title: The Rheb switch 2 segment is critical for signaling to target of rapamycin complex 1.
abstract: The small GTPase Rheb is a positive upstream regulator of the target of rapamycin (TOR) complex 1 in mammalian cells and can bind directly to TOR complex 1. To identify the regions of the Rheb surface most critical for signaling to TOR complex 1, we created a set of 26 mutants wherein clusters of 1-5 putative solvent-exposed residues were changed to alanine, ultimately changing 65 residues distributed over the entire Rheb surface. The signaling function of these mutants was assessed by their ability, in comparison to wild type Rheb, to restore the phosphorylation of S6K1(Thr389) when expressed transiently in amino acid-deprived 293T cells. The major finding is that two mutants situated in the Rheb switch 2 segment, Y67A/I69A and I76A/D77A, exhibit a near total loss of function, whereas extensive replacement of the switch 1 segment and other surface residues with alanines causes relatively little disturbance of Rheb rescue of S6K1 from amino acid withdrawal. This is surprising in view of the minimal impact of guanyl nucleotide on Rheb switch 2 configuration. The loss of function Rheb switch 2 mutants are well expressed and exhibit partial agonist function in amino acid-replete cells. They are unimpaired in their ability to bind GTP or mammalian (m)TOR in vivo or in vitro, and the mTOR polypeptides retrieved with these inactive Rheb mutants exhibit kinase activity in vitro comparable with mTOR bound to wild type Rheb. We conclude that Rheb signaling to mTOR in vivo requires a Rheb switch 2-dependent interaction with an element other than the three known polypeptide components of TOR complex 1.
authors.affiliation: Diabetes Unit and Medical Services and the Department of Molecular Biology, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: Long
authors.affiliation: Diabetes Unit and Medical Services and the Department of Molecular Biology, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Yenshou
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Diabetes Unit and Medical Services and the Department of Molecular Biology, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Sara
authors.initials: S
authors.lastname: Ortiz-Vega
authors.affiliation: Diabetes Unit and Medical Services and the Department of Molecular Biology, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Susann
authors.initials: S
authors.lastname: Busch
authors.affiliation: Diabetes Unit and Medical Services and the Department of Molecular Biology, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114. Electronic address: avruch@molbio.mgh.harvard.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Avruch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D009154:Mutation; D009479:Neuropeptides / Q000737:chemistry / Q000502:physiology*; D010455:Peptides / Q000737:chemistry; D011494:Protein Kinases / Q000378:metabolism*; D000076205:Ras Homolog Enriched in Brain Protein; D012568:Schizosaccharomyces / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014162:Transfection
chemical_list: D009479:Neuropeptides; D010455:Peptides; C490211:RHEB protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D006160:Guanosine Triphosphate; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins