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)

Antibodies recognising the products of alternatively spliced exons near the N-terminus of the leukocyte common antigen, CD45, have been widely used to distinguish populations of lymphocytes with different functional properties. These alternatively spliced regions contain a high content of serine and threonine residues (average 35%) and are heavily O-glycosylated. Despite evidence that the O-glycosylation contributes significantly to the antigenic character of this region of CD45, work with leukosialin and mucin glycoproteins leads to the prediction that the majority of epitopes in the N-terminal exons should be linear protein determinants. In this study the exons of CD45 were expressed in Escherichia coli as non-glycosylated proteins fused to glutathione S-transferase (GST). Fourteen out of 17 mAbs specific for human CD45R reacted with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD45, and four out of six mAbs specific for rat CD45R reacted with an equivalent rat protein. mAbs recognising the product of rat exon B are reported for the first time. Kinetic analysis of MRC OX22 antibody binding to spleen CD45 and to GST fusion proteins showed that the carbohydrate affected the kinetics of binding of antibodies to the protein backbone. In conclusion, heterogeneity in the glycosylation of heavily O-glycosylated cell surface proteins can affect interactions of these proteins both directly through the carbohydrate and indirectly through effects on the protein backbone.
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PMID:Antigenic determinants encoded by alternatively spliced exons of CD45 are determined by the polypeptide but influenced by glycosylation. 753 96

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

The human cytomegalovirus immediate-early gene product 2 (IE2) is able to transactivate homologous and heterologous promoters alone or augmented by immediate-early gene product 1 (IE1). IE2 has also been shown to autoregulate the major immediate-early promoter by directly binding to a cis repression signal located between the TATA box and the cap site. However, IE2 has not been shown to act directly through a specific DNA sequence in transactivating various promoters. To understand whether IE2 can be indirectly involved in DNA sequence-specific transactivation through interactions with other transcriptional factors, we performed a study of the interactions of IE2 with cellular proteins. In order to study these interactions, IE cDNAs were subcloned into a bacterial expression vector, pGEX2T, by polymerase chain reaction amplification to produce fusion proteins which were full-length as well as proteins which contained various functional domains. We were able to demonstrate IE2's ability to interact directly or indirectly with several cellular proteins ranging from > 200 to 14 kDa through glutathione S-transferase-fusion protein precipitation and far-Western analysis. These interactions have been mapped to domains within IE2 which are known to be necessary for either transactivation or both transactivation and autoregulation. All of the IE2-associated proteins are nuclear proteins, and a subset are phosphorylated. In vitro-synthesized 35S-IE2 protein and bacterially expressed IE2 fusion proteins were used to study IE2-IE2 interaction by binding assay and far-Western analysis. IE2-IE2 interactions were mapped to a domain containing a putative helix-turn-helix motif located near the C terminus of IE2, between amino acids 456 and 539. However, IE2 was unable to directly interact with either IE1, an alternatively spliced variant of IE2 (55 kDa), or IE2 deletion mutants that did not contain the multimerization domain.
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PMID:Human cytomegalovirus immediate-early gene 2 protein interacts with itself and with several novel cellular proteins. 839 23

Protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are both attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the previously identified spectrin-actin binding domain (10 kDa molecular mass) of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette, denoted 10k21 and 10k19,21, were able to bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette, 10k19 and 10k0, did not function in these assays. A bacterially expressed fusion protein with glutathione-S-transferase, designated GST-10k21, congealed spectrin-actin into a network in vitro as found with purified protein 4.1. Additionally, incorporation of GST-10k21 into mechanically weak, 4.1-deficient membranes increased mechanical strength of these membranes to normal. GST-10k19 did not function in these assays. These results show that the 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 are suggested to have different, yet to be defined functions.
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PMID:Mechanochemistry of the alternatively spliced spectrin-actin binding domain in membrane skeletal protein 4.1. 846 54

Max (Myc-associated factor X) is a basic helix-loop-helix/leucine zipper protein that has been shown to play a central role in the functional activity of c-Myc as a transcriptional activator. Max potentiates the binding of Myc-Max heterodimers through its basic region to its specific E-box Myc site (EMS), enabling c-Myc to transactivate effectively. In addition to the alternatively spliced exon a, several naturally occurring forms of alternatively spliced max mRNAs have been reported, but variant protein products from these transcripts have not been detected. Using Western blot (immunoblot) and immunoprecipitation analysis, we have identified a variant form of Max protein (16 to 17 kDa), termed dMax, in detergent nuclear extracts of murine B-lymphoma cells, normal B lymphocytes, and NIH 3T3 fibroblasts. Cloning and sequencing revealed that dMax contains a deletion spanning the basic region and helix 1 and the loop of the helix-loop-helix region, presumably as a result of alternative splicing of max RNA. S1 nuclease analysis confirmed the presence of the mRNA for dMax in cells. The dMax protein, prepared via in vitro transcription and translation, associated with bacterially synthesized Myc-glutathione S-transferase. Coimmunoprecipitation of dMax and c-Myc indicated their intracellular association. In vitro-synthesized dMax failed to bind EMS DNA, presumably because of the absence of the basic region. Coexpression of dMax inhibited EMS-mediated transactivation by c-Myc. Thus dMax, which can interact with c-Myc, appears to function as a dominant negative regulator, providing an additional level of regulation to the transactivation potential of c-Myc.
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PMID:Variant Max protein, derived by alternative splicing, associates with c-Myc in vivo and inhibits transactivation. 852 35

Differential association of regulatory B subunits with a core heterodimer, composed of a catalytic (C) and a structural (A) subunit, is an important mechanism that regulates protein phosphatase 2A (PP2A). We have isolated and characterized three novel cDNAs related to the B' subunit of bovine cardiac PP2A. Two human (B'alpha1 and B'alpha2) and a mouse (B'alpha3) cDNA encode for alternatively spliced variants of the B subunit. The deduced primary sequences of these clones contain 12 of 15 peptides derived from the purified bovine B' subunit. Differences between the deduced sequences of the B alpha splice variants and the cardiac peptide sequences suggest the existence of multiple isoforms of the B' subunit. Comparison of the protein and nucleotide sequences of the cloned cDNAs show that all three forms of B'alpha diverge at a common splice site near the 3'-end of the coding regions. Northern blot and reverse transcription-polymerase chain reaction analyses revealed that the B'alpha transcripts (4.3-4.4 kb) are widely expressed and very abundant in heart and skeletal muscle. The expressed human and mouse B'alpha proteins readily associated with the PP2A core enzyme in both in vitro and in vivo complex formation assays. Immunofluorescence microscopy revealed that epitope-tagged B'alpha was localized in both the cytosol and nuclei of transiently transfected cells. The efficiency of binding of all three expressed proteins to a glutathione S-transferase-A subunit fusion protein was greatly enhanced by the addition of the C subunit. Expression of the B'alpha subunits in insect Sf9 cells resulted in formation of AC.B'alpha heterotrimers with the endogenous insect A and C subunits. These results show that the B' subunit, which is the predominant regulatory subunit in cardiac PP2A, is a novel protein whose sequence is unrelated to other PP2A regulatory subunits. The nuclear localization of expressed B'alpha suggests that some variants of the B' subunit are involved in the nuclear functions of PP2A.
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PMID:Identification of a novel protein phosphatase 2A regulatory subunit highly expressed in muscle. 861 97

The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent Km of approximately 3.4 microM. Exchange of two tyrosine residues in the "activation loop" between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions.
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PMID:Dyrk, a dual specificity protein kinase with unique structural features whose activity is dependent on tyrosine residues between subdomains VII and VIII. 863 52

Protein tyrosine phosphorylation plays a crucial role in signaling from the receptor for erythropoietin (Epo), although the Epo receptor (EpoR) lacks the tyrosine kinase domain. We have previously shown that the Jak2 tyrosine kinase couples with the EpoR to transduce a growth signal. In the present study, we demonstrate that Lyn, a Src family tyrosine kinase, physically associates with the EpoR in Epo-dependent hematopoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of the EpoR and that both LynA and LynB, alternatively spliced forms of Lyn, bind with the membrane-proximal 91-amino acid region of the EpoR cytoplasmic domain. In vitro binding studies using GST-Lyn fusion proteins further showed that the Src homology (SH)-2 domain of Lyn specifically binds with the tyrosine-phosphorylated EpoR in lysate from Epo-stimulated cells, whereas the tyrosine kinase domain of Lyn binds with the unphosphorylated EpoR. Far-Western blotting and synthetic phosphopeptide competition assays further indicated that the Lyn SH2 domain directly binds to the tyrosine-phosphorylated EpoR, most likely through its interaction with phosphorylated Y-464 or Y-479 in the carboxy-terminal region of the EpoR. In vitro binding studies also demonstrated that the Lyn SH2 domain directly binds to tyrosine-phosphorylated Jak2. In vitro reconstitution experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of Stat5, mainly on Y-694, and activates the DNA-binding and transcription-activating abilities of Stat5. In agreement with this, Lyn enhanced the Stat5-dependent transcriptional activation when overexpressed in 32D/EpoR-Wt cells. In addition, Lyn was demonstrated to phosphorylate the EpoR and Stat5 on tyrosines in vitro. These results suggest that Lyn may play a role in activation of the Jak2/Stat5 and other signaling pathways by the EpoR.
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PMID:Lyn physically associates with the erythropoietin receptor and may play a role in activation of the Stat5 pathway. 957 10

The class I glutathione S-transferases (GSTs) of Anopheles gambiae are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes, aggst1-2, is intronless and has been described. In contrast, the two A. gambiae GST genes (aggst1alpha and aggst1beta) reported within are interrupted by introns. The gene aggst1alpha contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5' exon encoding the N termini of the GST protein spliced to one of four distinct 3' exons encoding the carboxyl termini. All four of the alternative transcripts of aggst1alpha are expressed in A. gambiae larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the aggst1beta gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.
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PMID:The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S-transferase family. 982 92

In this paper we report the genomic organization of the human microsomal GST-I gene. This gene spans 18 kb, and contains seven exons. Sequences that encode the 155 amino acid open reading frame are present in Exons II, III, IV, the 5'-untranslated region is present in Exons Ia, Ib, Ic, Id, and II, and the 3'-untranslated region is present in Exon IV. Exons Ia, Ib, Ic, Id, and III are alternatively spliced to generate at least six different mGST-I transcripts. The results of EST and PCR analysis show that most mGST-I transcripts terminate within Exon Ib, and primer extension analysis shows these transcripts initiate at three major sites located at 79, 81, and 88 nucleotides upstream of the ATG initiation codon. Sequences surrounding the putative initiation sites are G-C rich, and several Sp1 consensus binding sites were identified. Northern analysis shows that the human GST-I gene is preferentially expressed as a 1.0 kb transcript in liver, and in several other tissues. Finally, a comparison of the mGST-I and PIG12 sequences with those of FLAP, LTC4 synthase, mGST-II, and mGST-III suggests that these proteins are the related products of a dispersed microsomal GST gene superfamily.
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PMID:Microsomal GST-I: genomic organization, expression, and alternative splicing of the human gene. 1052 15


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