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)

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.
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PMID:Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin. 183 9

The PHO81 gene is thought to encode an inhibitor of the negative regulators (Pho80p and Pho85p) in the phosphatase (PHO) regulon. Transcription of PHO81 is regulated by Pi signals through the same PHO regulatory system. Elimination of the PHO81 promoter or its substitution by the GAL1 promoter revealed that stimulation of the PHO regulatory system requires both increased transcription of PHO81 and a Pi starvation signal. The predicted Pho81p protein contains 1,179 amino acids (aa) and has six repeats of an ankyrin-like sequence in its central region. The minimum amino acid sequence required for Pho81p function was narrowed down to a 141-aa segment (aa 584 to 724), which contains the fifth and sixth repeats of the ankyrin-like motif. The third to sixth repeats of the ankyrin-like motif of Pho81p have significant similarities to that of p16INK4, which inhibits activity of the human cyclin D-CDK4 kinase complex. Deletion analyses revealed that the N- and C-terminal regions of Pho81p behave as negative and positive regulatory domains, respectively, for the minimal 141-aa region. The negative regulatory activity of the N-terminal domain was antagonized by a C-terminal segment of Pho81p supplied in trans. All four known classes of PHO81c mutations that show repressible acid phosphatase activity in high-Pi medium affect the N-terminal half of Pho81p. An in vitro assay showed that a glutathione S-transferase-Pho81p fusion protein inhibits the Pho85p protein kinase. Association of Pho81p with Pho85p or with the Pho80p-Pho85p complex was demonstrated by the two-hybrid system.
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PMID:Functional domains of Pho81p, an inhibitor of Pho85p protein kinase, in the transduction pathway of Pi signals in Saccharomyces cerevisiae. 782 64

Central to spectrin's function is its association with the plasma membrane. The linking proteins ankyrin and protein 4.1 partly mediate this association, and their interactions with spectrin are well understood. Both beta I (erythrocyte) and beta II (fodrin, beta G) spectrin also associate with unknown protein receptors in crude membrane preparations by ankyrin and protein 4.1 independent mechanisms. As a first step to understanding this interaction, kinetic and equilibrium assays have been used to monitor which regions of beta I and beta II spectrin inhibit the binding of purified 125I-labeled bovine brain spectrin to demyelinated and NaOH-stripped bovine brain membranes. A series of 19 recombinant proteins spanning the entire sequence of beta II spectrin, including an alternatively spliced NH2-terminal isoform (beta II epsilon 2 spectrin), were prepared as glutathione S-transferase fusion proteins. Also prepared were peptides representing the alternatively spliced COOH-terminal domain found in beta I epsilon 2 spectrin ("muscle spectrin"). Two distinct sequence motifs inhibited the binding of native brain spectrin. Membrane association domain 1 (MAD1) was represented in all fusion peptides that included spectrin repeat 1. These peptides slowed the kinetics of brain spectrin binding and inhibited up to 46% of the maximal binding under the conditions of these assays (apparent Ki < or = 0.2 microM). Peptides representative of repeats 2-17 of beta II spectrin were devoid of inhibitory activity. The second membrane association domain (MAD2) was identified in penultimate COOH-terminal sequences (domain III) of both beta II and beta I epsilon 2 spectrin. These sequences were absent in beta I epsilon 1 (erythrocyte) spectrin. MAD2 competitively inhibited over 80% of brain spectrin binding in these assays, with an apparent Ki < or = 0.1 microM. Direct binding studies confirmed that both MAD1 and MAD2 peptides associated with membranes with affinities comparable to their inhibition constants. Sequence comparisons suggest that MAD1 is created by the insertion of two non-homologous sequence motifs into repeat 1, extending it from 106 to 122 amino acids. Similarly, MAD2 encompasses a putative site of beta gamma-heterotrimeric G-protein binding called the pleckstrin homology domain, and MAD2 may in fact be the pleckstrin homology domain although this has not been rigorously proven. Collectively these studies identify two novel functional motifs in spectrin that mediate ankyrin independent association with membranes. We hypothesize that these motifs and their still to be discovered ligands play a primary role in the nascent assembly and stabilization of an ordered and polarized spectrin skeleton.
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PMID:Beta II-spectrin (fodrin) and beta I epsilon 2-spectrin (muscle) contain NH2- and COOH-terminal membrane association domains (MAD1 and MAD2). 796 88

Ankyrin has a spectrin-binding region within a central 62-kDa chymotryptic peptide. We examined the spectrin binding ability of a series of smaller ankyrin fragments and recombinant peptides within the 62-kDa domain using a ligand blot assay. The smallest proteolytic fragment that bound was a 12-kDa tryptic peptide starting at amino acid 1068. Peptides containing this region expressed as glutathione S-transferase fusion products also bound spectrin and suggested that residues 1101-1192 were important. In contrast, a fusion protein containing residues 826-898 did not bind spectrin, a surprising finding since this region is known to influence binding affinity. Proteins that bound spectrin on ligand blots also competed for binding in solution, but did so with one-tenth the affinity of the native peptide. Comparing the 62-kDa domains of erythrocyte and brain ankyrins (species that bind spectrin but with 10-fold differences in affinity), the NH2-terminal regions are 0-40% identical, while the regions (1136-1160) common to all binding peptides are 80-90% identical. We hypothesize that the highly conserved region contains an important spectrin-binding site, while the poorly conserved region controls the binding affinity. We speculate that this unique NH2-terminal region is what gives different members of the ankyrin family their signature set of affinities, and accordingly their distinctive cellular localization.
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PMID:A highly conserved region of human erythrocyte ankyrin contains the capacity to bind spectrin. 822 93

To identify the molecular pathways that guide cardiac ventricular chamber specification, maturation and morphogenesis, we have sought to characterize factors that regulate the expression of the ventricular myosin light chain-2 gene, one of the earliest markers of ventricular regionalization during mammalian cardiogenesis. Previously, our laboratory identified a 28 bp HF-la/MEF-2 element in the MLC-2v promoter region, which confers cardiac ventricular chamber-specific gene expression during murine cardiogenesis, and showed that the ubiquitous transcription factor YB-1 binds to the HF-la site in conjunction with a co-factor. In a search for interacting co-factors, a nuclear ankyrin-like repeat protein CARP (cardiac ankyrin repeat protein) was isolated from a rat neonatal heart cDNA library by yeast two-hybrid screening, using YB-1 as the bait. Co-immunoprecipitation and GST-CARP pulldown studies reveal that CARP forms a physical complex with YB-1 in cardiac myocytes and immunostaining shows that endogenous CARP is localized in the cardiac myocyte nucleus. Co-transfection assays indicate that CARP can negatively regulate an HF-1-TK minimal promoter in an HF-1 sequence-dependent manner in cardiac myocytes, and CARP displays a transcriptional inhibitory activity when fused to a GAL4 DNA-binding domain in both cardiac and noncardiac cell context. Northern analysis revealed that carp mRNA is highly enriched in the adult heart, with only trace levels in skeletal muscle. During murine embryogenesis, endogenous carp expression was first clearly detected as early as E8.5 specifically in heart and is regulated temporally and spatially in the myocardium. Nkx2-5, the murine homologue of Drosophila gene tinman was previously shown to be required for heart tube looping morphogenesis and ventricular chamber-specific myosin light chain-2 expression during mammalian heart development. In Nkx2-5(-/-)embryos, carp expression was found to be significantly and selectively reduced as assessed by both whole-mount in situ hybridizations and RNase protection assays, suggesting that carp is downstream of the homeobox gene Nkx2-5 in the cardiac regulatory network. Co-transfection assays using a dominant negative mutant Nkx2-5 construct with CARP promoter-luciferase reporter constructs in cardiac myocytes confirms that Nkx2-5 either directly or indirectly regulates carp at the transcriptional level. Finally, a carp promoter-lacZ transgene, which displays cardiac-specific expression in wild-type and Nkx2-5(+/-) background, was also significantly reduced in Nkx2-5(-/-) embryos, indicating that Nkx2-5 either directly or indirectly regulates carp promoter activity during in vivo cardiogenesis as well as in cultured cardiac myocytes. Thus, CARP is a YB-1 associated factor and represents the first identified cardiac-restricted downstream regulatory gene in the homeobox gene Nkx2-5 pathway and may serve as a negative regulator of HF-1-dependent pathways for ventricular muscle gene expression.
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PMID:CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2-5 homeobox gene pathway. 904 61

IkappaB alpha retains the transcription factor NF-kappaB in the cytoplasm, thus inhibiting its function. Various stimuli inactivate IkappaB alpha by triggering phosphorylation of the N-terminal residues Ser32 and Ser36. Phosphorylation of both serines is demonstrated directly by phosphopeptide mapping utilizing calpain protease, which cuts approximately 60 residues from the N terminus, and by analysis of mutants lacking one or both serine residues. Phosphorylation is followed by rapid proteolysis, and the liberated NF-kappaB translocates to the nucleus, where it activates transcription of its target genes. Transfer of the N-terminal domain of IkappaB alpha to the ankyrin domain of the related oncoprotein Bcl-3 or to the unrelated protein glutathione S-transferase confers signal-induced phosphorylation on the resulting chimeric proteins. If the C-terminal domain of IkappaB alpha is transferred as well, the resulting chimeras exhibit both signal-induced phosphorylation and rapid proteolysis. Thus, the signal response of IkappaB alpha is controlled by transferable N-terminal and C-terminal domains.
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PMID:The signal response of IkappaB alpha is regulated by transferable N- and C-terminal domains. 915

The ankyrin 33-residue repeating motif, an L-shaped structure with protruding beta-hairpin tips, mediates specific macromolecular interactions with cytoskeletal, membrane, and regulatory proteins. The association between ankyrin and alpha-Na,K-ATPase, a ubiquitous membrane protein critical to vectorial transport of ions and nutrients, is required to assemble and stabilize Na,K-ATPase at the plasma membrane. alpha-Na,K-ATPase binds both red cell ankyrin (AnkR, a product of the ANK1 gene) and Madin-Darby canine kidney cell ankyrin (AnkG, a product of the ANK3 gene) utilizing residues 142-166 (SYYQEAKSSKIMESFK NMVPQQALV) in its second cytoplasmic domain. Fusion peptides of glutathione S-transferase incorporating these 25 amino acids bind specifically to purified ankyrin (Kd = 118 +/- 50 nM). The three-dimensional structure (2.6 A) of this minimal ankyrin-binding motif, crystallized as the fusion protein, reveals a 7-residue loop with one charged hydrophilic face capping a double beta-strand. Comparison with ankyrin-binding sequences in p53, CD44, neurofascin/L1, and the inositol 1,4,5-trisphosphate receptor suggests that the valency and specificity of ankyrin binding is achieved by the interaction of 5-7-residue surface loops with the beta-hairpin tips of multiple ankyrin repeat units.
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PMID:Structure of the ankyrin-binding domain of alpha-Na,K-ATPase. 966 35

Biological, molecular, and epidemiological data have demonstrated that human T cell leukemia virus type 1 (HTLV-1) encoded Tax protein plays a central role in the initiation of T cell malignancy. The 40-kDa Tax oncoprotein serves as a potent transcriptional activator that induces viral gene expression driven by the HTLV-1 long terminal repeats and also stimulates multiple cellular genes involved in T cell activation, cell cycle regulation, and gene activation. Since Tax has been shown to interact directly and indirectly with the NF-kappa B/I kappa B regulatory proteins, we examined the significance of an in vivo association between Tax and the I kappa B alpha inhibitor. Using GST affinity chromatography, Tax was shown to interact with the I kappa B alpha ankyrin repeats which are essential for interaction with the NF-kappa B/Rel proteins. In vivo, using I kappa B alpha mutants and co-immunoprecipitation, a preferential interaction between HTLV-1 Tax and N-terminally hypophosphorylated I kappa B alpha was detected. Tax also enhanced binding of I kappa B alpha to the proteasome subunit HsN3, resulting in a Tax-enhanced, constitutive degradation of wild-type and mutated forms of I kappa B alpha in the absence of phosphorylation and ubiquitination. Binding of I kappa B alpha to proteasome subunit HC9 was also observed, but this interaction occurred independently of Tax. Taken together, these results suggest a role for Tax as a viral chaperone resulting in the enhanced constitutive turnover of I kappa B alpha. The association of Tax with hypophosphorylated I kappa B alpha may prevent I kappa B alpha from binding to NF-kappa B and also target I kappa B alpha to the proteasome for degradation via a phosphorylation-independent pathway.
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PMID:Association between HTLV-1 Tax and I kappa B alpha is dependent on the I kappa B alpha phosphorylation state. 987 28

Since the structures of several ankyrin-repeat proteins including the INK4 (inhibitor of cyclin-dependent kinase 4) family have been reported recently, the detailed structures and the functional roles of the loops have drawn considerable interest. This paper addresses the potential importance of the loops of ankyrin-repeat proteins in three aspects. First, the solution structure of p18INK4C was determined by NMR, and the loop structures were analyzed in detail. The loops adapt nascent antiparallel beta-sheet structures, but the positions are slightly different from those in the crystal structure. A detailed comparison between the solution structures of p16 and p18 has also been presented. The determination of the p18 solution structure made such detailed comparisons possible for the first time. Second, the [1H,15N]HSQC NMR experiment was used to probe the interactions between p18INK4C and other proteins. The results suggest that p18INK4C interacts very weakly with dna K and glutathione S-transferase via the loops. The third aspect employed site-specific mutagenesis and functional assays. Three mutants of p18 and 11 mutants of p16 were constructed to test functional importance of loops and helices. The results suggest that loop 2 is likely to be part of the recognition surface of p18INK4C or p16INK4A for CDK4, and they provide quantitative functional contributions of specific residues. Overall, our results enhance understanding of the structural and functional roles of the loops in INK4 tumor suppressors in particular and in ankyrin-repeat proteins in general.
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PMID:Tumor suppressor INK4: determination of the solution structure of p18INK4C and demonstration of the functional significance of loops in p18INK4C and p16INK4A. 1007 45

Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.
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PMID:Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling. 1033 Apr 11


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