Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(DeltaM) and EF-Tu(DeltaC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N-terminal domain, they had lost a typical feature of EF-Tu, the >100 times stronger affinity for GDP than for GTP. EF-Tu(DeltaM) and EF-Tu(DeltaC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(DeltaC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was >90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF-Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(DeltaC).GTP, showing the same kind of effect as on EF-Tu.GTP, but they were little active on EF-Tu(DeltaM). GTP. Like EF-Tu(DeltaC).GTP, EF-Tu(DeltaM).GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.
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PMID:Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands. 942 69

Activation of phospholipase D1 (PLD1) by Arf has been implicated in vesicle transport and membrane trafficking. PLD1 has also been shown to be associated with the small GTPase RalA, which functions downstream from Ras in a Ras-RalA GTPase cascade that facilitates intracellular signal transduction. Although PLD1 associates directly with RalA, RalA has no effect upon the activity of PLD1. However, PLD1 precipitated from cell lysates with immobilized glutathione S-transferase-RalA fusion protein is active. This suggests the presence of an additional activating factor in the active RalA-PLD1 complexes. Because Arf stimulates PLD1, we looked for the presence of Arf in the active RalA-PLD1 complexes isolated from v-Src- and v-Ras-transformed cell lysates. Low levels of Arf protein were detected in RalA-PLD1 complexes; however, if guanosine 5'-[gamma-thio]triphosphate was added to activate Arf and stimulate translocation to the membrane, high levels of Arf were precipitated by RalA from cell lysates. Interestingly, deletion of 11 amino-terminal amino acids unique to Ral GTPases, which abolished the ability of RalA to precipitate PLD activity, prevented the association between RalA and Arf. Brefeldin A, which inhibits Arf GDP-GTP exchange, inhibited PLD activity in v-Src- and v-Ras-transformed cells but not in the nontransformed cells, suggesting that the association of Arf with RalA is required for the increased PLD activity induced by v-Src and v-Ras. These data implicate Arf in the transduction of intracellular signals activated by v-Src and mediated by the Ras-RalA GTPase cascade. Because both Arf and PLD1 stimulate vesicle formation in the Golgi, these data raise the possibility that vesicle formation and trafficking may play a role in the transduction of intracellular signals.
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PMID:Functional association between Arf and RalA in active phospholipase D complex. 952 Apr 17

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of alphao and alphai1 was done. G184S alphao and G183S alphai1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alphao and alphai1 to compete with fluorescein isothiocyanate-alphao binding to glutathione S-transferase-RGS4. The G --> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alphao and alphai1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.
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PMID:A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins. 958 6

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.
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PMID:Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle. 972 79

Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S-transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3'-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin.
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PMID:Mixed-lineage kinase 2-SH3 domain binds dynamin and greatly enhances activation of GTPase by phospholipid. 974 20

The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin-1 and dynamin-2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathione S-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin-1 and dynamin-2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission.
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PMID:The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding. 976 10

Elongation factor 3 (EF-3) is an essential requirement for translation in fungi. We previously reported activation of EF-3-ATPase by yeast ribosomes. EF-3 interacts with both ribosomal subunits and shows high affinity for 60S subparticles. Translational inhibitors alpha-sarcin, ricin and auto-immune antibodies to GTPase-activation center inhibit binding of EF-2 but not of EF-3 to yeast ribosomes. EF-2 competes with EF-3 for the ribosomal binding sites and inhibits EF-3-ATPase activity. Neomycin relieves the inhibitory effect of EF-2 on EF-3 function. The apparent competition between EF-2 and EF-3 may represent binding of these two proteins to specific conformational states of the ribosome. EF-3 stimulates ternary complex binding to yeast ribosomes. Neither the binding of EF-3 to ribosomes, nor the ribosome-dependent EF-3-ATPase activity are influenced by EF-1 alpha. Three lines of experimental evidence suggest a direct interaction between EF-1 alpha and EF-3. A polyclonal antibody to EF-3 immunoprecipitates EF-1 alpha along with EF-3. EF-1 alpha co-migrates with GST-EF-3 on glutathione-Sepharose columns. ELISA tests demonstrate an interference of EF-3/anti-EF-3 interaction by EF-1 alpha but not by EF-2. These results strongly suggest that the stimulatory effect of EF-3 on the ternary complex binding to yeast ribosomes involves a direct interaction between EF-1 alpha and EF-3.
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PMID:Competition and cooperation amongst yeast elongation factors. 999 Mar 16

Lysophosphatidic acid (LPA) is the prototypic G-protein-coupled receptor agonist that activates the Ras-mitogen-activated protein (MAP) kinase cascade through pertussis toxin (PTX)-sensitive Gi and enhanced tyrosine kinase activity. We recently detected a 100 kDa protein (p100) that binds to the C-terminal SH3 domain of growth-factor-receptor-bound protein 2 (Grb2) and becomes tyrosine phosphorylated in a PTX-sensitive manner in LPA-treated Rat-1 cells [Kranenburg, Verlaan, Hordijk and Moolenaar (1997) EMBO J. 16, 3097-3105]. Through glutathione S-transferase-Grb2 affinity purification and microsequencing, we have now identified p100 as dynamin-II, a GTPase that regulates clathrin-mediated endocytosis. We show that in Rat-1 cells, Grb2-bound dynamin-II is rapidly tyrosine phosphorylated in response to LPA in a PTX-sensitive manner. Thus, tyrosine phosphorylation of Grb2-bound dynamin-II may be a critical event in Gi-mediated activation of the Ras-MAP kinase cascade in fibroblasts.
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PMID:Gi-mediated tyrosine phosphorylation of Grb2 (growth-factor-receptor-bound protein 2)-bound dynamin-II by lysophosphatidic acid. 1008 21

ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of the Sec7 domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay. Similarly, full-length [35S]mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy. Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes. These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.
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PMID:The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D. 1009 63

Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.
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PMID:Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling. 1033 Apr 11


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