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

Marshall, Christopher B.; Ho, Jason; Buerger, Claudia; Plevin, Michael J.; Li, Guangyao; Li, Zhihong; Ikura, Mitsuhiko; Stambolic, Vuk

doi:10.1126/scisignal.2000029

Sci. Signal.

2009

DOI: 10.1126/scisignal.2000029

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

Christopher B. Marshall

Jason Ho

Claudia Buerger

et al.

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-cataly… Show more

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Cited by 62 publications

(93 citation statements)

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“…We recently developed an NMR-based, real-time assay to measure the kinetics of GTP hydrolysis by the small GTPase Rheb by monitoring nucleotide-dependent changes in the NMR spectra of the GTPase protein (10). In this report, the NMR GTPase assay was extended to monitor intrinsic and PDZ-RhoGEF-mediated RhoA nucleotide exchange using native GDP and GTP as substrates.…”

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confidence: 99%

Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

Gasmi-Seabrook

Marshall

Cheung

et al. 2010

Journal of Biological Chemistry

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Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMRbased assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PH PRG ). Using the non-hydrolyzable analogue guanosine-5-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 ؋ 10 ؊4 min ؊1 . This reaction is markedly accelerated to 1179 ؋ 10 ؊4 min ؊1 in the presence of DH-PH PRG at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PH PRG , together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PH PRG . Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles.The small GTPase 3 RhoA, a member of the Ras superfamily, plays a crucial role in cellular processes including proliferation, movement, cell shape, as well as cell-cell and cell-matrix interactions (1). RhoA acts as a molecular switch, cycling between the inactive GDP-and activated GTP-bound states (Fig. 1A). Akin to other small GTPases, RhoA contains a phosphate binding loop (P-loop) and two switch regions that undergo conformational changes upon nucleotide cycling and mediate interactions with effector proteins. Together these three regions interact with the nucleotide phosphate groups and a magnesium ion that is required for high affinity nucleotide binding (low nM to sub nM K d ). GTPase-activating proteins catalyze nucleotide hydrolysis, thus inactivating GTPases, whereas guanine nucleotide exchange factors (GEFs) activate GTPases by stimulating nucleotide exchange (2). PDZ-RhoGEF, like its homologues LARG and p115-RhoGEF, is specific to RhoA and is activated by the G␣ 12/13 subunit of G-protein coupled receptors via its regulator of G-protein signaling domain. Interestingly, PDZ-RhoGEF single nucleotide polymorphisms have been associated with type II diabetes (3) and lung cancer (4).RhoGEFs are multidomain proteins (5), most of which contain a conserved catalytic Dbl homology (DH) ...

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confidence: 99%

Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

Gasmi-Seabrook

Marshall

Cheung

et al. 2010

Journal of Biological Chemistry

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“…S2b) (10). We have demonstrated that the real-time NMR methodology can be successfully used to assay nucleotide exchange in RhoA (11).…”

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confidence: 99%

“…The spectra were processed with NMRPipe (14), and the peak heights were analyzed with Sparky (15) via Gaussian line fitting. Residues from switches I and II, P-loop, ␤3 and ␤4, and the ␣3 helix that exhibit distinct well resolved peaks in each nucleotide-bound state were used as reporters of the reaction rates for each of the three GTPases, as described previously for Rheb (10).…”

mentioning

confidence: 99%

“…according to previous protocols (10,12,13). Tuberous sclerosis 2 (TSC2) GAP domain (residues 1525-1742: hereafter termed TSC2GAP) and the DH-PH fragment of PDZ-RhoGEF (residues 713-1081: hereafter termed DH-PH PRG ) were prepared using pGEX2T and pGEX4T1 vectors, respectively (10,13).…”

mentioning

confidence: 99%

“…Tuberous sclerosis 2 (TSC2) GAP domain (residues 1525-1742: hereafter termed TSC2GAP) and the DH-PH fragment of PDZ-RhoGEF (residues 713-1081: hereafter termed DH-PH PRG ) were prepared using pGEX2T and pGEX4T1 vectors, respectively (10,13). Catalytic domain constructs of the Son of Sevenless (SOS, Cdc25 residues 566 -1049: hereafter referred to as Cdc25 SOS ) and the GTPase-activating domain of human GTPase-activating protein p120GAP (residues 715-1047: hereafter referred to as RasGAP) were prepared as His-tagged proteins from the pET15b vector.…”

mentioning

confidence: 99%

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Real-time NMR Study of Three Small GTPases Reveals That Fluorescent 2′(3′)-O-(N-Methylanthraniloyl)-tagged Nucleotides Alter Hydrolysis and Exchange Kinetics

Mazhab-Jafari

Marshall

Smith

et al. 2010

Journal of Biological Chemistry

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The Ras family of small GTPases control diverse signaling pathways through a conserved "switch" mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2(3)-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is ϳ10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay.

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NMR analysis of the backbone dynamics of the small GTPase Rheb and its interaction with the regulatory protein FKBP38

2017

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Ras homolog enriched in brain (Rheb) is a small GTPase that regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) and, thereby, cell growth and metabolism. Here we show that cycling between the inactive GDP‐ and the active GTP‐bound state modulates the backbone dynamics of a C‐terminal truncated form, RhebΔCT, which is suggested to influence its interactions. We further investigated the interactions between RhebΔCT and the proposed Rheb‐binding domain of the regulatory protein FKBP38. The observed weak interactions with the GTP‐analogue‐ (GppNHp‐) but not the GDP‐bound state, appear to accelerate the GDP to GTP exchange, but only very weakly compared to a genuine GEF. Thus, FKBP38 is most likely not a GEF but a Rheb effector that may function in membrane targeting of Rheb.

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

Cited by 62 publications

References 71 publications

Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

Real-time NMR Study of Three Small GTPases Reveals That Fluorescent 2′(3′)-O-(N-Methylanthraniloyl)-tagged Nucleotides Alter Hydrolysis and Exchange Kinetics

NMR analysis of the backbone dynamics of the small GTPase Rheb and its interaction with the regulatory protein FKBP38

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