Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR.
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.
Marshall
Christopher B
CB
Division of Signaling Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada M5G 2M9.
Ho
Jason
J
Buerger
Claudia
C
Plevin
Michael J
MJ
Li
Guang-Yao
GY
Li
Zhihong
Z
Ikura
Mitsuhiko
M
Stambolic
Vuk
V
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*
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