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)

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP-80, have anchoring sites for cAMP-dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell-specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1-s (HK1-S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell-specific member of the mu-class glutathione S-transferase family (GSTM5) and an intermediate filament-like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida.
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PMID:Fibrous sheath of mammalian spermatozoa. 1267 26

Insulin stimulates glucose transport by promoting translocation of GLUT4 proteins from the perinuclear compartment to the cell surface. It has been previously suggested that the microtubule-associated motor protein kinesin, which transports cargo toward the plus end of microtubules, plays a role in translocating GLUT4 vesicles to the cell surface. In this study, we investigated the role of Rab4, a small GTPase-binding protein, and the motor protein KIF3 (kinesin II in mice) in insulin-induced GLUT4 exocytosis in 3T3-L1 adipocytes. Photoaffinity labeling of Rab4 with [gamma-(32)P]GTP-azidoanilide showed that insulin stimulated Rab4 GTP loading and that this insulin effect was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 or expression of dominant-negative protein kinase C-lambda (PKC-lambda). Consistent with previous reports, expression of dominant-negative Rab4 (N121I) decreased insulin-induced GLUT4 translocation by 45%. Microinjection of an anti-KIF3 antibody into 3T3-L1 adipocytes decreased insulin-induced GLUT4 exocytosis by 65% but had no effect on endocytosis. Coimmunoprecipitation experiments showed that Rab4, but not Rab5, physically associated with KIF3, and this was confirmed by showing in vitro association using glutathione S-transferase-Rab4. A microtubule capture assay demonstrated that insulin stimulation increased the activity for the binding of KIF3 to microtubules and that this activation was inhibited by pretreatment with the PI3-kinase inhibitor LY294002 or expression of dominant-negative PKC-lambda. Taken together, these data indicate that (i) insulin signaling stimulates Rab4 activity, the association of Rab4 with kinesin, and the interaction of KIF3 with microtubules and (ii) this process is mediated by insulin-induced PI3-kinase-dependent PKC-lambda activation and participates in GLUT4 exocytosis in 3T3-L1 adipocytes.
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PMID:Insulin-induced GLUT4 translocation involves protein kinase C-lambda-mediated functional coupling between Rab4 and the motor protein kinesin. 1283 75

The ADP-ribosylation factor 6 (ARF6) small GTPase functions as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganization and membrane ruffling. The formation of active ARF6GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ARF6 activation. Using cytohesin 2 as bait in yeast two-hybrid screening, we have isolated a cDNA encoding a protein termed interaction protein for cytohesin exchange factors 1 (IPCEF1). Using yeast two-hybrid and glutathione S-transferase pull-down assays coupled with deletion mutational analysis, the specific domains required for the cytohesin 2-IPCEF1 interaction were mapped to the coiled-coil domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1. IPCEF1 also interacts with the other members of the cytohesin family of ARF GEFs, suggesting that the interaction with IPCEF1 is highly conserved among the cytohesin family of ARF GEFs. The interaction of cytohesin 2 and IPCEF1 in mammalian cells was demonstrated by immunoprecipitation. Immunofluorescence analysis revealed that IPCEF1 co-localizes with cytohesin 2 to the cytosol in unstimulated cells and translocates to the plasma membrane via binding to cytohesin 2 in epidermal growth factor-stimulated cells. However, a deletion mutant of IPCEF1 that lacks the cytohesin 2 binding site failed to co-migrate with cytohesin 2 to the membrane in stimulated cells. The functional significance of the IPCEF1-cytohesin 2 interaction is demonstrated by showing that IPCEF1 increases the in vitro and in vivo stimulation of ARFGTP formation by cytohesin 2.
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PMID:Interaction protein for cytohesin exchange factors 1 (IPCEF1) binds cytohesin 2 and modifies its activity. 1292 Jan 29

Nonreceptor tyrosine kinase Abl is an actin-binding protein and a key regulator of neuronal axonal development. Although Abl family kinases also are localized in dendrites and are implicated in postsynaptic functions, it is not clear how Abl kinases regulate dendritic morphogenesis. Using a developing hippocampal culture as a model, we found that the inhibition of Abl kinases by STI571 leads to a remarkable simplification of dendritic branching similar to the phenotype caused by an increased activity of small GTPase RhoA. Time-lapse microscopic imaging reveals a prominent reduction of dendritic branching. In contrast, neurons expressing a constitutively active v-abl construct (CA-Abl) show an exuberant microtubule-associated protein 2-positive (MAP2-positive) dendrite outgrowth, suggesting that Abl modulates dendritic growth. Biochemical assays using a glutathione S-transferase pull-down method to determine GTP-bound active Rho GTPases demonstrate that Abl inhibition increases RhoA activity but has no effect on the activity of Rac1 or Cdc42. At the cellular level the alteration of Abl also changes actin organization consistent with RhoA inhibition. Suppression of the RhoA downstream effector Rho kinase reverses STI571-induced dendritic simplification, demonstrating that activity of the Rho pathway is responsible for the Abl-induced changes in dendrogenesis. Furthermore, CA-Abl-induced neurite outgrowth is blocked by the expression of a constitutively active RhoA construct. The CA-Abl phenotype is not affected by destabilization of microtubules but is reversed partially when actin filaments are stabilized with jasplakinolide. Together, these studies support a critical role for Abl kinases in regulating dendrogenesis by inducing actin cytoskeletal rearrangements in cooperation with Rho GTPases.
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PMID:Abl tyrosine kinase promotes dendrogenesis by inducing actin cytoskeletal rearrangements in cooperation with Rho family small GTPases in hippocampal neurons. 1545 25

Although the causal relationship between chronic inflammation and carcinogenesis has long been discussed, the molecular basis of the relation is poorly understood. In the present study, we focused on reactive oxygen species (ROS) and their signals under inflammatory conditions leading to the carcinogenesis of epithelial cells and found that repeated treatment with a low dose of H(2)O(2) (0.2 mmol/L) for periods of 2 to 4 days caused a phenotypic conversion of mouse NMuMG mammary epithelial cells from epithelial to fibroblast-like as in malignant transformation. The phenotypic conversion included the dissolution of cell-cell contacts, redistribution of E-cadherin in the cytoplasm, and up-regulation of a set of integrin family members (integrin alpha2, alpha6, and beta3) and matrix metalloproteinases (MMPs; MMP-3, -10, and -13), as analyzed using Northern blot analysis and quantitative reverse transcription-PCR. Gelatin zymography indicated post-transcriptional activation of gelatinases, including MMP-2 and -9. In parallel, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 were activated, which contributed to the induction of MMP-13, and a glutathione S-transferase pull-down assay showed the activation of a small GTPase, Rac1. Surprisingly, the prolonged oxidative treatment was sufficient to induce all of the aforementioned events. Most importantly, depending on the MMP activities, the epithelial cells exposed to oxidative conditions eventually acquired invasiveness in a reconstituted model system with a Matrigel invasion chamber containing normal fibroblasts at the bottom, providing the first substantial evidence supporting the direct role of ROS signals in the malignant transformation of epithelial cells.
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PMID:Invasive potential induced under long-term oxidative stress in mammary epithelial cells. 1549 71

Rabaptin-5 is an effector for the small GTPase Rab5, a regulator of the early steps in endocytosis. In addition, Rabaptin-5 interacts with the small GTPase Rab4 that has been implicated in recycling from early endosomes to the cell surface. Recently we have identified a ubiquitous transcript encoding the Rabaptin-5 isoform, Rabaptin-5delta. To evaluate the interaction properties of Rabaptin-5delta with the small GTPases Rab4 and Rab5, we have applied protein interaction assays using the yeast two-hybrid system and a glutathione S-transferase pull-down assay. We found that unlike Rabaptin-5, that interacts with both GTPases in GTP-bound conformations, Rabaptin-5delta interacts only with GTP-bound Rab5, and does not interact with Rab4, presumably due to a disrupted Rab4 binding site. Immunofluorescence microscopy analysis carried out to address the localization of Rabaptin-5delta relative to GTP-bound Rab4 and Rab5 in BHK-21 cells supported these data. Our data suggests that while Rabaptin-5 was proposed to act as a molecular linker between Rab5 and Rab4, to coordinate endocytic and recycling traffic, Rabaptin-5delta is involved only in the Rab5-driven events.
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PMID:The Rab5 effector Rabaptin-5 and its isoform Rabaptin-5delta differ in their ability to interact with the small GTPase Rab4. 1563 30

The small GTPase rab1 plays a role in vesicle trafficking between ER and the Golgi complex. Recently, MICAL-1 was identified as new rab1 interacting protein. In this study, we show an interaction between two additional members of the MICAL family and rab1 in a yeast two-hybrid approach and GST pulldown experiments. We present data about the previously uncharacterized MICAL-3 concerning tissue distribution, size, and cellular localization. Furthermore, we investigated the connection between MICAL proteins and the cytoskeleton. Using the microtubule depolymerizing drug nocodazole we detected a link between MICAL-1 and -3, and the microtubule cytoskeleton.
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PMID:The MICAL proteins and rab1: a possible link to the cytoskeleton? 1569 64

Our initial characterization of Rac3, a close relative of the small GTPase Rac1, established its ability to promote membrane ruffling, transformation, and activation of c-jun transcriptional activity. The finding that Rac3 is transforming, and its similarity to Rac1, a protein that has a well-established connection to many processes important for cancer progression, prompted further investigation into Rac3 transformation. We used effector domain mutants (EDMs) to explore the relationship among Rac signaling, transformation, and effector usage. All Rac3 EDMs tested (N26D, F37L, Y40C, and N43D) retained the ability to promote membrane ruffling and focus formation. In contrast, only the N43D mutant promoted anchorage independence. This differs from Rac1, where both N26D and N43D mutants were impaired in both types of transformation. To learn more about the signaling pathways involved, we did luciferase reporter assays and glutathione S-transferase pull-down assays for effector binding. We found evidence for a functional link between activation of phospholipase Cbeta2 by Rac3 and signaling to the serum response factor (SRF). Surprisingly, we also found that Rac3 binds poorly to the known Rac1 effectors mixed lineage kinases 2 and 3 (MLK2 and MLK3). Transcription of cyclin D1 was the only pathway that correlated with growth in soft agar. Our experiments show that activation of membrane ruffling and transcriptional activation of c-jun, SRF, or E2F are not sufficient to promote anchorage-independent growth mediated by Rac3. Instead, multiple effector pathways are required for Rac3 transformation, and these overlap partially but not completely with those used by Rac1.
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PMID:Rac3-mediated transformation requires multiple effector pathways. 1626 12

Hypoxia is a crucial factor in tumor aggressiveness and resistance to treatment, particularly in glioma. Our previous results have shown that inhibiting the small GTPase RhoB increased oxygenation of U87 human glioblastoma xenografts, in part, by regulating angiogenesis. We investigated here whether RhoB might also control a signaling pathway that would permit glioma cells to adapt to hypoxia. We first showed that silencing RhoB with siRNA induced degradation and inhibition of the transcriptional activity of the hypoxia-inducible factor by the proteasome in U87 hypoxic cells. This RhoB-dependent degradation of hypoxia-inducible factor-1alpha in hypoxic conditions was mediated by the Akt/glycogen synthase kinase-3beta pathway. While investigating how hypoxia could activate this signaling pathway, using the GST-Rhotekin RBD pulldown assay, we showed the early activation of RhoB by reactive oxygen species under hypoxic conditions and, subsequently, its participation in the ensuing cellular adaptation to hypoxia. Overall, therefore, our results have not only highlighted a new signaling pathway for hypoxia controlled by the small GTPase RhoB, but they also strongly implicate RhoB as a potentially important therapeutic target for decreasing tumor hypoxia.
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PMID:Activation of RhoB by hypoxia controls hypoxia-inducible factor-1alpha stabilization through glycogen synthase kinase-3 in U87 glioblastoma cells. 1639 64

In this chapter, we describe various approaches that allow us to study interactions between the small GTPase Rab4a and its two effectors, Rabip4 and CD2AP. Two complementary approaches, one using the yeast two-hybrid system and the other using a GST pull-down assay, are described. We document the studies of the localization of these proteins by cellular fractionation. Finally, we develop cellular imaging techniques to study the morphology of vesicular structures containing Rab4a. We show that the coexpression of Rab4a with its effectors affects Rab4a-containing structures, giving a clear indication of their interaction in the mammalian cellular context.
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PMID:CD2AP, Rabip4, and Rabip4': analysis of interaction with Rab4a and regulation of endosomes morphology. 1647 81


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