Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity.
Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated that TSC2 displays GAP (GTPase-activating protein) activity specifically towards the small G protein Rheb and inhibits its ability to stimulate the mTOR signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, because Rheb has high basal GTP levels and TSC2 does not have the catalytic arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to function as a substrate for TSC2 GAP. In addition, we identified asparagine residues essential for TSC2 GAP activity. We demonstrated a novel catalytic mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that farnesylation and membrane localization of Rheb is not essential for Rheb to stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but may function as a regulatory component in the TSC1/TSC2 complex. Our data further demonstrate that GAP activity is essential for the cellular function of TSC2 to inhibit S6K phosphorylation.
Li
Yong
Y
Life Sciences Institute, Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
Inoki
Ken
K
Guan
Kun-Liang
KL
D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009479:Neuropeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017368:Protein Prenylation; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D000076205:Ras Homolog Enriched in Brain Protein; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014402:Tuberous Sclerosis / Q000235:genetics / Q000378:metabolism; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins
D020690:GTPase-Activating Proteins; D009479:Neuropeptides; D011506:Proteins; C490211:RHEB protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D011994:Recombinant Proteins; D012097:Repressor Proteins; C000624650:TSC1 protein, human; C000624653:TSC2 protein, human; 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