Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts

Marshall, Christopher B.; Meiri, David; Smith, Matthew J.; Mazhab-Jafari, Mohammad T.; Gasmi-Seabrook, Geneviève M.C.; Rottapel, Robert; Stambolic, Vuk; Ikura, Mitsuhiko

doi:10.1016/j.ymeth.2012.06.014

Cited by 42 publications

(34 citation statements)

References 117 publications

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“…To measure GEF activity in lysates of mammalian cells, real-time NMR GEF assays were performed 6,17 . Nucleotide exchange assays were carried out at 20°C with 0.4 mM 15 N-labelled RhoA (residues 1-181) in NMR buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 2 mM Tris (2-carboxyethyl) phosphine, 10% D 2 O, pH 7.0) using a Bruker 600 MHz NMR spectrometer equipped with a 1.7-mm microcryoprobe.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1

et al. 2014

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Section: Methodsmentioning

confidence: 99%

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1

et al. 2014

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“…Protein Preparation-Mus musculus Rheb (residues 1-169) WT and G63A and G63V mutants and TSC2 GAP domain (residues 1,525-1,742) were expressed as glutathione S-transferase (GST) fusions from the pGEX2T vector in Escherichia coli (BL21 strain) as described previously (13). Briefly, bacteria were grown in minimal M9 media supplemented with either 14 NH 4 Cl or 15 NH 4 Cl at 37°C to A 600 ϭ 0.6, and protein expression was initiated with 0.25 mM isopropyl-1-thio-␤-D-galactopyranoside at 15°C.…”

Section: Methodsmentioning

confidence: 99%

Structure-guided Mutation of the Conserved G3-box Glycine in Rheb Generates a Constitutively Activated Regulator of Mammalian Target of Rapamycin (mTOR)

Mazhab-Jafari

Marshall

et al. 2014

Journal of Biological Chemistry

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Background: GTPases regulate cellular signaling by cycling between GDP-(inactive) and GTP-(active) bound states. Results: Rheb GTPase cycling was manipulated by structure-guided mutagenesis of an ultraconserved residue. Conclusion: Constitutively activated or inactive Rheb mutants were generated by substitutions that displace the hydrolytic water or ␥-phosphate, respectively. Significance: These mutants offer new research tools, and the approach may be extended to other GTPases.

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“…In this article we apply our recently developed GTPase assay (18) to directly measure RAS cycling defects and the regulatory impact of intact, multidomain RASopathy proteins in mammalian cell extracts. We characterize how opposing intrinsic properties integrate with regulators to hyperactivate oncogenic RAS.…”

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NMR-based functional profiling of RASopathies and oncogenic RAS mutations

Smith

Neel

Ikura

2013

Proc. Natl. Acad. Sci. U.S.A.

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Defects in the RAS small G protein or its associated network of regulatory proteins that disrupt GTPase cycling are a major cause of cancer and developmental RASopathy disorders. Lack of robust functional assays has been a major hurdle in RAS pathway-targeted drug development. We used NMR to obtain detailed mechanistic data on RAS cycling defects conferred by oncogenic mutations, or full-length RASopathy-derived regulatory proteins. By monitoring the conformation of wild-type and oncogenic RAS in real-time, we show that opposing properties integrate with regulators to hyperactivate oncogenic RAS mutants. Q61L and G13D exhibited rapid nucleotide exchange and an unexpected susceptibility to GAP-mediated hydrolysis, in direct contrast with G12V, indicating different approaches must be taken to inhibit these oncoproteins. An NMR methodology was established to directly monitor RAS cycling by intact, multidomain proteins encoded by RASopathy genes in mammalian cell extracts. By measuring GAP activity from tumor cells, we demonstrate how loss of neurofibromatosis type 1 (NF1) increases RAS-GTP levels in NF1-derived cells. We further applied this methodology to profile Noonan Syndrome (NS)-derived SOS1 mutants. Combining NMR with cell-based assays allowed us to differentiate defects in catalysis, allosteric regulation, and membrane targeting of individual mutants, while revealing a membrane-dependent compensatory effect that attenuates dramatic increases in RAS activation shown by Y337C, L550P, and I252T. Our NMR method presents a precise and robust measure of RAS activity, providing mechanistic insights that facilitate discovery of therapeutics targeted against the RAS signaling network.nuclear magnetic resonance | real-time bioassay | guanine nucleotide exchange factor inhibition R AS functions downstream of membrane-bound receptors to control cell proliferation, differentiation, and survival pathways crucial to development. Deregulated RAS signaling leads to disorders ranging from cancer to developmental syndromes termed RASopathies (1). RAS and its associated signaling network represent extremely attractive therapeutic targets, yet there has been minimal success at exploiting these for drug development (2, 3).RAS exists in two distinct conformations dependent on the state of bound nucleotide. Following stimulation, a GDP-to-GTP exchange is catalyzed by guanine nucleotide exchange factors (GEFs), transmitting downstream signals through RAS-GTP to diverse effector proteins (4). Inactivation via GTP hydrolysis is assisted by GTPase-activating proteins (GAPs), which enhance the slow intrinsic activity of the enzyme. Disorders stemming from aberrations in RAS GTPase cycling (Fig. 1A) are driven by abnormally high levels of activated RAS. Single amino acid mutations in RAS proteins are found in a remarkable 30% of human tumors, often in those with high-risk clinical features (1). Oncogenic mutations are most common at three loci, considered "hotspots" for transformation: Gly12, Gly13, and Gln61. The most frequently found ...

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Probing the GTPase cycle with real-time NMR: GAP and GEF activities in cell extracts

Cited by 42 publications

References 117 publications

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1

Structure-guided Mutation of the Conserved G3-box Glycine in Rheb Generates a Constitutively Activated Regulator of Mammalian Target of Rapamycin (mTOR)

NMR-based functional profiling of RASopathies and oncogenic RAS mutations

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