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)

Potential signaling substrates for the insulin-like growth factor I (IGF-I) receptor are SH2 domain proteins including the p85 subunit of phosphatidylinositol 3-kinase, the tyrosine phosphatase Syp, GTPase activating protein (GAP), and phospholipase C-gamma (PLC-gamma). In this study, we demonstrate an association between the IGF-I receptor and p85, Syp, and GAP, but not with PLC-gamma in lysates of cells overexpressing the human IGF-I receptor. We further investigated these interactions using glutathione S-transferase (GST) fusion proteins containing the amino-terminal SH2 domains of p85 or GAP, or both SH2 domains of Syp or PLC-gamma to precipitate the IGF-I receptor from purified receptor preparations and from whole cell lysates. p85-, Syp-, and GAP-GSTs precipitated the IGF-I receptor, whereas the PLC-gamma-GST did not. Using phosphopeptides corresponding to IGF-I receptor phosphorylation sites, we determined that the p85- and Syp-GST association with the IGF-I receptor could be inhibited by a carboxyl-terminal peptide containing pY1316 and that the GAP-GST association could be inhibited by a NPXY domain peptide. The GAP-GST binding site was confirmed by showing that a mutant IGF-I receptor with a deletion of the NPXY domain including tyrosine 950 was poorly precipitated by the GAP-GST. We conclude that p85 and Syp may bind directly to the IGF-I receptor at tyrosine 1316, and that GAP may bind to the IGF-I receptor at and PLC-gamma was not evident. p85, Syp, and GAP are potential modulators of IGF-I receptor signal transduction.
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PMID:Localization of the insulin-like growth factor I receptor binding sites for the SH2 domain proteins p85, Syp, and GTPase activating protein. 764 82

Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from Arabidopsis thaliana. The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPT/rab type. To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into ara cDNAs. Mutant proteins were expressed in E. coli as GST-chimeric molecules and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro. The results indicate that four conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding. A point mutation of Asn at position 72 for ARA-2, or 71 for ARA-4, to Ile decreased GTP-binding and a point mutation of Gln at position 126 for ARA-2, or 125 for ARA-4, to Leu suppressed GTP-hydrolysis activity. Furthermore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells.
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PMID:In vitro mutation analysis of Arabidopsis thaliana small GTP-binding proteins and detection of GAP-like activities in plant cells. 801 29

The GTPase activating protein, p120GAP, contains an amino acid sequence motif called the Ca2+-dependent lipid binding domain (CaLB) which mediates a protein-protein interaction between p120GAP and annexin VI and also binds to negatively charged phospholipids. Because membrane association of p120GAP is important for the regulation of p21 Ras activity, we have studied the roles played by Ca2+, phospholipids and annexin VI in the membrane association of p120GAP. Here we demonstrate that a truncated CaLB domain GST fusion protein (GSTGAP618-632), lacking the ability to bind to phospholipids, is able to bind to rat fibroblast membranes in a Ca2+- and concentration-dependent manner. In addition, this fusion protein also binds to annexin VI in an amino acid sequence specific but Ca2+ independent manner. Also, when bound to annexin VI in the presence of Ca2+, this fusion protein has the ability to co-bind to phosphatidylserine vesicles. Thus, annexin VI may simultaneously mediate an interaction with p120GAP and also an interaction with membrane phospholipids. This may in part explain the mechanism by which p120GAP associates with membranes in response to Ca2+ elevation and suggests the potential importance of annexin VI in the regulation of p21 Ras and the role CaLB domains may play in the specific recognition of cellular membranes.
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PMID:Investigating the role played by protein-lipid and protein-protein interactions in the membrane association of the p120GAP CaLB domain. 1040 Mar 17

Endothelin-1 (ET-1) can stimulate insulin-responsive glucose transporter (GLUT4) translocation in 3T3-L1 adipocytes (Wu-Wong, J. R., Berg, C. E., Wang, J., Chiou, W. J., and Fissel, B. (1999) J. Biol. Chem. 274, 8103-8110), and in the current study, we have evaluated the signaling pathway leading to this response. First, we inhibited endogenous Galpha(q/11) function by single-cell microinjection using anti-Galpha(q/11) antibody or RGS2 protein (a GTPase activating protein for Galpha(q)) followed by immunostaining to quantitate GLUT4 translocation in 3T3-L1 adipocytes. ET-1-stimulated GLUT4 translocation was markedly decreased by 70 or 75% by microinjection of Galpha(q/11) antibody or RGS2 protein, respectively. Pretreatment of cells with the Galpha(i) inhibitor (pertussis toxin) or microinjection of a Gbetagamma inhibitor (glutathione S-transferase-beta-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1-induced GLUT4 translocation, indicating that Galpha(q/11 )mediates ET-1 signaling to GLUT4 translocation. Next, we found that ET-1-induced GLUT4 translocation was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitors wortmannin or LY294002, but not by the phospholipase C inhibitor U-73122. ET-1 stimulated the PI 3-kinase activity of the p110alpha subunit (5.5-fold), and microinjection of anti-p110alpha or PKC-lambda antibodies inhibited ET-stimulated GLUT4 translocation. Finally, we found that Galpha(q/11) formed immunocomplexes with the type-A endothelin receptor and the 110alpha subunit of PI 3-kinase and that ET-1 stimulation enhances tyrosine phosphorylation of Galpha(q/11). These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Galpha(q/11) and PI 3-kinase; and 2) Galpha(q/11) can transmit signals from the ET(A) receptor to the p110alpha subunit of PI 3-kinase, as does insulin, subsequently leading to GLUT4 translocation.
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PMID:Endothelin-1-induced GLUT4 translocation is mediated via Galpha(q/11) protein and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. 1055 59

We have characterized the cDNA for a Rho GTPase activating protein (GAP) mapping to chromosome 13q12. The cDNA was characterized by determining the complete sequence of a 4.8 kb cDNA clone that represents the 5' untranslated region (UTR), the translated region, and the 3' UTR. The protein has a sterile alpha-motif (SAM), a distinct GAP domain, and a conserved START (StAR related lipid transfer) domain. The cDNA has 5 instability motifs (ATTTA) in the 3' UTR and one motif in the translated region between GAP and START domains. The RhoGAP transcript is truncated in some breast carcinoma cell lines and it has low expression in other breast cancer cell lines as compared to a normal breast cell line. We have previously observed the absence of RhoGAP transcript in a breast tumor specimen. A GST-fusion of the RhoGAP was tested for its specificity on RhoA, Cdc42, and Rac1. The protein was most active for RhoA. Transfection of RhoGAP into MCF7 cells significantly inhibited cell growth. The introduction of the RhoGAP construct into MDAMB231 cells that had previously been transfected with a p21 construct did not affect cell proliferation, indicating the involvement of p21 in Rho-mediated proliferation of cancer cells. NIH3T3 cells overexpressing RhoGAP showed considerable inhibition of stress fiber formation. Several cDNAs were identified as RhoGAP interactors by using the yeast two-hybrid assay system. These cDNAs correspond to SWI/SNF, alpha-tubulin, HMG CoA reductase, and TAX1 binding protein (TAX1BP1). The interaction with HMG CoA reductase may partially explain the growth inhibition of breast carcinoma cells by statin class of cholesterol lowering drugs. The biological significance of the interacting proteins is discussed in the context of their involvement in tumorigenesis. Our results indicate that loss of RhoGAP or its altered activity suppresses the growth of breast tumor cells. The presence of various motifs in RhoGAP and its interaction with several other proteins suggest that the protein may regulate Rho signaling in multiple ways and possibly function in a Rho-independent manner.
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PMID:Chromosome 13q12 encoded Rho GTPase activating protein suppresses growth of breast carcinoma cells, and yeast two-hybrid screen shows its interaction with several proteins. 1498 79

Centaurin-alpha(1) is a phosphatidylinositol 3,4,5-trisphosphate binding protein as well as a GTPase activating protein (GAP) for the ADP-ribosylation factor (ARF) family of small GTPases. To further understand its cellular function, we screened a rat brain cDNA library using centaurin-alpha(1) as bait to identify centaurin-alpha(1) interacting proteins. The yeast two-hybrid screen identified a novel kinesin motor protein as a centaurin-alpha(1) binding partner. The motor protein, termed KIF13B, encoded by a single approximately 9.5-kb transcript, is widely expressed with high levels observed in brain and kidney. Yeast two-hybrid and GST pull-down assays showed that the interaction between centaurin-alpha(1) and KIF13B is direct and mediated by the GAP domain of centaurin-alpha(1) and the stalk domain of KIF13B. Centaurin-alpha(1) and KIF13B form a complex in vivo and the KIF13B interaction appears to be specific to centaurin-alpha(1) as other members of the ARF GAP family did not show any binding activity. We also show that KIF13B and centaurin-alpha(1) colocalize at the leading edges of the cell periphery whereas a deletion mutant of centaurin-alpha(1) that lacks the KIF13B binding site, failed to colocalize with KIF13B in vivo. Finally, we demonstrate that KIF13B binding suppresses the ARF6 GAP activity of centaurin-alpha(1) in intact cells. Together, our data suggest a mechanism where direct binding between centaurin-alpha(1) and KIF13B could concentrate centaurin-alpha(1) at the leading edges of cells, thus modulating ARF6 function.
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PMID:Centaurin-alpha1 interacts directly with kinesin motor protein KIF13B. 1592 60

The human EVI5 protein carries a TBC domain indicative of Rab GTPase activating protein (GAP) activity, and an extensive coiled-coil motif in the C-terminal region. EVI5 is ubiquitously expressed in adult, fetal, and cancer tissues and exists as two mRNA species resulting from differential use of polyadenylation signals. Western blot analysis suggests that different molecular weight protein species are probably generated by posttranslational modification. FPLC analysis demonstrates that EVI5 protein can form dimers and confocal microscopy indicates that EVI5, in addition to a diffuse localization in the nucleus, also preferentially localizes to the pericentriolar material in interphase cells. Immunoprecipitation and GST pull-down experiments demonstrate that EVI5 exists in complexes with both alpha- and gamma-tubulin. Both interactions are localized to the N-terminal part of the EVI5 protein. Thus, EVI5 is a novel centrosomal protein with a complex expression pattern and subcellular localization, possibly involved in centrosome stability and dynamics.
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PMID:EVI5 is a novel centrosomal protein that binds to alpha- and gamma-tubulin. 1603 5

During endocytosis of the activated platelet-derived growth factor (PDGF) receptor, phosphatidylinositol 3-kinase (PI3K) remains associated with the receptor. We found that the p85 alpha subunit of PI3 kinase binds directly to Rab5 and possesses GTPase activating protein (GAP) activity toward Rab5. Rab5 is a small monomeric GTPase involved in regulating vesicle fusion events during receptor-mediated endocytosis. We used two methods to characterize the direct binding between Rab5 in various nucleotide-bound states and the p85 protein. In the first assay, the ability of p85 to bind to Rab5 is measured using an enzyme-linked immunosorbent assay (ELISA). The second assay is a glutathione S-transferase (GST) pull-down approach in which GST-Rab5 proteins in various nucleotide-bound states are allowed to bind p85. In both instances, bound p85 is detected using anti-p85 antibodies.
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PMID:Measurement of the interaction of the p85alpha subunit of phosphatidylinositol 3-kinase with Rab5. 1647 18

EhMLBP is an essential Entamoeba histolytica protein that binds preferentially to methylated long interspersed nuclear elements and rDNA. In an effort to identify more EhMLBP DNA substrates, we developed an affinity-based technique in which the C-terminal DNA binding domain of EhMLBP (GST-CterEhMLBP) was used as the ligand. Bioinformatic analysis of the DNA sequences that were isolated by this affinity method revealed the presence of a 29-nucleotide consensus motif that includes a stretch of ten adenines. Gel retardation analysis showed that EhMLBP binds to the consensus motif with a preference for its methylated form. Four DNA sequences, namely those that encoded either dihydrouridine synthetase, RAP GTPase activating protein, serine/threonine protein kinase or leucine-rich repeat containing protein (LRPP) were then selected for further analysis. In vivo binding of EhMLBP to these genes was confirmed by chromatin immunoprecipitation. The presence of methylated cytosines was detected in DNA encoding LRPP and to a lower extent in the other genes. EhMLBP binds preferentially to the methylated forms of these DNA targets. The ability of the consensus motif to compete with EhMLBP binding to its DNA substrates indicates that the adenine stretch is involved in the mechanism of DNA recognition. The results of this investigation extend our existing knowledge on the number of DNA sequences that are recognized by EhMLBP and reinforce the notion that this protein is an innate methylated DNA binding protein in E. histolytica.
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PMID:Insights into the mechanism of DNA recognition by the methylated LINE binding protein EhMLBP of Entamoeba histolytica. 1945 Jul 28

Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.
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PMID:An alteration in ELMOD3, an Arl2 GTPase-activating protein, is associated with hearing impairment in humans. 2403 9


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