title: Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR.
abstract: Tuberous sclerosis complex 2 (TSC2), whose gene is frequently mutated in tuberous sclerosis, increases the guanosine triphosphatase (GTPase) activity of the small heterotrimeric GTP-binding protein (G protein) Rheb, thus resulting in the decreased activity of the mammalian target of rapamycin (mTOR), the master regulator of cell growth. Here, we describe the development of a nuclear magnetic resonance (NMR)-based, quantitative, real-time assay to explore the molecular mechanism of the intrinsic and TSC2-catalyzed GTPase activity of Rheb. We confirmed that TSC2 accelerated GTP hydrolysis by Rheb 50-fold through an "asparagine-thumb" mechanism to substitute for the nonfunctional "catalytic" glutamine of Rheb and we determined that catalysis was enthalpy driven. Most, but not all, of the disease-associated GTPase-activating protein (GAP) domain mutants of TSC2 that we examined affected its enzymatic activity. This method can now be applied to study the function and regulation of other GTPases.
authors.affiliation: Division of Signaling Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada M5G 2M9.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Marshall
authors.forename: Jason
authors.initials: J
authors.lastname: Ho
authors.forename: Claudia
authors.initials: C
authors.lastname: Buerger
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Plevin
authors.forename: Guang-Yao
authors.initials: GY
authors.lastname: Li
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Li
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
authors.forename: Vuk
authors.initials: V
authors.lastname: Stambolic
mesh_terms: D002384:Catalysis; D004252:DNA Mutational Analysis; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D009154:Mutation; D009479:Neuropeptides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D000076205:Ras Homolog Enriched in Brain Protein; D013816:Thermodynamics; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D009479:Neuropeptides; C490211:RHEB protein, human; D000076205:Ras Homolog Enriched in Brain Protein; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins