTOR protein, human; C513100:tor2 protein, S pombe; C412832:Rhb1 protein, S pombe