Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1.
AuthorsKehlenbach RH, Assheuer R, Kehlenbach A, Becker J, Gerace L
JournalJ Biol Chem
PubMed ID11278834
'Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) ... More
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
Molecular basis for Rac2 regulation of phagocyte NADPH oxidase.
AuthorsDiebold BA, Bokoch GM
JournalNat Immunol
PubMed ID11224519
'A Rac GTPase-regulated multiprotein NADPH oxidase is critical for the formation of reactive oxygen species (ROS) in phagocytic leukocytes and other nonphagocytic cells. NADPH oxidase reduces molecular oxygen to form superoxide anion in a two-step process. Electrons are initially transferred from NADPH to cytochrome b-associated FAD, then to cytochrome b ... 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
Isomerization couples chemistry in the ATP sulfurylase-GTPase system.
AuthorsWei J, Leyh TS
JournalBiochemistry
PubMed ID10320361
'ATP sulfurylase catalyzes and couples the free energies of two reactions: GTP hydrolysis and the synthesis of activated sulfate, or APS. The GTPase active site undergoes changes during its catalytic cycle that are driven by events that occur at the APS-forming active site, which is located in a separate subunit. ... More
Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein.
AuthorsSchwemmle M, Richter MF, Herrmann C, Nassar N, Staeheli P
JournalJ Biol Chem
PubMed ID7539430
'MxA is an interferon-induced 76-kDa GTPase that inhibits the multiplication of several RNA viruses. Deleting seven amino acids from the COOH terminus reduced the GTPase activity of purified MxA to 1.4%. MxA mutants with COOH-terminal deletions of 63 or more amino acids lost all ability to hydrolyze GTP and failed ... More
Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase.
AuthorsHerrmann C, Martin GA, Wittinghofer A
JournalJ Biol Chem
PubMed ID7852367
The Ras-binding domain (RBD) of human Raf-1 was purified from Escherichia coli, and its interaction with Ras was investigated. Its dissociation constant with p21ras.guanyl-5'-yl imidodiphosphate was found to be 18 nM, with a slight preference for H-ras over K- and N-ras. Oncogenic forms bind with slightly lower affinity. The affinity ... More
Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor.
AuthorsHerrmann C, Horn G, Spaargaren M, Wittinghofer A
JournalJ Biol Chem
PubMed ID8636102
The interactions of H-Ras, R-Ras, and Rap1A with the Ras-binding domains (RBD) of the c-Raf kinase and of the Ral guanine nucleotide exchange factor (RGF) was studied biochemically in solution. From deletion cloning the RGF-RBD was defined as a 97-amino acid-long fragment from the C-terminal end of the human RGF, ... 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
Elucidation of binding determinants and functional consequences of Ras/Raf-cysteine-rich domain interactions.
AuthorsWilliams JG, Drugan JK, Yi GS, Clark GJ, Der CJ, Campbell SL
JournalJ Biol Chem
PubMed ID10777480
Raf-1 is a critical downstream target of Ras and contains two distinct domains that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been shown to be essential for Ras-mediated translocation of Raf-1 to the plasma membrane, whereas the second site, in the Raf-1 cysteine-rich domain (Raf-CRD), has been ... More
Engineering modular protein interaction switches by sequence overlap.
AuthorsSallee NA, Yeh BJ, Lim WA
JournalJ Am Chem Soc
PubMed ID17381089
Many cellular signaling pathways contain proteins whose interactions change in response to upstream inputs, allowing for conditional activation or repression of the interaction based on the presence of the input molecule. The ability to engineer similar regulation into protein interaction elements would provide us with powerful tools for controlling cell ... 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
Rac "insert region" is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65.
AuthorsFreeman JL, Abo A, Lambeth JD
JournalJ Biol Chem
PubMed ID8702687
The small GTPase Rac assembles with the cytosolic p47(phox) and p67(phox) and the membrane-associated flavocytochrome b558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to ... 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
Identification of a putative effector for Cdc42Hs with high sequence similarity to the RasGAP-related protein IQGAP1 and a Cdc42Hs binding partner with similarity to IQGAP2.
AuthorsMcCallum SJ, Wu WJ, Cerione RA
JournalJ Biol Chem
PubMed ID8702968
Cdc42 is a Ras-related GTP-binding protein that has been implicated in the regulation of the actin cytoskeleton and cell morphology. In this study, we have identified a protein with a molecular mass approximately 180 kDa from rabbit liver cytosol (designated p180), which binds preferentially to the GTP- and guanosine 5'-3-O-(thio)triphosphate-bound ... More
Rac binding to p67(phox). Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase.
AuthorsNisimoto Y, Freeman JL, Motalebi SA, Hirshberg M, Lambeth JD
JournalJ Biol Chem
PubMed ID9228059
Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b558 with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Herein, the interaction between Rac and p67(phox) is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH ... More
Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy.
AuthorsNomanbhoy TK, Cerione R
JournalJ Biol Chem
PubMed ID8626553
The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how GDI interactions with Rho-like proteins mediate these ... More
The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride.
AuthorsGraham DL, Eccleston JF, Lowe PN
JournalBiochemistry
PubMed ID9893994
The Rho family of small GTP-binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be involved in catalysis. Here, the role of this arginine has been elucidated by mutagenesis followed by determination ... More
Use of a fluorescence spectroscopic readout to characterize the interactions of Cdc42Hs with its target/effector, mPAK-3.
AuthorsLeonard DA, Satoskar RS, Wu WJ, Bagrodia S, Cerione RA, Manor D
JournalBiochemistry
PubMed ID9033409
The family of p21-activated kinases (PAKs) has been shown to contain a domain that can independently bind to the Ras-like proteins Cdc42Hs and Rac. We have expressed a 72 amino acid recombinant form of this p21-binding domain (PBD) from mPAK-3 in Escherichia Coli for use in structure-function studies. The protein ... 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
Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry.
AuthorsRudolph MG, Linnemann T, Grunewald P, Wittinghofer A, Vetter IR, Herrmann C
JournalJ Biol Chem
PubMed ID11292826
Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different ... More
Covalent Inhibitors Allosterically Block the Activation of Rho Family Proteins and Suppress Cancer Cell Invasion.
Authors
JournalAdv Sci (Weinh)
PubMed ID32714746
Biochemical characterization of purified mammalian ARL13B protein indicates that it is an atypical GTPase and ARL3 guanine nucleotide exchange factor (GEF).