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Sci. Signal., 27 January 2009
Vol. 2, Issue 55, p. ra3
[DOI: 10.1126/scisignal.2000029]

RESEARCH

Characterization of the Intrinsic and TSC2-GAP–Regulated GTPase Activity of Rheb by Real-Time NMR

Christopher B. Marshall1,2, Jason Ho1, Claudia Buerger1,2, Michael J. Plevin1,2, Guang-Yao Li1,2, Zhihong Li1,2, Mitsuhiko Ikura1,2*, and Vuk Stambolic1,2*

1 Division of Signaling Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada M5G 2M9.
2 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M5G 2M9.

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.

* To whom correspondence should be addressed. E-mail: vuks{at}uhnres.utoronto.ca (V.S.) and mikura{at}uhnresearch.ca (M.I.)

Citation: C. B. Marshall, J. Ho, C. Buerger, M. J. Plevin, G.-Y. Li, Z. Li, M. Ikura, V. Stambolic, Characterization of the Intrinsic and TSC2-GAP–Regulated GTPase Activity of Rheb by Real-Time NMR. Sci. Signal. 2, ra3 (2009).

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