title: Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts.
abstract: The Ras superfamily of small GTPases is a large family of switch-like proteins that control diverse cellular functions, and their deregulation is associated with multiple disease processes. When bound to GTP they adopt a conformation that interacts with effector proteins, whereas the GDP-bound state is generally biologically inactive. GTPase activating proteins (GAPs) promote hydrolysis of GTP, thus impeding the biological activity of GTPases, whereas guanine nucleotide exchange factors (GEFs) promote exchange of GDP for GTP and activate GTPase proteins. A number of methods have been developed to assay GTPase nucleotide hydrolysis and exchange, as well as the activity of GAPs and GEFs. The kinetics of these reactions are often studied with purified proteins and fluorescent nucleotide analogs, which have been shown to non-specifically impact hydrolysis and exchange. Most GAPs and GEFs are large multidomain proteins subject to complex regulation that is challenging to reconstitute in vitro. In cells, the activities of full-length GAPs or GEFs are typically assayed indirectly on the basis of nucleotide loading of the cognate GTPase, or by exploiting their interaction with effector proteins. Here, we describe a recently developed real-time NMR method to assay kinetics of nucleotide exchange and hydrolysis reactions by direct monitoring of nucleotide-dependent structural changes in an isotopically labeled GTPase. The unambiguous readout of this method makes it possible to precisely measure GAP and GEF activities from extracts of mammalian cells, enabling studies of their catalytic and regulatory mechanisms. We present examples of NMR-based assays of full-length GAPs and GEFs overexpressed in mammalian cells.
authors: Ontario Cancer Institute and The Campbell Family Cancer Research Institute, University Health Network, 101 College Street, Rm 4-804 Toronto Medical Discovery Tower, MaRS Building, Toronto, ON, Canada M5G 1L7.
authors: Christopher B
authors: CB
authors: Marshall
authors: David
authors: D
authors: Meiri
authors: Matthew J
authors: MJ
authors: Smith
authors: Mohammad T
authors: MT
authors: Mazhab-Jafari
authors: Geneviève M C
authors: GM
authors: Gasmi-Seabrook
authors: Robert
authors: R
authors: Rottapel
authors: Vuk
authors: V
authors: Stambolic
authors: Mitsuhiko
authors: M
authors: Ikura
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000737:chemistry*; D004398:Dyneins / Q000737:chemistry; D057075:Enzyme Assays; D005456:Fluorescent Dyes / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D020690:GTPase-Activating Proteins / Q000737:chemistry*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry; D009479:Neuropeptides / Q000737:chemistry; D000076205:Ras Homolog Enriched in Brain Protein; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000737:chemistry
chemical_list: D002457:Cell Extracts; D005456:Fluorescent Dyes; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D009479:Neuropeptides; C490211:RHEB protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D011994:Recombinant Proteins; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D004398:Dyneins; D020559:Monomeric GTP-Binding Proteins