Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1alpha (PP1alpha). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo (32)P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1alpha. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1alpha phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1alpha phosphatase, whose enzymatic activity is regulated by Ras.
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PMID:Protein phosphatase 1alpha is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis. 1081 15

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein that contains a serine-threonine protein kinase (PK) activity (Nelson, J. W., Zhu, J. , Smith, C. C., Kulka, M., and Aurelian, L. (1996) J. Biol. Chem. 271, 17021-17027). Phylogenetic analyses indicated that ICP10 PK belongs to a distinct subfamily of growth factor receptor serine-threonine PKs that are characterized by their ability to function with a limited number of conserved catalytic motifs (Hunter, J. C. R., Smith, C. C., and Aurelian, L. (1995) Int. J. Onc. 7, 515-522). Here, we report the isolation and characterization of a novel gene, designated H11, that contains an open reading frame of 588 nucleotides, which encodes a protein similar to ICP10 PK. The H11 protein has Mn(2+)-dependent serine-threonine-specific PK activity as determined with a GST-H11 fusion protein and by immununocomplex PK/immunoblotting assays of 293 cells transfected with a H11 eukaryotic expression vector. PK activity is ablated by mutation of Lys(113) within the presumtive catalytic motif II (invariant Lys). 293 cells stably transfected with H11 acquire anchorage-independent growth. Endogenous H11 RNA and the H11 phosphoprotein are expressed in melanoma cell lines and primary melanoma tissues at levels higher than in normal melanocytes and in benign nevi. Melanoma cell proliferation is inhibited by treatment with antisense oligonucleotides that inhibit H11 translation, suggesting that H11 expression is associated with cell growth.
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PMID:A novel human gene similar to the protein kinase (PK) coding domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) codes for a serine-threonine PK and is expressed in melanoma cells. 1083 16

huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D. , Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.
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PMID:Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue of Cdc7 bY Cdks. 1084 77

The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.
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PMID:In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase. 1090 49

The tumour suppressor p53 protein integrates multiple signals regulating cell cycle progression and apoptosis. This regulation is mediated by several kinases that phosphorylate specific residues in the different functional domains of the p53 molecule. The human VRK1 protein is a new kinase related to a poxvirus kinase, and more distantly to the casein kinase 1 family. We have characterized the biochemical properties of human VRK1 from HeLa cells. VRK1 has a strong autophosphorylating activity in several Ser and Thr residues. VRK-1 phosphorylates acidic proteins, such as phosvitin and casein, and basic proteins such as histone 2b and myelin basic protein. Because some transcription factors are regulated by phosphorylation, we tested as substrates the N-transactivation domains of p53 and c-Jun fused to GST. Human c-Jun is not phosphorylated by VRK1. VRK1 phosphorylates murine p53 in threonine 18. This threonine is within the p53 hydrophobic loop (residues 13-23) required for the interaction of p53 with the cleft of its inhibitor mdm-2. The VRK1 C-terminus domain (residues 268-396) that contains a nuclear localization signal targets the protein to the nucleus, as determined by using fusion proteins with the green fluorescent protein. We conclude that VRK1 is an upstream regulator of p53 that belongs to a new signalling pathway.
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PMID:The human vaccinia-related kinase 1 (VRK1) phosphorylates threonine-18 within the mdm-2 binding site of the p53 tumour suppressor protein. 1095 72

Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17. 5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.
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PMID:Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis. 1098 45

A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".
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PMID:Mapping the functional domains of elongation factor-2 kinase. 1101

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.
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PMID:Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. 1115 51

beta-arrestins have been shown to play important roles in regulation of signaling and desensitization of opioid receptors in many in vivo studies. The current study was carried out to measure the direct interaction of beta-arrestins with two functional intracellular domains, the third intracellular loop (I3L) and the carboxyl terminus (CT), of delta-, mu-, and kappa-opioid receptors (DOR, MOR, and KOR, respectively). Results from the pull-down assay using glutathione S-transferase fusion proteins demonstrated that beta-arrestins (1 and 2) were able to bind to the I3L of DOR and to the CT of DOR and KOR. Surface plasmon resonance measurement gave similar results with typical dissociation equilibrium constant (K(D)) values in the micromolar range. The site-directed mutagenesis experiment further revealed that certain specific serine/threonine residues in these receptor domains play a critical role in their interaction with beta-arrestins. Taken together, our data clearly indicated that beta-arrestins interact differentially with the functional domains of different opioid receptors; this may provide a possible molecular basis for differential regulation of opioid receptors by beta-arrestins.
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PMID:Direct and differential interaction of beta-arrestins with the intracellular domains of different opioid receptors. 1125 20

Cyclin A1 is tissue-specifically expressed during spermatogenesis, but it is also highly expressed in acute myeloid leukemia (AML). Its pathogenetic role in AML and in the cell cycle of leukemic blasts is unknown. B-myb is essential for G1/S transition and has been shown to be phosphorylated by the cyclin A2/cdk2 complex. Here it is demonstrated that cyclin A1 interacts with the C-terminal portion of B-myb as shown by glutathione S-transferase (GST) precipitation. This interaction is confined to cyclin A1 because binding could not be detected between cyclin A2 and B-myb. Also, cdk2 was not pulled down by GST-B-myb from U937 lysates. In addition, co-immunoprecipitation of cyclin A1 and B-myb in leukemic cells evidenced protein interaction in vivo. Baculovirus-expressed cyclin A1/cdk2 complexes were able to phosphorylate human as well as murine B-myb in vitro. Tryptic phosphopeptide mapping revealed that cyclin A1/cdk2 complexes phosphorylated the C-terminal part of B-myb at several sites including threonine 447, 490, and 497 and serine 581. These phosphorylation sites have been demonstrated to be important for the enhancement of B-myb transcriptional activity. Further studies showed that cyclin A1 cooperated with B-myb to transactivate myb binding site containing promoters including the promoter of the human cyclin A1 gene. Taken together, the data suggest that cyclin A1 is a tissue-specific regulator of B-myb function and activates B-myb in leukemic blasts. (Blood. 2001;97:2091-2097)
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PMID:Cyclin A1 directly interacts with B-myb and cyclin A1/cdk2 phosphorylate B-myb at functionally important serine and threonine residues: tissue-specific regulation of B-myb function. 1126 76


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