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)

We have cloned a protein tyrosine kinase, MATK, which is expressed abundantly in megakaryocytes and the brain. We investigated whether MATK participates in the c-Kit ligand/stem cell factor (KL/SCF) signaling pathway in the megakaryocytic cell line CMK. After KL/SCF stimulation, five major proteins of molecular masses of 145, 113, 92, 76, and 63 kDa were rapidly and transiently tyrosine-phosphorylated in a time-dependent manner, peaking within 5 min, and returning to basal levels within 60 min. To study the role of MATK in the KL/SCF signaling pathway, glutathione S-transferase (GST) fusion proteins containing SH2 and SH3 domains of MATK were cloned, expressed in Escherichia coli, and purified. MATK-SH2, but not MATK-SH3, precipitated the tyrosine-phosphorylated c-Kit (molecular mass of 145 kDa) in KL/SCF-stimulated CMK cells. Other GST fusion proteins containing the SH2 domain of p85 of phosphatidylinositol 3-kinase, phospholipase C gamma-1, and ras-GAP also precipitated c-Kit. The tyrosine-phosphorylated c-Kit was co-immunoprecipitated with anti-MATK and anti-p85 antibodies in KL/SCF-stimulated CMK cells, but not in granulocyte-macrophage colony stimulating factor or interleukin-6-stimulated cells, suggesting receptor specificity. These results indicate that MATK associates with the c-Kit receptor following specific stimulation by KL/SCF via its SH2 domain and likely participates in transduction of growth signals induced by this cytokine in megakaryocytes.
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PMID:The MATK tyrosine kinase interacts in a specific and SH2-dependent manner with c-Kit. 753 44

Stem cell factor (SCF) is a cytokine critical for normal hematopoiesis. The receptor for SCF is c-Kit, a receptor tyrosine kinase. Our laboratory is interested in delineating critical components of the SCF signal transduction pathway in hematopoietic tissue. The present study examines activation of Src family members in response to SCF. Stimulation of cell lines as well as normal progenitor cells with SCF rapidly increased tyrosine phosphorylation of the Src family member Lyn. Peak responses were noted 10-20 min after SCF treatment, and phosphorylation of Lyn returned to basal levels 60-90 min after stimulation. SCF also induced increases in Lyn kinase activity in vitro. Lyn coimmunoprecipitated with c-Kit, and studies with GST fusion proteins demonstrated that Lyn readily associated with the juxtamembrane region of c-Kit. Treatment of cells with either Lyn antisense oligonucleotides or PP1, a Src family inhibitor, resulted in dramatic inhibition of SCF-induced proliferation. These data demonstrate that SCF rapidly activates Lyn and suggest that Lyn is critical in SCF-induced proliferation in hematopoietic cells.
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PMID:Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells. 934 Nov 98

Interaction of stem cell factor (SCF), a haematopoietic growth factor, with the receptor tyrosine kinase c-kit leads to autophosphorylation of c-kit as well as tyrosine phosphorylation of various substrates. Little is known about the role of the JAK/STAT pathway in signal transduction via receptor tyrosine kinases, although this pathway has been well characterized in cytokine receptor signal transduction. We recently found that the Janus kinase Jak2 associates with c-kit and that SCF induces rapid and transient phosphorylation of Jak2. Here we present evidence that SCF activates the transcription factor Stat1. Phosphorylated c-kit co-immunoprecipitates with Stat1 within 1 min of SCF stimulation of the human cell line MO7e. Co-precipitation experiments using glutathione S-transferase fusion proteins indicate that association with c-kit is mediated by the Stat1 SH2 domain. Stat1 is rapidly tyrosine-phosphorylated in response to SCF in MO7e cells, the murine cell line FDCP-1 and normal progenitor cells. SCF-induced phosphorylation of Jak2 and Stat1 was also observed in murine 3T3 fibroblasts stably transfected with full-length human c-kit receptor. Furthermore c-kit directly phosphorylates Stat1 fusion proteins in in vitro kinase assays. Electrophoretic mobility-shift assays with nuclear extracts from SCF-stimulated cell lines and normal progenitor cells indicate that activated Stat1 binds the m67 oligonucleotide, a high-affinity SIE promoter sequence. These results demonstrate that Stat1 is activated in response to SCF, and suggest that Stat1 is a component of the SCF signal-transduction pathway.
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PMID:Stat1 associates with c-kit and is activated in response to stem cell factor. 935 37

To protect bone marrow cells from the toxicity of chemotherapy, a multidrug resistant gene or a dihydrofolate reductase gene has been introduced into stem cells. These genes, however, are not capable of conferring refractoriness to alkylating agents (AA), which are some of the most commonly used agents in chemotherapy regimens. In the present study, an attempt was made to endow human stem cell (CD34+ cells) with resistance to cyclophosphamide, a well-known AA, and adriamycin (ADM) by transducing the glutathione-S-transferase pi (GST-pi) gene whose product is thought to detoxify AA by conjugating them with glutathione and to remove a toxic peroxide formed by ADM. The gene transduction was carried out retrovirally with a virus titer of 1 x 10(5) FFU/ml, employing a recombinant fibronectin fragment; transduction efficiency was extremely low without the fragment. Incubation with interleukin-6 and stem cell factor enhanced the expression of fibronectin ligands VLA4 and VLA5 on CD34+ cells. This enhanced expression of VLA4 and VLA5 was considered to facilitate a close contact of the CD34+ cell to the retroviral vector via fibronectin fragments and the subsequent transduction process. The GST-pi gene-transduced CD34+ cells formed almost 3- and 2.5-fold more CFU-GM than neo gene-transduced CD34+ cells in the presence of 2.5 microg/ml of 4-hydroperoxycyclophosphamide (4-HC), an active form of cyclophosphamide, and 30 ng/ml ADM, respectively. The transfectants formed an appreciable number of colonies, even at higher concentrations of these drugs (5.0 microg/ml of 4-HC, 50 ng/ml of ADM) whereas neo gene-transduced or nontransduced CD34+ cells formed no colonies at all, indicating the possibility of selecting out the transfectants by exposing them to these anticancer drugs. Thus, we were able to demonstrate that transduction of the GST-pi gene confers resistance to cyclophosphamide as well as to ADM, and therefore this approach can be applied clinically for high-dose chemotherapy.
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PMID:Fibronectin fragment-facilitated retroviral transfer of the glutathione-S-transferase pi gene into CD34+ cells to protect them against alkylating agents. 938 56

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit-SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3-c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation.
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PMID:SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain. 952 81

Hematopoietic progenitor cells from Fanconi anemia (FA) group C (FA-C) patients display hypersensitivity to the apoptotic effects of gamma interferon (IFN-gamma) and constitutively express a variety of IFN-dependent genes. Paradoxically, however, STAT1 activation is suppressed in IFN-stimulated FA cells, an abnormality corrected by transduction of normal FANCC cDNA. We therefore sought to define the specific role of FANCC protein in signal transduction through receptors that activate STAT1. Expression and phosphorylation of IFN-gamma receptor alpha chain (IFN-gammaRalpha) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in coimmunoprecipitation experiments STAT1 did not dock at the IFN-gammaR of FA-C cells, an abnormality corrected by transduction of the FANCC gene. In addition, glutathione S-transferase fusion genes encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of IFN-gamma-stimulated B cells and IFN-, granulocyte-macrophage colony-stimulating factor- and stem cell factor-stimulated MO7e cells. Kinetic studies revealed that the initial binding of FANCC was to nonphosphorylated STAT1 but that subsequently the complex moved to the receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and STAT1-DNA complexes were not detected in nuclear extracts of FA-C cells. We also determined that the IFN-gamma hypersensitivity of FA-C hematopoietic progenitor cells does not derive from STAT1 activation defects because granulocyte-macrophage CFU and erythroid burst-forming units from STAT1(-/-) mice were resistant to IFN-gamma. However, BFU-E responses to SCF and erythropoietin were suppressed in STAT(-/-) mice. Consequently, because the FANCC protein is involved in the activation of STAT1 through receptors for at least three hematopoietic growth and survival factor molecules, we reason that FA-C hematopoietic cells are excessively apoptotic because of an imbalance between survival cues (owing to a failure of STAT1 activation in FA-C cells) and apoptotic and mitogenic inhibitory cues (constitutively activated in FA-C cells in a STAT1-independent fashion).
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PMID:The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors. 1084 98

Paclitaxel and carboplatin chemotherapy is reported to be a platelet-sparing drug combination. This study investigated potential mechanisms for this observation by studying the effects of paclitaxel and carboplatin on (1) normal donor and chemotherapy patient-derived erythroid (burst-forming units-erythroid [BFU-E]), myeloid (colony-forming units-granulocyte/macrophage [CFU-GM]), and megakaryocyte (CFU-Meg) progenitor cell growth; (2) P-glycoprotein (P-gp) protein and glutathione S-transferase (GST) messenger RNA (mRNA) expression; (3) serum thrombopoietin (Tpo), stem cell factor (SCF), interleukin-6 (IL-6), IL-11, IL-1beta, IL-8, and tumor necrosis factor-alpha levels in patients treated with paclitaxel and carboplatin; and (4) stromal cell production of Tpo and SCF after paclitaxel and carboplatin exposure. CFU-Meg were more resistant to paclitaxel alone, or in combination with carboplatin, than CFU-GM and BFU-E. Although all progenitors expressed P-gp protein and GST mRNA, verapamil treatment significantly, and selectively, increased the toxicity of paclitaxel and carboplatin to CFU-Meg, suggesting an important role for P-gp in megakaryocyte drug resistance. Compared to normal controls, serum Tpo levels in patients receiving paclitaxel and carboplatin were significantly elevated 5 hours after infusion and remained elevated at day 7 (287% +/- 63% increase, P <.001). Marrow stroma was shown to be the likely source of this Tpo. It is concluded here that P-gp-mediated efflux of paclitaxel, and perhaps GST-mediated detoxification of carboplatin, results in relative sparing of CFU-Meg, which may then respond to locally high levels of stromal cell-derived Tpo. The confluence of these events might lead to the platelet-sparing phenomenon observed in patients treated with paclitaxel and carboplatin chemotherapy.
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PMID:Investigating the platelet-sparing mechanism of paclitaxel/carboplatin combination chemotherapy. 1115 79

Macrophage colony-stimulating factor receptor (M-CSF-R) is a tyrosine kinase that regulates proliferation, differentiation, and cell survival during monocytic lineage development. Upon activation, M-CSF-R dimerizes and autophosphorylates on specific tyrosines, creating binding sites for several cytoplasmic SH2-containing signaling molecules that relay and modulate the M-CSF signal. Here we show that M-CSF-R interacts with suppressor of cytokine signaling 1 (Socs1), a negative regulator of various cytokine and growth factor signaling pathways. Using the yeast two-hybrid system, in vitro glutathione S-transferase-M-CSF-R pull-down, and in vivo coimmunoprecipitation experiments, we demonstrated a direct interaction between the SH2 domain of Socs1 and phosphorylated tyrosines 697 or 721 of the M-CSF-R kinase insert region. Moreover, Socs1 is tyrosine-phosphorylated in response to M-CSF. Ectopic expression of Socs1 in FDC-P1/MAC and EML hematopoietic cell lines decreased their growth rates in the presence of limiting concentrations of M-CSF. However, Socs1 expression did not totally suppress long term cell growth in the presence of saturating M-CSF concentrations, in contrast to other cytokines such as stem cell factor and interleukin 3. Taken together, these results suggest that Socs1 is an M-CSF-R-binding partner involved in negative regulation of proliferation signaling and that it differentially affects cytokine receptor signals.
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PMID:Suppressor of cytokine signaling 1 interacts with the macrophage colony-stimulating factor receptor and negatively regulates its proliferation signal. 1129 60

The mammalian forkhead transcription factors, FOXO3a (FKHRL1), FOXO1a (FKHR) and FOXO4 (AFX) are negatively regulated by PKB/Akt kinase. In the present study we examined the engagement of forkhead family of transcription factors in erythropoietin (Epo)- and stem cell factor (SCF)-mediated signal transduction. Our data show that all three forkhead family members, FOXO3a, FOXO1a and FOXO4 are phosphorylated in human primary erythroid progenitors. Experiments performed to determine various upstream signaling pathways contributing to phosphorylation of forkhead family members show that only PI-3-kinase pathway is required for inactivation of FOXO3a. Our data also demonstrate that during Epo deprivation FOXO3a interacts with the transcriptional coactivator p300 and such interaction is disrupted by stimulation of cells with Epo. To determine the domains in FOXO3a, mediating its interaction with p300, we performed GST pull-down assays and found that the N-terminus region containing the first 52 amino acids was sufficient for binding p300. Finally, our data demonstrate that FOXO3a and FOXO1a are acetylated during growth factor deprivation and such acetylation is reversed by stimulation with Epo. Thus mammalian forkhead transcription factors are involved in Epo and SCF signaling in primary erythroid progenitors and may play a role in the induction of apoptotic and mitogenic signals.
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PMID:Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells. 1189 84

The adaptor protein Lnk is expressed in haemopoietic cells and plays a critical role in haemopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in haemopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including an SH2 (Src homology 2) domain which binds phosphotyrosine residues in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors [e.g. Mpl, EpoR (erythropoietin receptor), c-Kit]. Therefore inhibition of the binding of Lnk to cytokine receptors might lead to enhanced downstream signalling of the receptor and thereby to improved haemopoiesis in response to exposure to cytokines (e.g. erythropoietin in anaemic patients). This hypothesis led us to define the exact binding site of Lnk to the stem cell factor receptor c-Kit. Pull-down experiments using GST (glutathione transferase)-fusion proteins of the different domains of c-Kit showed that Lnk almost exclusively binds to the phosphorylated juxtamembrane domain. Binding of Lnk to the juxtamembrane domain was abolished by point mutation of Tyr(568) and was competed by peptides with a phosphotyrosine residue at position 568. Co-immunoprecipitation with full-length wild-type or Y568F mutant c-Kit and Lnk confirmed these results, thus showing the importance of this phosphorylated tyrosine residue. Lnk bound directly to c-Kit without requiring other interacting partners. The identification of the binding site of Lnk to c-Kit will be useful to discover inhibitory molecules that prevent the binding of these two proteins, thus making haemopoietic cells more sensitive to growth factors.
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PMID:Adaptor protein Lnk associates with Tyr(568) in c-Kit. 1858 18


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