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 beta spectrin genes each produce two alternate transcripts the longer of which has a approximately 210 amino acid C-terminal extension including a pleckstrin homology (PH) domain and also an uncharacterized membrane binding site. GST constructs including the entire or the N-terminal segment of the beta I sigma II spectrin PH domain bind to crude and extracted brain membranes, to protein free brain lipid and to vesicles containing phosphatidylinositol-4,5-bisphosphate. This PH domain also binds radiolabelled inositol-1,4,5-trisphosphate (IP3) and preincubation with IP3 inhibits binding to extracted brain membranes. We conclude that membrane binding of the beta I sigma II spectrin C-terminal region is by means of a direct interaction between the N-terminal region of the PH domain and membrane lipids and does not require membrane protein. The PH domain of the beta-adrenergic receptor kinase showed different binding properties in every assay employed, showing that different PH domains may have different membrane binding specificity.
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PMID:The association of the C-terminal region of beta I sigma II spectrin to brain membranes is mediated by a PH domain, does not require membrane proteins, and coincides with a inositol-1,4,5 triphosphate binding site. 750 42

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

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

Although a role for the beta gamma-subunits of heterotrimeric G proteins (G beta gamma) in signal transduction by several cellular systems has been established, the structural features of cellular proteins interacting with G beta gamma have yet to be fully elucidated. The G beta gamma-binding region of beta-adrenergic receptor kinase (beta ARK), a cytosolic enzyme recruited to the membrane receptor substrate by G beta gamma, has been localized to the carboxyl terminus of the enzyme. Here, we demonstrate that the amino terminus of phosducin, a 33-kDa G beta gamma-binding retinal phosphoprotein, contains sequences homologous with the G beta gamma-binding domain of beta ARK. Accordingly, a glutathione S-transferase-fusion protein containing only the amino-terminal 105 amino acids of phosducin displayed G beta gamma binding ability. This domain of phosducin contains a protein kinase A (PKA) phosphorylation site, and upon phosphorylation, the binding of full-length phosducin to G beta gamma is reduced. In addition, transient expression of phosducin in COS-7 cells significantly inhibits G beta gamma-mediated phosphoinositide hydrolysis. This inhibitory effect is completely reversed by pretreatment of cells with dibutyryl cAMP, an activator of PKA. Thus, the binding of G beta gamma to phosducin can be regulated by PKA-phosphorylation in an intact cell model system.
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PMID:Determination of the G beta gamma-binding domain of phosducin. A regulatable modulator of G beta gamma signaling. 796 75

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.
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PMID:Binding of G protein beta gamma-subunits to pleckstrin homology domains. 814 1

The beta gamma subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes. Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227) and beta-adrenergic (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267) receptors. Beta gamma mediates the membrane targeting of the beta-adrenergic receptor kinase (beta ARK), in response to receptor activation, through a specific beta ARK-beta gamma interaction. This process utilizes the membrane-anchoring properties of the isoprenylated gamma subunit of beta gamma. In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of beta ARK which binds beta gamma and thereby results in membrane translocation. We studied the ability of beta gamma to enhance the enzymatic activity of a series of truncated mutants of bovine beta ARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of beta ARK to bind beta gamma subunits, and the ability of synthetic peptides comprised of beta ARK sequences to inhibit beta gamma activation of beta ARK1. We find that the minimal beta gamma binding domain of beta ARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus. A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited beta gamma activation of beta ARK1, with an IC50 of 76 microM. The identification of this "beta gamma binding domain" on beta ARK and the development of peptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of beta gamma activation of other G protein-effector systems.
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PMID:The binding site for the beta gamma subunits of heterotrimeric G proteins on the beta-adrenergic receptor kinase. 846 35

Phosphatidylinositol 4,5-bisphosphate (PIP2) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP2, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidyicholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP2 for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP2. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP2 that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP2. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP2 in the vesicles and correlated well with the extent of the enzyme activation. Further-more, it was found that a recombinant peptide of the pleckstrin homology domain of beta-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP2, inhibited the PIP2-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP2, owing to its specific interaction with PIP2, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP2 effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP2 serves as an anchor to localize PLD on plasma membranes in vivo.
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PMID:Partially purified RhoA-stimulated phospholipase D activity specifically binds to phosphatidylinositol 4,5-bisphosphate. 876 89

Phosducin-like protein (PhLP) has recently been identified as a ubiquitous inhibitor of G-protein betagamma-subunit (G betagamma)-mediated signaling, with an affinity about 5-fold lower than that of phosducin. The G betagamma binding site of phosducin has been suggested to be contained in its N-terminus. A region corresponding to this N-terminus is lacking in PhLP, suggesting that PhLP must utilize a different mode of G betagamma binding. To map the G betagamma binding site in PhLP, a series of deletion mutants were constructed, expressed in E. coli as glutathione S-transferase (GST) fusion proteins, and the purified fusion proteins were examined for their ability to attenuate G(o) GTPase activity. Progressive N-terminal truncations of PhLP caused only minor reductions in potency, whereas the complementary N-terminal PhLP fragments turned out to be inactive. We further identified a short C-terminal segment comprising residues 168 to 195 that inhibited G0 GTPase activity similar in efficacy and potency to full-length PhLP. This C-terminal fragment was also capable of antagonizing a second G betagamma-mediated function, the enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase. Taken together, these data indicate that PhLP interacts with G betagamma via a short C-terminal binding site which is distinct from that identified previously in phosducin.
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PMID:Identification of a C-terminal binding site for G-protein betagamma-subunits in phosducin-like protein. 901 96

We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by a protein kinase of approximately 40 kDa purified from porcine cerebellum (Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) J. Biol Chem. 271, 3907-3916). This kinase, called muscarinic receptor kinase (MRK), is distinct from second messenger-regulated protein kinases and from beta-adrenergic receptor kinase and other members of the G-protein-coupled receptor kinase family. In the present study we propose that MRK is casein kinase 1alpha (CK1alpha) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1alpha. 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1alpha expressed in Sf9 cells is able to phosphorylate both casein and the bacterial fusion protein, Ex-m3, that contains a portion of the third intracellular loop of the m3-muscarinic receptor downstream of glutathione S-transferase. 4) Partially purified CK1alpha increased the level of muscarinic receptor phosphorylation in an agonist-sensitive manner when reconstituted with membranes from Chinese hamster ovary-m3 cells expressing the human recombinant m3-muscarinic receptor. 5) Partially-purified CK1alpha phosphorylated rhodopsin, contained in urea-treated bovine rod outer segment membranes, and the extent of phosphorylation was increased in the presence of light. These data demonstrate that the kinase previously called MRK is CK1alpha, and that CK1alpha offers an alternative protein kinase pathway from that of the G-protein-coupled receptor kinase family for the stimulus-dependent phosphorylation of the m3-muscarinic receptor, rhodopsin, and possibly other G-protein-coupled receptors.
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PMID:Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1alpha. 925 10

Pleckstrin homology (PH) domains are discrete structural modules present in numerous proteins involved in signal transduction processes. In the case of the beta-adrenergic receptor kinase (betaARK), PH domain-mediated binding of two ligands, the betagamma subunits of heterotrimeric G proteins (Gbetagamma) and phosphatidylinositol 4,5-bisphosphate (PIP2), has been shown to be required for the kinase function. In this study, the ability of Gbetagamma and PIP2 to affect membrane localization of betaARK is used to define the ligand binding characteristics of the betaARK PH domain. The binding of these ligands to the PH domain of the intact kinase is shown to be cooperative, Gbetagamma increasing the affinity of the PH domain for PIP2. Notably, although PIP2-dependent membrane association of betaARK is observed at high concentrations of this lipid, in the absence of Gbetagamma, no receptor phosphorylation is observed. Peptides derived from the receptor intracellular loop inhibit the receptor phosphorylation without affecting the membrane translocation of the kinase complex, suggesting that betaARK activity does not necessarily correlate with the amount of betaARK associated with the membrane. These results point to a distinct role for each PH domain ligand in betaARK-mediated receptor phosphorylation. Strikingly, the ligand binding characteristics of the isolated betaARK PH domain fused to glutathione S-transferase are very different from those of the PH domain of the intact kinase. Thus, in contrast to the native protein, the isolated PH domain binds Gbetagamma and PIP2 independently and with no apparent cooperativity. That protein environment plays an important role in determining the ligand binding characteristics of a particular PH domain highlights the potential risks of inferring mechanisms from studies of isolated PH domains.
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PMID:Binding of multiple ligands to pleckstrin homology domain regulates membrane translocation and enzyme activity of beta-adrenergic receptor kinase. 939 5


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