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
; 289(18): 12195-201, 2014 May 02.
Article
em En
| MEDLINE
| ID: mdl-24648513
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.
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01-internacional
Base de dados:
MEDLINE
Assunto principal:
Neuropeptídeos
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Proteínas Monoméricas de Ligação ao GTP
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Serina-Treonina Quinases TOR
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Glicina
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Mutação
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Canadá