Structure-guided mutation of the conserved G3-box glycine in Rheb generates a constitutively activated regulator of mammalian target of rapamycin (mTOR)

Mohammad T Mazhab-Jafari; Christopher B Marshall; Jason Ho; Noboru Ishiyama; Vuk Stambolic; Mitsuhiko Ikura

doi:10.1074/jbc.C113.543736

Structure-guided mutation of the conserved G3-box glycine in Rheb generates a constitutively activated regulator of mammalian target of rapamycin (mTOR)

J Biol Chem. 2014 May 2;289(18):12195-201. doi: 10.1074/jbc.C113.543736. Epub 2014 Mar 19.

Authors

Mohammad T Mazhab-Jafari¹, Christopher B Marshall, Jason Ho, Noboru Ishiyama, Vuk Stambolic, Mitsuhiko Ikura

Affiliation

¹ From the Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre and, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.

Abstract

Constitutively activated variants of small GTPases, which provide valuable functional probes of their role in cellular signaling pathways, can often be generated by mutating the canonical catalytic residue (e.g. Ras Q61L) to impair GTP hydrolysis. However, this general approach is ineffective for a substantial fraction of the small GTPase family in which this residue is not conserved (e.g. Rap) or not catalytic (e.g. Rheb). Using a novel engineering approach, we have manipulated nucleotide binding through structure-guided substitutions of an ultraconserved glycine residue in the G3-box motif (DXXG). Substitution of Rheb Gly-63 with alanine impaired both intrinsic and TSC2 GTPase-activating protein (GAP)-mediated GTP hydrolysis by displacing the hydrolytic water molecule, whereas introduction of a bulkier valine side chain selectively blocked GTP binding by steric occlusion of the γ-phosphate. Rheb G63A stimulated phosphorylation of the mTORC1 substrate p70S6 kinase more strongly than wild-type, thus offering a new tool for mammalian target of rapamycin (mTOR) signaling.

Keywords: Constitutively Active; Crystal Structure; Enzyme Catalysis; G3-box; Protein Engineering; Rheb; Small GTPases; TSC2GAP; mTOR Complex (mTORC).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alanine / chemistry
Alanine / genetics
Alanine / metabolism
Amino Acid Motifs / genetics
Animals
Binding Sites / genetics
Cells, Cultured
Crystallography, X-Ray
Glycine / chemistry
Glycine / genetics*
Glycine / metabolism
Guanosine Triphosphate / chemistry
Guanosine Triphosphate / metabolism
HeLa Cells
Humans
Hydrolysis
Mechanistic Target of Rapamycin Complex 1
Mice
Models, Molecular
Monomeric GTP-Binding Proteins / chemistry
Monomeric GTP-Binding Proteins / genetics*
Monomeric GTP-Binding Proteins / metabolism
Multiprotein Complexes / genetics
Multiprotein Complexes / metabolism
Mutation*
Neuropeptides / chemistry
Neuropeptides / genetics*
Neuropeptides / metabolism
Phosphorylation
Protein Binding / genetics
Protein Structure, Tertiary
Ras Homolog Enriched in Brain Protein
Ribosomal Protein S6 Kinases, 70-kDa / metabolism
Signal Transduction / genetics
TOR Serine-Threonine Kinases / genetics*
TOR Serine-Threonine Kinases / metabolism

Substances

Multiprotein Complexes
Neuropeptides
RHEB protein, human
Ras Homolog Enriched in Brain Protein
Guanosine Triphosphate
MTOR protein, human
Mechanistic Target of Rapamycin Complex 1
Ribosomal Protein S6 Kinases, 70-kDa
TOR Serine-Threonine Kinases
Monomeric GTP-Binding Proteins
Alanine
Glycine

Associated data

PDB/4O25
PDB/4O2L
PDB/4O2R