Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67(phox) and the small GTPase Rac1.
AuthorsAlloul N, Gorzalczany Y, Itan M, Sigal N, Pick E
JournalBiochemistry
PubMed ID11724569
'Activation of the superoxide (O2(-))-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b(559) with the cytosolic proteins p47(phox) and p67(phox) and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67(phox). This hypothesis was tested ... More
A novel function for Cyclin A2: control of cell invasion via RhoA signaling.
AuthorsArsic N, Bendris N, Peter M, Begon-Pescia C, Rebouissou C, Gadéa G, Bouquier N, Bibeau F, Lemmers B, Blanchard JM,
JournalJ Cell Biol
PubMed ID22232705
'Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration ... More
Low affinity interactions of GDPbetaS and ribose- or phosphoryl-substituted GTP analogues with the heterotrimeric G protein, transducin.
'We have examined the effects of three commonly used classes of guanine nucleotide analogues on the retinal G protein, transducin (Gt), and found them to be quite different from those that might be expected from results with other GTP-binding proteins. The most surprising results were with guanosine 5''-O-(2-thiodiphosphate) (GDPbetaS); rather ... More
Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function.
AuthorsLin B, Skidmore JM, Bhatt A, Pfeffer SM, Pawloski L, Maddock JR
JournalMol Microbiol
PubMed ID11251813
'The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide-binding and exchange properties of CgtA are different from those ... More
Molecular role for the Rab binding platform of guanine nucleotide dissociation inhibitor in endoplasmic reticulum to Golgi transport.
AuthorsWu SK, Luan P, Matteson J, Zeng K, Nishimura N, Balch WE
JournalJ Biol Chem
PubMed ID9756941
'Guanine nucleotide dissociation inhibitor (GDI) regulates the recycling of Rab GTPases involved in vesicle targeting and fusion. We have analyzed the requirement for conserved amino acid residues in the binding of Rab1A and the function of GDI in transport of cargo between the endoplasmic reticulum (ER) and the Golgi apparatus. ... More
Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly.
AuthorsGorzalczany Y, Sigal N, Itan M, Lotan O, Pick E
JournalJ Biol Chem
PubMed ID11007780
'The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome ... More
Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.
AuthorsAlexandrov K, Simon I, Yurchenko V, Iakovenko A, Rostkova E, Scheidig AJ, Goody RS
JournalEur J Biochem
PubMed ID10491170
'Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein ... More
Kinetics of interaction of Rab5 and Rab7 with nucleotides and magnesium ions.
AuthorsSimon I, Zerial M, Goody RS
JournalJ Biol Chem
PubMed ID8702787
'We describe here the kinetics of the interaction of GTP and GDP with the small GTP-binding proteins Rab5 and Rab7. It was possible to make use of the intrinsic fluorescence of these proteins, since Rab5 contains two and Rab7 three tryptophan residues, respectively. With both enzymes, there is a significant ... More
New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes.
AuthorsHiratsuka T
JournalBiochim Biophys Acta
PubMed ID6132622
'The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3''-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence ... More
Solution dynamics of p21ras proteins bound with fluorescent nucleotides: a time-resolved fluorescence study.
AuthorsHazlett TL, Moore KJ, Lowe PN, Jameson DM, Eccleston JF
JournalBiochemistry
PubMed ID8257693
'The solution dynamics of normal and transforming p21ras proteins in both the GTP- and GDP-bound forms were examined with time-resolved fluorescence spectroscopy. The fluorescent 2''(3'')-O-(N-methylanthraniloyl) derivatives (mant derivatives) of GTP, dGTP, and GDP and the aminocoumarin and fluorescein derivatives of GTP and GDP were synthesized and used as reporter groups. ... More
RanBP10 is a cytoplasmic guanine nucleotide exchange factor that modulates noncentrosomal microtubules.
AuthorsSchulze H, Dose M, Korpal M, Meyer I, Italiano JE, Shivdasani RA,
JournalJ Biol Chem
PubMed ID18347012
'Microtubule spindle assembly in mitosis is stimulated by Ran.GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including ... More
Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm.
AuthorsLenzen C, Cool RH, Prinz H, Kuhlmann J, Wittinghofer A
JournalBiochemistry
PubMed ID9585556
'Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, ... More
Nucleotide binding by the erythrocyte transglutaminase/Gh protein, probed with fluorescent analogs of GTP and GDP.
AuthorsMurthy SN, Lorand L
JournalProc Natl Acad Sci U S A
PubMed ID10869438
'GTP is known to be a potent inhibitor of the protein crosslinking activity of transglutaminase (TG), probably the most abundant G protein in the human red cell. Nucleotide binding to TG was examined by fluorescence spectroscopy and anisotropy in mixtures of TG with methylanthraniloyl analogs of GTP and GDP. A ... More
Fluorescence methods for monitoring interactions of Rab proteins with nucleotides, Rab escort protein, and geranylgeranyltransferase.
AuthorsAlexandrov K, Scheidig AJ, Goody RS
JournalMethods Enzymol
PubMed ID11210530
Fluorescence approaches to the study of the p21ras GTPase mechanism.
AuthorsEccleston JF, Moore KJ, Brownbridge GG, Webb MR, Lowe PN
JournalBiochem Soc Trans
PubMed ID1889625
The use of ribose-modified guanine nucleotides and tryptophan mutants of p21ras, neither of which have significant effect on the kinetic mechanism of the p21ras GTPase and the GAP-activated p21ras GTPase, will now allow a detailed kinetic study of how GAP and other regulatory proteins interact with p21ras. This will lead ... More
Stimulation of the GTPase activity of translation elongation factor G by ribosomal protein L7/12.
AuthorsSavelsbergh A, Mohr D, Wilden B, Wintermeyer W, Rodnina MV
JournalJ Biol Chem
PubMed ID10625623
Elongation factors (EFs) Tu and G are GTPases that have important functions in protein synthesis. The low intrinsic GTPase activity of both factors is strongly stimulated on the ribosome by unknown mechanisms. Here we report that isolated ribosomal protein L7/12 strongly stimulates GTP hydrolysis by EF-G, but not by EF-Tu, ... More
Correlation between self-association modes and GTPase activation of dynamin.
The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized ... More
The importance of structural transitions of the switch II region for the functions of elongation factor Tu on the ribosome.
AuthorsKnudsen C, Wieden HJ, Rodnina MV
JournalJ Biol Chem
PubMed ID11304547
Elongation factor Tu (EF-Tu) undergoes a large conformational transition when switching from the GTP to GDP forms. Structural changes in the switch I and II regions in the G domain are particularly important for this rearrangement. In the switch II region, helix alpha2 is flanked by two glycine residues: Gly(83) ... More
Analysis of guanine nucleotide binding and exchange kinetics of the Escherichia coli GTPase Era.
AuthorsSullivan SM, Mishra R, Neubig RR, Maddock JR
JournalJ Bacteriol
PubMed ID10852878
Era is an essential Escherichia coli guanine nucleotide binding protein that appears to play a number of cellular roles. Although the kinetics of Era guanine nucleotide binding and hydrolysis have been described, guanine nucleotide exchange rates have never been reported. Here we describe a kinetic analysis of guanine nucleotide binding, ... More
Substrate and product structural requirements for binding of nucleotides to H-ras p21: the mechanism of discrimination between guanosine and adenosine nucleotides.
AuthorsRensland H, John J, Linke R, Simon I, Schlichting I, Wittinghofer A, Goody RS
JournalBiochemistry
PubMed ID7819254
The interaction of the protein product of the H-ras oncogene with a series of nucleoside di- and triphosphates has been examined to investigate the tolerance of the active site to departures from the GTP or GDP structures. Nucleotides which bind relatively strongly could be used as competitors of GDP in ... More
The mechanism of Ras GTPase activation by neurofibromin.
Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage ... More
Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain.
AuthorsSheahan KL, Cordero CL, Satchell KJ
JournalEMBO J
PubMed ID17464284
Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved ... More
Interaction of guanine nucleotides with the signal recognition particle from Escherichia coli.
AuthorsJagath JR, Rodnina MV, Lentzen G, Wintermeyer W
JournalBiochemistry
PubMed ID9799502
The bacterial signal recognition particle (SRP) is an RNA-protein complex. In Escherichia coli, the particle consists of a 114 nt RNA, a 4.5S RNA, and a 48 kDa GTP-binding protein, Ffh. GDP-GTP exchange on, and GTP hydrolysis by, Ffh are thought to regulate SRP function in membrane targeting of translating ... More
Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1.
AuthorsKlebe C, Bischoff FR, Ponstingl H, Wittinghofer A
JournalBiochemistry
PubMed ID7819259
The guanine nucleotide dissociation and GTPase reactions of Ran, a Ras-related nuclear protein, have been investigated using different fluorescence techniques to determine how these reactions are stimulated by the guanine nucleotide exchange factor RCC1 and the other regulatory protein, RanGAP1 (GTPase-activating protein). The intrinsic GTPase of Ran is one-tenth of ... More
Individual rate constants for the interaction of Ras proteins with GTPase-activating proteins determined by fluorescence spectroscopy.
AuthorsAhmadian MR, Hoffmann U, Goody RS, Wittinghofer A
JournalBiochemistry
PubMed ID9109662
Individual rate constants for the interaction of H-, K-, and N-Ras with GAP-334 and NF1-333 were determined using fluorescent derivatives of guanine nucleotides at the active site of the Ras proteins. Stopped-flow experiments with NF1-333 show a fast concentration-dependent initial phase corresponding to the binding reaction followed by a slower ... More
Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome.
AuthorsWilden B, Savelsbergh A, Rodnina MV, Wintermeyer W
JournalProc Natl Acad Sci U S A
PubMed ID16940356
The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP-GTP ... More
Fluorescent guanine nucleotide analogs and G protein activation.
AuthorsRemmers AE, Posner R, Neubig RR
JournalJ Biol Chem
PubMed ID8188654
The N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Both MANT-GTP gamma S (mGTP gamma S) and MANT-GTP (mGTP) displayed a magnesium-dependent increase in fluorescence upon binding to bovine brain G(o). A much greater increase in MANT-guanine nucleotide fluorescence was observed with ... More
The Caulobacter crescentus CgtA protein displays unusual guanine nucleotide binding and exchange properties.
AuthorsLin B, Covalle KL, Maddock JR
JournalJ Bacteriol
PubMed ID10482526
The Caulobacter crescentus CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30 degrees C and displayed equilibrium binding constants of 1.2 and 0.5 ... More
The importance of two conserved arginine residues for catalysis by the ras GTPase-activating protein, neurofibromin.
AuthorsSermon BA, Lowe PN, Strom M, Eccleston JF
JournalJ Biol Chem
PubMed ID9545275
Ras proteins are guanine-nucleotide binding proteins that have a low intrinsic GTPase activity that is enhanced 10(5)-fold by the GTPase-activating proteins (GAPs) p120-GAP and neurofibromin. Comparison of the primary sequences of RasGAPs shows two invariant arginine residues (Arg1276 and Arg1391 of neurofibromin). In this study, site-directed mutagenesis was used to ... More
Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.
AuthorsNeal SE, Eccleston JF, Webb MR
JournalProc Natl Acad Sci U S A
PubMed ID2185475
2'(3')-O-(N-Methyl)anthraniloylguanosine 5'-triphosphate (mantGTP) is a fluorescent analogue of GTP that has similar properties to the physiological substrate in terms of its binding constant and the kinetics of its interactions with p21NRAS, the NRAS protooncogene product. There is a 3-fold increase in fluorescence intensity when mantGTP binds to p21NRAS. The rate ... More
Inhibition of GTP binding to Rac2 by peroxynitrite: potential role for tyrosine modification.
AuthorsRohn TT, Nelson LK, Davis AR, Quinn MT
JournalFree Radic Biol Med
PubMed ID10381206
Peroxynitrite is a potent oxidant generated by the reaction of nitric oxide (*NO) and superoxide anion (O2*-), and both can be produced in inflammatory tissues. In the present studies, we analyzed the effects of peroxynitrite treatment on the GTP-binding activity of Rac2, a low molecular weight GTP-binding protein important in ... More
Dynamic and equilibrium studies on the interaction of Ran with its effector, RanBP1.
AuthorsKuhlmann J, Macara I, Wittinghofer A
JournalBiochemistry
PubMed ID9315840
Ran, a small nuclear GTP-binding protein, is one of the most abundant Ras-related proteins in eucaryotic cells. Ran is essential for nucleo-cytoplasmatic transport and is primarily localized in the nucleus and at the nuclear pore complex. Here, we characterize the kinetics and equilibrium of the interaction between Ran and RanBP1 ... More
Structural basis for the inhibition of mammalian membrane adenylyl cyclase by 2 '(3')-O-(N-Methylanthraniloyl)-guanosine 5 '-triphosphate.
AuthorsMou TC, Gille A, Fancy DA, Seifert R, Sprang SR
JournalJ Biol Chem
PubMed ID15591060
Membrane-bound mammalian adenylyl cyclase (mAC) catalyzes the synthesis of intracellular cyclic AMP from ATP and is activated by stimulatory G protein alpha subunits (Galpha(s)) and by forskolin (FSK). mACs are inhibited with high potency by 2 '(3')-O-(N-methylanthraniloyl) (MANT)-substituted nucleotides. In this study, the crystal structures of the complex between Galpha(s).GTPgammaS ... More
Mechanism of GTP hydrolysis by p21N-ras catalyzed by GAP: studies with a fluorescent GTP analogue.
AuthorsMoore KJ, Webb MR, Eccleston JF
JournalBiochemistry
PubMed ID8338843
The mechanism of the hydrolysis of GTP by p21N-ras and its activation by the catalytic domain of p120 GTPase activating protein (GAP) have been studied using a combination of chemical and fluorescence measurements with the fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl)GTP (mantGTP). Since the concentration of active p21 is important in these ... More
Delineation of the Cdc42/Rac-binding domain of p21-activated kinase.
AuthorsThompson G, Owen D, Chalk PA, Lowe PN
JournalBiochemistry
PubMed ID9601050
p21-activated kinases (PAKs) serve as effector proteins for the GTP-binding proteins Cdc42 and Rac. They are serine/threonine kinases containing the Cdc42/Rac interactive binding (CRIB) motif. The main aim of this study was to define the minimal domain of alphaPAK required for Cdc42/Rac binding. Eight stable PAK fragments of varying lengths, ... More
Conformational states of the nuclear GTP-binding protein Ran and its complexes with the exchange factor RCC1 and the effector protein RanBP1.
AuthorsGeyer M, Assheuer R, Klebe C, Kuhlmann J, Becker J, Wittinghofer A, Kalbitzer HR
JournalBiochemistry
PubMed ID10471274
It has been shown before by (31)P NMR that Ras bound to the nonhydrolyzable GTP analogue guanosine 5'-O-(beta, gamma-imidotriphosphate) (GppNHp) exists in two conformations which are rapidly interconverting with a rate constant of 3200 s-1 at 30 degrees C [Geyer, M., et al. (1996) Biochemistry 35, 10308-10320]. Here we show ... More
Interaction of GTPase activating proteins (GAPs) with p21ras measured by a novel fluorescence anisotropy method. Essential role of Arg-903 of GAP in activation of GTP hydrolysis on p21ras.
AuthorsBrownbridge GG, Lowe PN, Moore KJ, Skinner RH, Webb MR
JournalJ Biol Chem
PubMed ID8496156
Ras GTPase activating proteins (GAPs) contain an invariant motif, -FLR-, within the most conserved region of their catalytic domains. Certain mutations in this motif have greatly reduced activity (Skinner, R. H., Bradley, S., Brown, A. L., Johnson, N. J., Rhodes, S., Stammers, D. K., and Lowe, P. N. (1991) J. ... More
Kinetics of the interaction of translation factor SelB from Escherichia coli with guanosine nucleotides and selenocysteine insertion sequence RNA.
AuthorsThanbichler M, Bock A, Goody RS
JournalJ Biol Chem
PubMed ID10781605
The kinetics of the interaction of GTP and GDP with SelB, the specific translation factor for the incorporation of selenocysteine into proteins, have been investigated using the stopped-flow method. Useful signals were obtained using intrinsic (i.e. tryptophan) fluorescence, the fluorescence of methylanthraniloyl derivatives of nucleotides, or fluorescence resonance energy transfer ... More
Spontaneous nucleotide exchange in low molecular weight GTPases by fluorescently labeled gamma-phosphate-linked GTP analogs.
Regulated guanosine nucleotide exchange and hydrolysis constitute the fundamental activities of low molecular weight GTPases. We show that three guanosine 5'-triphosphate analogs with BODIPY fluorophores coupled via the gamma phosphate bind to the GTPases Cdc42, Rac1, RhoA, and Ras and displace guanosine 5'-diphosphate with high intrinsic exchange rates in the ... More
Partial G protein activation by fluorescent guanine nucleotide analogs. Evidence for a triphosphate-bound but inactive state.
AuthorsRemmers AE, Neubig RR
JournalJ Biol Chem
PubMed ID8617747
N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Previously, we showed that MANT fluorescence intensity when bound to G protein was related to the degree of G protein activation where MANT-guanosine-5'-O-(3-thiotriphosphate) (mGTP gammaS) had the highest fluorescence followed by mGTP and mGDP, ... More
The kinetic mechanism of Ran--nucleotide exchange catalyzed by RCC1.
AuthorsKlebe C, Prinz H, Wittinghofer A, Goody RS
JournalBiochemistry
PubMed ID7548002
The interaction of Ran, a Ras-related nuclear GTP-binding protein, with its guanine nucleotide exchange factor RCC1 has been studied by equilibrium and transient kinetic measurements using fluorescent nucleotides. The four-step mechanism of catalyzed nucleotide exchange involves the formation of ternary complexes consisting of Ran, RCC1, and GXP as well as ... More
Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf.
Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway. In this study, we have characterized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and ... More
Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors.
Authors
JournalCancer Cell
PubMed ID30300582
The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice.
Authors
JournalPLoS Pathog
PubMed ID25658451
Nod factor receptor complex phosphorylates GmGEF2 to stimulate ROP signaling during nodulation.
Authors
JournalCurr Biol
PubMed ID34216556
ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses.
Authors
JournalProc Natl Acad Sci U S A
PubMed ID26912459
An ancient P-loop GTPase in rice is regulated by a higher plant-specific regulatory protein.