Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR.
Sci Signal
; 2(55): ra3, 2009 Jan 27.
Article
em En
| MEDLINE
| ID: mdl-19176517
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.
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Base de dados:
MEDLINE
Assunto principal:
Neuropeptídeos
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Espectroscopia de Ressonância Magnética
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Proteínas Monoméricas de Ligação ao GTP
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Proteínas Supressoras de Tumor
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GTP Fosfo-Hidrolases
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article