Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb.
BACKGROUND
Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear.


RESULTS
We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently.


CONCLUSIONS
We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.
Tee
Andrew R
AR
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Manning
Brendan D
BD
Roux
Philippe P
PP
Cantley
Lewis C
LC
Blenis
John
J
D048868:Adaptor Proteins, Signal Transducing; D001681:Biological Assay; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010957:Plasmids; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D000076205:Ras Homolog Enriched in Brain Protein; D012097:Repressor Proteins / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014402:Tuberous Sclerosis / Q000235:genetics* / Q000378:metabolism; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C087000:EIF4EBP1 protein, human; C111931:ELF4 protein, human; D020690:GTPase-Activating Proteins; D009479:Neuropeptides; D010750:Phosphoproteins; D011506:Proteins; C490211:RHEB protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D012097:Repressor Proteins; C000624650:TSC1 protein, human; C000624653:TSC2 protein, human; D014157:Transcription Factors; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C546842:MTOR protein, human; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins