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)

Chk2 is a key player of the DNA damage signalling pathway. To identify new regulators of this kinase, we performed a yeast two-hybrid screen and found that Chk2 associated with the B' regulatory subunit of protein phosphatase PP2A. In vitro GST-Chk2 pulldowns demonstrated that B'gamma isoforms bound to Chk2 with the strongest apparent affinity. This was confirmed in cellulo by co-immunoprecipitation after overexpression of the respective partners in HEK293 cells. The A and C subunits of PP2A were present in the complexes, suggesting that Chk2 was associated with a functionnal PP2A. In vitro kinase assays showed that B'gamma3 was a potent Chk2 substrate. This phosphorylation increased the catalytic phosphatase activity of PP2A measured on MAP kinase-phosphorylated myelin basic protein as well as on autophosphorylated Chk2. Finally, we demonstrated that overexpressing B'gamma3 in HEK293 suppressed the phosphorylation of Chk2 induced by a genotoxic treatment, suggesting that PP2A may counteract the action of the checkpoint kinase in living cells.
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PMID:Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A via its B' regulatory subunit. 1538 Jun 17

The activity of NF-kappaB is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-kappaB and the functional role of these phosphorylations are still being uncovered. Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-kappaB. Serine 468 lies within a GSK-3beta consensus site, and recombinant GSK-3beta specifically phosphorylates a GST-p65-(354-551) fusion protein at Ser(468) in vitro. In intact cells, phosphorylation of endogenous Ser(468) of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3beta inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-kappaB activation. Collectively our results suggest that a GSK-3beta-PP1-dependent mechanism regulates phosphorylation of p65 NF-kappaB at Ser(468) in unstimulated cells and thereby controls the basal activity of NF-kappaB.
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PMID:Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity. 1546 28

The neural cell adhesion molecule (NCAM) is implicated in important functions during development and maintenance of the nervous system. Two of the three major isoforms, NCAM 140 and NCAM 180, are transmembrane glycoproteins with large cytoplasmic domains of different length. The purpose of this study was to identify novel intracellular binding partners of NCAM 140 and NCAM 180. We expressed both cytoplasmic domains, as well as cytoplasmic fragments of NCAM, as fusion proteins in Escherichia coli and used them for ligand affinity chromatography or glutathione S-transferase (GST) pull-down assays. By peptide mass fingerprinting Western blot analysis, or both, we identified PLCgamma, LANP, syndapin, PP1, and PP2A as binding partners for both NCAM 140 and NCAM 180, whereas TOAD-64 was identified as a NCAM 180-specific interacting protein. Furthermore, we were able to show that binding of these novel binding proteins, as well as the previously described interaction partners ROK alpha (rho A binding kinase alpha) and alpha- and beta-tubulin, bind to specific cytosolic sequences of NCAM. For this purpose, we performed GST pull-down experiments using cytosolic fragments of NCAM as GST-fusion proteins and cytosolic- or cytoskeleton-enriched protein fractions of rat brain.
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PMID:Novel cytosolic binding partners of the neural cell adhesion molecule: mapping the binding domains of PLC gamma, LANP, TOAD-64, syndapin, PP1, and PP2A. 1586 39

G(q) protein-coupled receptor stimulation increases sarcolemmal Na(+)/H(+) exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A(1) receptor stimulation inhibits this response through a G(i) protein-mediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A(1) receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the alpha(1)-adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A(c)) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate G(i) proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A(c) and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2A(c) dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A(1) receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve G(i) protein-mediated translocation of PP2A(c) and NHE1 dephosphorylation.
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PMID:A novel role for protein phosphatase 2A in receptor-mediated regulation of the cardiac sarcolemmal Na+/H+ exchanger NHE1. 1670 1

A trimeric protein phosphatase 2A (PP2A(T55)) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55 kDa, respectively. The 54 kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein. The 55 kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST-NRX, PP2A(C) and PP2A(D) was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST-NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A.
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PMID:Interaction of nucleoredoxin with protein phosphatase 2A. 1676 67

Integrin alpha3beta1 is a receptor for the extracellular matrix component laminin 5. To elucidate possible signaling pathways induced by integrin alpha3beta1, we looked for proteins that interact with the cytoplasmic part of the alpha3A integrin subunit. We identified several multifunctional proteins by affinity chromatography and subsequent MALDI-TOF-MS and focused on the inhibitor 1 of serine/threonine phosphatase PP2A (I1PP2A, synonym: lanp) which also plays a role during the development of the mouse cerebellum. I1PP2A/lanp colocalizes with the alpha3A integrin subunit in differentiated PC12 cells in the cell body and in neurites as well as in Purkinje cells of mouse cerebellum. Overexpression of GFP-I1PP2A/lanp in PC12 cells leads to markedly reduced neurite length on laminin 5 after induction with nerve growth factor. By affinity chromatography the protein phosphatase PP1 can also be identified as a alpha3A/cyto-binding protein. PP1 and integrin alpha3beta1 can be pulled down by GST-I1PP2A/lanp from cell lysates of differentiated and undifferentiated PC12 cells. The phosphatase binds to the cytoplasmic membrane-proximal conserved GFFKR motif of the alpha integrin subunit, whereas I1PP2A/lanp requires a longer sequence for binding. PP1 but not PP2A is able to dephosphorylate precipitated integrin alpha3beta1 in vitro. Furthermore, PP1 releases phosphate from T1046 of phosphopeptides that mimic the phosphorylation consensus sequence in the cytoplasmic part of the alpha3A integrin subunit. These data suggest that I1PP2A/lanp forms a complex with PP1 and the alpha3A integrin subunit and might possibly regulate the phosphorylation status of integrin alpha3beta1 and/or integrin downstream targets.
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PMID:Integrin alpha3beta1 interacts with I1PP2A/lanp and phosphatase PP1. 1701 59

The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-alpha catalytic subunit expressed as a histidine fusion protein (6xHis-PP1alpha) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). ICP34.5 recruits PP1 to dephosphorylate the alpha subunit of eukaryotic translational initiation factor 2 (also known as eIF-2alpha); possibly the ASFV DP71L protein has a similar function.
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PMID:The MyD116 African swine fever virus homologue interacts with the catalytic subunit of protein phosphatase 1 and activates its phosphatase activity. 1721 79

The pore-forming subunit of the large-conductance Ca(2+)-dependent K(+) (Slo1) channel is encoded by one gene. However, the functional properties of Slo1 channels are diverse in part because of their numerous regulatory mechanisms including posttranslational modification and alternative splicing. In particular, multiple splice variants of the pore-forming subunit have been reported but their significance is only beginning to be elucidated. Here we examined the cell biological properties of the three common C-terminal isoforms that differ in the last 8 (Slo1_ERL and Slo1_VYR) or 61 residues (Slo1_DEC). We found that Slo1_DEC, the longest isoform, shows dramatically reduced surface expression compared to that of Slo1_ERL or Slo1_VYR. Immunocytochemistry revealed that a large fraction of Slo1_DEC remains localized in endoplasmic reticulum (ER). Using a GST fusion protein containing the Slo1_DEC-specific sequence, affinity purification was carried out to isolate interacting proteins. The identified proteins include protein phosphatase 2A (PP2A-A), actin, and tubulin. The PP2A-A interaction is specific to Slo1_DEC and causes a significant reduction of phosphorylation in Slo1_DEC but not Slo1_ERL or Slo1_VYR. The results together support the notion that Slo1_DEC nucleates isoform-specific protein complexes and possesses a cis element(s) for regulating trafficking of the Slo1 channels.
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PMID:Differential trafficking of carboxyl isoforms of Ca2+-gated (Slo1) potassium channels. 1730 27

Members of the Casein Kinase 1 (CK1) family are implicated in the regulation of a variety of physiological processes like development and circadian rhythm, as well as in diseases like cancer and Alzheimer's disease. From that perspective, CK1 family members are interesting targets for potential chemotherapy. We describe here a rapid and efficient method for the purification of CK1 by affinity chromatography on an immobilised fragment of axin. Axin is a scaffolding protein that interacts with a multitude of proteins, amongst them APC, GSK-3, beta-catenin, CK1alpha, delta, and epsilon, and PP2A. A GST-tagged axin peptide (residues 495-684) was produced in Escherichia coli and either immobilised on glutathione agarose beads or purified and immobilised on CNBr-activated sepharose 4B. These "GST-axin" matrices were found to selectively bind native CK1alpha and CK1epsilon from porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to efficiently purify large amounts of active native CK1 for screening purposes. This single step purification method also provides a convenient tool to follow the status of the axin-binding CK1 isoforms alpha, delta, and epsilon (protein levels, composition of isoforms, kinase activity) under different physiological settings.
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PMID:Purification of CK1 by affinity chromatography on immobilised axin. 1743 49

KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.
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PMID:Physical association of GPR54 C-terminal with protein phosphatase 2A. 1897 1


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