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)

The adamalysins (ADAMs) are transmembrane glycoproteins involved in cell adhesion and proteolytic ectodomain processing of cytokines and adhesion molecules. Many ADAM cytoplasmic domains are proline-rich and have potential phosphorylation sites. We show here that the cytoplasmic domain of ADAM15, metargidin, can interact specifically with Src family protein-tyrosine kinases (PTKs) and the adaptor protein Grb2 in hematopoietic cells (Jurkat, THP-1, U937, and K562 cell lines). Src homology 3 domains from several Src family PTKs including Lck, Fyn, Abl, and Src associate with ADAM15 in vitro. Dephosphorylation of cell extracts resulted in decreased association of ADAM15 with Src family PTK SH3 domains, indicating that phosphorylation influences ADAM15 interactions with its binding partners. This was confirmed in vitro for Hck, Lck, and Grb2, which showed enhanced association with tyrosine-phosphorylated glutathione S-transferase-ADAM15 cytoplasmic domain compared with unphosphorylated protein. In contrast, binding of MAD2 to ADAM15 was slightly reduced by phosphorylation of the ADAM. Immunoprecipitation of ADAM15 from Jurkat cells confirmed the association with Lck in vivo, and upon PMA stimulation, the phosphorylation level of ADAM15 was increased. Cotransfection of ADAM15 and Hck showed Hck-dependent phosphorylation of ADAM15 in vivo. Hck, and to a lesser extent Lck, phosphorylated the ADAM15 cytoplasmic domain in vitro in immune complex kinase assays. Binding of ADAM15 cytoplasmic domain to Hck and Lck was also shown by Far Western analysis. In contrast to Hck, Lck activity was not required for binding to ADAM15, as shown by treatment of cells with PP1. Deletion and point mutation analysis of the ADAM15 cytoplasmic domain confirmed the importance of the proline-rich motifs for Grb2 and Lck binding and indicated the regulatory nature of Tyr(715) and Tyr(735). These data demonstrate selective, phosphorylation-dependent interactions of ADAM15 with Src family PTKs and Grb2, which highlight the potential for integration of ADAM functions and cellular signaling.
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PMID:Phosphorylation-dependent interactions between ADAM15 cytoplasmic domain and Src family protein-tyrosine kinases. 1174 29

Protein phosphatase type 1 catalytic subunit (PP1c) is a serine/threonine phosphatase involved in the dephosphorylation of many proteins in eukaryotic cells. It associates with several known targeting or regulatory subunits that directly regulate PP1c activity toward specific substrates. The recently identified Phosphatase Nuclear Targeting Subunit (PNUTS) binds to PP1c and inhibits PP1 activity toward phosphorylase a. One of the substrates of PP1c has been shown to be the cell cycle regulatory protein, Retinoblastoma (pRb). In this study, we show that PNUTS dissociates from PP1c under mildly hypoxic cell growth conditions that lead to an increase of PP1c activity toward pRb. We developed an assay that measures pRb-directed PP1c activity and show that a GST-PNUTS fusion protein inhibits phosphatase activity toward pRb when using PP1c from cell lysates, GST-PP1c, or purified PP1c. These studies suggest that PNUTS is involved in the regulation of PP1c activity toward pRb.
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PMID:PNUTS (phosphatase nuclear targeting subunit) inhibits retinoblastoma-directed PP1 activity. 1227 Jan 15

The catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c) was shown to bind trithorax (TRX) in the yeast two-hybrid system. Interaction between PP1c and TRX was confirmed in vivo by co-immunoprecipitation from Drosophila extracts. An amino-terminal fragment of TRX, containing a putative PP1c-binding motif, was shown to be sufficient for binding to PP1c by in vitro glutathione S-transferase pull-down assays using recombinant protein and fly extracts expressing epitope tagged PP1c. Disruption of the PP1c-binding motif abolished binding, indicating that this motif is necessary for interaction with PP1. On polytene chromosomes, PP1c is found at many discrete bands, which are widely distributed along the chromosomes. Many of the sites that stain strongly for PP1c correspond to sites of TRX, consistent with a physical association of PP1c with chromatin-bound TRX. Homeotic transformations of haltere to wing in flies mutant for trx are dominantly suppressed by PP1c mutants, indicating that PP1c not only binds TRX, but is a physiologically relevant regulator of TRX function in vivo.
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PMID:Trithorax interacts with type 1 serine/threonine protein phosphatase in Drosophila. 1252 22

Cell surface receptor membrane localization is strongly dependent on protein-protein interactions often involving regulation by phosphorylation/dephosphorylation of the intracellular domains of membrane proteins. The present study was carried out to identify metabotropic glutamate receptor (mGluR) 3 regulatory binding proteins. Using the yeast two-hybrid technique, we found that the 50-aa C-terminal cytoplasmic tail of mGluR3 interacts specifically with protein phosphatase 2Calpha (PP2Calpha). This interaction was confirmed by GST pull-down and coimmunoprecipitation assays. mGluR3 interacts with PP2Calpha, beta, gamma, and delta isoforms; however, among the mGluR family only mGluR3 interacted with PP2C. The minimal interacting domain of mGluR3 comprised residues 836-855. Alignment between mGluR3 and mGluR2, a closely related group II receptor, indicated that this domain is not conserved between the two receptors. The mGluR3 cytoplasmic C-terminal tail contains one phosphorylation site for protein kinase A (Ser-845), but the phosphatase that dephosphorylates this site has not been previously identified. We find that PP2C, but not PP1, PP2A, or PP2B, dephosphorylates the mGluR3 cytoplasmic tail in vitro. The dephosphorylated form of the mGluR3 cytoplasmic tail, but not the equivalent region of mGluR2, inhibited PP2C assayed by using [32P]casein as a substrate. However, phosphorylation of the mGluR3 cytoplasmic tail at Ser-845 inhibits the interaction with PP2C. These results indicate distinct functions for mGluR2 and mGluR3 and suggest a dynamic regulation of mGluR3 by PP2C.
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PMID:Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3. 1466 50

Myosin phosphatase (PP1M) is composed of the delta isoform of the PP1 catalytic subunit (PP1cdelta), the myosin phosphatase target subunit (MYPT), and a 20 kDa subunit. Western blots detected higher amounts of the MYPT1 isoform compared to MYPT2 in whole brain extracts. The localization of MYPT1 was studied in rat brain and in primary cell cultures of neurons using specific antibodies. Analysis of lysates of brain regions for MYPT1 and PP1M by Western blots using anti-MYPT1 antibodies and by phosphatase assays with myosin as substrate suggested a ubiquitous distribution. Immunohistochemistry of tissue sections revealed that MYPT1 was distributed in all areas of the brain, with staining observed in many different cell types. Depending on the method used for fixation, the MYPT1 appeared with varying intensity in nuclei, in nucleoli, and in the cytoplasm. In primary hippocampal cultures, MYPT1 was identified by confocal microscopy in the cytoplasm and in the nucleus, whereas a predominantly cytoplasmic localization was found in cochlear nucleus cells. In cultured cells, MYPT1 and PP1cdelta colocalized with synaptophysin. PP1M activity was high in synaptosomes isolated from the cerebral cortex, but was relatively low in the postsynaptic densities. The interaction of MYPT1 with synaptophysin and with known partners (Rho-kinase, PP1cdelta) in brain extracts was shown by immunoprecipitation with anti-MYPT1. Pull-down assays from synaptosomes, using GST-MYPT1, also confirmed these interactions. In conclusion, the widespread cellular and subcellular localization of MYPT1 implies that PP1M may play an important role in the dephosphorylation of key regulatory proteins in neuronal cells.
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PMID:Localization of myosin phosphatase target subunit 1 in rat brain and in primary cultures of neuronal cells. 1533 50

Type 1 Ser/Thr protein phosphatase (PP1) has many roles in Drosophila: regulating diverse processes from chromatin condensation to transforming growth factor-beta signaling. The presence of four PP1 genes, PP1alpha87B, PP1beta9C, PP1alpha96A, and PP1alpha13C, encoding very similar proteins complicates analysis of their particular functions. Here, we report that the minor PP1 isoform PP1beta9C binds in vitro and in vivo and genetically interacts with Trithorax (TRX), the archetypal member of the Trx-G family of epigenetic regulators in Drosophila. Direct binding was demonstrated by GST pull-down experiments and PP1beta9C/TRX interaction in vivo was confirmed by coimmune precipitation from Drosophila embryonic extracts. PP1beta9C was found to be present at all TRX sites on the polytene chromosomes. Flies homo- and hemizygous for loss-of-function alleles of PP1beta9C exhibited specific wing defects when combined with various trx mutants, which indicates that PP1beta9C and TRX cooperate in Drosophila wing development.
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PMID:PP1beta9C interacts with Trithorax in Drosophila wing development. 1536 10

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

Signal Transducers and Activators of Transcription (STATs) are transcription factors shown to be activated by G protein-coupled receptors. In the present study, we demonstrate that acute morphine or [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) exposure of COS-7 cells transiently transfected with the micro-opioid receptor and STAT5A, leads to receptor-dependent tyrosine phosphorylation of STAT5A. Activation of HEK293 cells, stably expressing the micro-opioid receptor with micro-opioid agonists results in the transcriptional activation of a STAT-responsive reporter gene. Pertussis toxin has no effect on the level of STAT5A phosphorylation, while the Src inhibitor PP1 abolishes opioid-dependent STAT5A phosphorylation. All three opioid receptor subtypes -micro, delta and kappa- share the conserved motif YXXL (amino-acids 336-339 for the micro-opioid receptor), known to be critical for STAT5A/5B binding. Co-immunoprecipitation and pull-down experiments using a GST-carboxyl-terminal tail of the micro-opioid receptor and rat brain, or COS-7 cell cytosolic extracts, demonstrate the direct binding of STAT5A to this region. Mutation of the Y336 to alanine does not prevent STAT5A binding, whereas deletion of the entire putative STAT5A binding site YXXL abolishes STAT5A interaction to the carboxyl-terminal tail of the micro-opioid receptor. Collectively, our results demonstrate the association of STAT5A with the micro-opioid receptor and reveal novel signalling pathways in the regulation of transcription by the micro-opioid receptor.
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PMID:STAT5A interacts with and is phosphorylated upon activation of the mu-opioid receptor. 1585 95

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

Protein phosphatase 1delta (PP1delta) localizes to focal adhesions and associates with the focal adhesion kinase (FAK). In the present work we used deletion mutants of PP1delta and FAK to detect their reciprocally interacting domains. Dissection of PP1delta indicated 194-260 as the shortest FAK-interacting domain among those tested. Domain 194-260 encompasses several sites involved in catalysis, indirectly confirming that FAK is a PP1 substrate. Mutation of one of these sites, R220 (R220S or R220Q), did not abolish but on the contrary increased the ability of 194-260 to pull-down FAK. Such property might be exploited to detect new potential PP1 substrates. Among the FAK deletion mutants, only the C-terminal domain (684-1053, also known as FRNK) pulled-down a significant amount of PP1. The PP1 eluted from a GST-FRNK affinity column displayed Mr of 35,000 when analyzed by gel-filtration on FPLC Superose 12, indicating the presence of an isolated PP1 catalytic subunit.
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PMID:Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase. 1601 Sep 75


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