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)

Tumoral biological markers of breast cancer expand the predictive value of conventional prognostic factors, such as tumor size, axillary lymph node status, and histoprognostic grade. They include tumor estrogen and progesterone receptor levels, flow cytometric DNA analysis, to convey a prognostic value. Expression of the product of the gene pS2, which reflects the functional integrity of the estradiol receptor, indicates a good prognosis. In contrast, presence of growth factor receptors, such as the EGF receptor, or amplification of the HER2/neu or INT2 oncogene indicate a poor prognosis. Study of protein gp 170 and GST-pi predicts the response of tumors to chemotherapy, while the study of the potential doubling time (Tpot) provides an indication of the renewal capacity of the tumor. Markers of tumor invasiveness and metastatic potential include proteases (activators and inhibitors) produced either by tumor cells or by the cells of the stroma, gene nm 23, and membrane fatty acids. The place of the last markers in patients' treatment is not known yet. The knowledge of the tumor biological parameters along with clinical features should provide an accurate prediction of the aggressiveness of the tumor, allowing the best adjustment of treatment with the expected behavior of the disease.
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PMID:[Intratumoral biological markers in breast cancers]. 148 91

The 21 kDa Ras proteins are well known for their regulatory role in oncogenic, mitogenic, and developmental signaling pathways. Less well understood are the downstream signal transduction cascades initiated by Ras in response to external stimuli. Only recently have many diverse studies in lower eukaryotes and vertebrates converged to demonstrate that Ras directly regulates multiple signaling pathways. In most eukaryotes, Ras functions as a positive regulator of an ERK/MAPK signal transduction cascade through the activation of a MEKK. In mammalian cells the primary Ras-responsive MEKK is the protein kinase Raf. Although Raf remains the most significant mediator of Ras signaling in most model systems, it does not explain all the biochemical responses observed in cells with activated Ras proteins. Yeast two hybrid and GST-fusion protein binding studies have identified new proteins distinct from Raf that could interact with Ras in other signaling pathways. In addition to Raf, other potential Ras target proteins include MEKK1, PI(3)K, p120GAP, ralGDS, and PKC zeta. This review will attempt to summarize the current literature of accepted and potential Ras-dependent signaling proteins in both lower eukaryotes and vertebrates.
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PMID:Ras target proteins in eukaryotic cells. 755 21

The beta gamma subunits of heterotrimeric G proteins (G beta gamma) play a variety of roles in cellular signaling, one of which is membrane targeting of the beta-adrenergic receptor kinase (beta ARK). This is accomplished via a physical interaction of G beta gamma and a domain within the carboxyl terminus of beta ARK which overlaps with a pleckstrin homology (PH) domain. The PH domain of beta ARK not only binds G beta gamma but also interacts with phosphatidylinositol 4,5-bisphosphate (PIP2). Based on previous mapping of the G beta gamma binding region of beta ARK, and conserved residues within the PH domain, we have constructed a series of mutants in the carboxyl terminus of beta ARK in order to determine important residues involved in G beta gamma and PIP2 binding. To examine the effects of mutations on G beta gamma binding, we employed three different methodologies: direct G beta gamma binding to GST fusion proteins; the ability of GST fusion proteins to inhibit G beta gamma-mediated beta ARK translocation to rhodopsin-enriched rod outer segments; and the ability of mutant peptides expressed in cells to inhibit G beta gamma-mediated inositol phosphate accumulation. Direct PIP2 binding was also assessed on mutant GST fusion proteins. Ala residue insertion following Trp643 completely abolished the ability of beta ARK to bind G beta gamma, suggesting that a proper alpha-helical conformation is necessary for the G beta gamma.beta ARK interaction. In contrast, this insertional mutation had no effect on PIP2 binding. Both G beta gamma binding and PIP2 binding were abolished following Ala replacement of Trp643, suggesting that this conserved residue within the last subdomain of the PH domain is crucial for both interactions. Other mutations also produced differential effects on the physical interactions of the beta ARK carboxyl terminus with G beta gamma and PIP2. These results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of beta ARK, including Trp643, Leu647, and residues Lys663-Arg669, are critical for G beta gamma binding while Trp643 and residues Asp635-Glu639 are important for the PH domain to form the correct structure for binding to PIP2.
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PMID:Mutational analysis of the pleckstrin homology domain of the beta-adrenergic receptor kinase. Differential effects on G beta gamma and phosphatidylinositol 4,5-bisphosphate binding. 762 21

Ret is a receptor protein tyrosine kinase that has been implicated in the development of the enteric nervous, endocrine, and renal systems. Mutations associated with multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B) have been shown to activate the intrinsic kinase and transforming ability of ret (Santoro, M., Carlomagno, F., Romano, A., Bottaro, D. P., Dathan, N. A., Grieco, M., Fusco, A., Vecchio, G., Matoskova, B., Kraus, M. H., and Paolo DiFiore, P. (1995) Science 267, 381-383). Using the cytoplasmic domain of Ret as bait in a yeast two-hybrid screen of a mouse embryonic library, it was discovered that the src homology 2 (SH2) domain containing protein Grb10 bound Ret. Grb10 belongs to an emerging family of SH2 containing adapter proteins, the prototypical member being Grb7. Using glutathione S-transferase fusion proteins, it was demonstrated that the SH2 domain of Grb10 specifically interacted with Ret. Additionally, using an EGFR/Ret chimera, it was shown that Grb10 bound Ret in an activation dependent manner in vivo. This is the first description of a receptor protein tyrosine kinase that utilizes Grb10 as a signaling intermediate.
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PMID:The Ret receptor protein tyrosine kinase associates with the SH2-containing adapter protein Grb10. 766 56

The c-fes proto-oncogene product is expressed predominantly in hematopoietic cells of the myeloid lineage and has been implicated in the regulation of myeloid differentiation. The c-fes locus encodes a 93-kDa protein tyrosine kinase (p93c-fes) that possesses several structural features characteristic of the cytoplasmic class of protein tyrosine kinases, including a consensus sequence for autophosphorylation surrounding Tyr-713 and a src homology 2 (SH2) domain. To assess the effect of each of these potential regulatory sites on p93c-fes protein tyrosine kinase activity, we specifically deleted the c-fes SH2 domain using the polymerase chain reaction and replaced Tyr-713 with phenylalanine by oligonucleotide-directed mutagenesis (Y713F mutant). The resulting mutants were expressed in Escherichia coli and assayed for changes in protein tyrosine kinase activity using an immune complex kinase assay. Both mutations produced a marked decrease in the rate and extent of autophosphorylation and phosphorylation of the model substrate, enolase. To test whether the c-fes SH2 domain could interact with the autophosphorylated kinase domain, the SH2 domain was expressed as a fusion protein with glutathione S-transferase and immobilized on glutathione-agarose. The recombinant c-fes SH2 domain precipitated p93c-fes as readily as a monoclonal antibody. Binding of the SH2 domain to p93c-fes was completely dependent upon autophosphorylation, as a kinase-defective mutant of p93c-fes was not precipitated by the SH2 domain. High-affinity binding was also observed with recombinant SH2 domains from v-src and v-fps, raising the possibility of protein-protein interactions between various members of the cytoplasmic PTK family. These results indicate that the c-fes SH2 domain and consensus autophosphorylation site (Tyr-713) play major roles in the positive regulation of p93c-fes tyrosine kinase activity, possibly through intramolecular interaction.
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PMID:Regulation of the human c-fes protein tyrosine kinase (p93c-fes) by its src homology 2 domain and major autophosphorylation site (Tyr-713). 768 63

Antigenic cross-linking of the high affinity IgE receptor (Fc epsilon R1) on mast cells results in protein tyrosine kinase activation. The object of the present study was to explore the regulation of the SH2 and SH3 domain containing adapter molecule Grb2 by Fc epsilon R1-stimulated PTK signal transduction pathways. Affinity purification of in vivo Grb2 complexes together with in vitro experiments with Grb2 glutathione S-transferase fusion proteins were used to analyze Grb2 complexes in the mast cell line RBL2H3. The data show that in RBL2H3 cells several different proteins are complexed to the SH3 domains of Grb2. These include the p21ras guanine nucleotide exchange factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for Fc epsilon R1-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins which suggests that Grb2 may be a pleiotropic adapter. Two Grb2 SH2-binding proteins were also characterized in RBL2H3 cells; the adapter Shc and a 33-kDa molecule. Shc is constitutively tyrosine phosphorylated in unstimulated cells and Fc epsilon R1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for Fc epsilon R1-activated PTKs and binds to Grb2 SH2 domains in Fc epsilon R1 activated but not quiescent cells. The beta subunit of the Fc epsilon R1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2-binding protein described in the present report is not the Fc epsilon R1 beta chain and its identity is unknown. The present report thus demonstrates that there are multiple Grb2 containing protein complexes in mast cells of which a subset are Fc epsilon R1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to Fc epsilon R1 ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain. We propose that protein complex formation by Grb2 is an important consequence of Fc epsilon R1 cross-linking and that this may be a signal transduction pathway which acts synergistically with calcium/PKC signals to bring about optimal mast cell end function.
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PMID:Regulation of the adapter molecule Grb2 by the Fc epsilon R1 in the mast cell line RBL2H3. 772 78

Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.
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PMID:A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase. 778 77

G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.
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PMID:Lipid-mediated regulation of G protein-coupled receptor kinases 2 and 3. 789 Jul 2

The ETS domain family of transcription factors is comprised of several important proteins that are involved in controlling key cellular events such as proliferation, differentiation, and development. One such protein, Elk-1, regulates the activity of the c-fos promoter in response to extracellular stimuli. Elk-1 is representative of a subgroup of ETS domain proteins that utilize a bipartite recognition mechanism that is mediated by both protein-DNA and protein-protein interactions. In this study, we have overexpressed, purified, and characterized the ETS DNA-binding domain of Elk-1 (Elk-93). Elk-93 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and purified to homogeneity from both the soluble and insoluble fractions using a two-column protocol. A combination of CD, NMR, and fluorescence spectroscopy demonstrates that Elk-93 represents an independently folded domain of mixed alpha/beta structure in which the three conserved tryptophans appear to contribute to the hydrophobic core of the protein. Moreover, DNA binding studies demonstrate that Elk-93 binds DNA with both high affinity (Kd approximately 0.85 x 10(-10)M) and specificity. Circular permutation analysis indicates that DNA binding by Elk-93 does not induce significant bending of the DNA. Our results are discussed with respect to predictive models for the structure of the ETS DNA-binding domain.
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PMID:Characterization of the Elk-1 ETS DNA-binding domain. 789 Jul 10

Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.
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PMID:[Regulation of G protein-coupled receptor kinase activity]. 795 13


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