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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity. The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex. In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments. A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts. Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTR(His10)) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni(2+)-nitrilotriacetic acid-agarose. Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTR(His10) after dithiobis (sulfosuccinimidyl propionate) exposure. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel.
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PMID:Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C. 1050 64

Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 +/- 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 +/- 10% per 100 meq/l). Increases in membrane PP2A activity (270 +/- 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 +/- 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes.
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PMID:Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes. 1056 85

Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.
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PMID:Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density. 1064 97

The tumor suppressor gene PTEN encodes a 55-kDa enzyme that hydrolyzes both protein phosphotyrosyl and 3-phosphorylated inositol phospholipids in vitro. We have found that the latter activity is physiologically relevant in intact T cells. Expression of active PTEN lead to a 50% loss of transfected cells due to increased apoptosis, which was completely prevented by coexpression of a constitutively active, membrane-bound form of protein kinase B. A mutant of PTEN selectively lacking lipid phosphatase activity, but retaining protein phosphatase activity, had no effects on cell number. Active (but not mutant) PTEN also decreased TCR-induced activation of the mitogen-activated protein kinase ERK2 (extracellular signal-related kinase 2), as seen after inhibition of phosphatidylinositol 3-kinase. Our data indicate that PTEN is a phosphatidylinositol 3-phosphatase in T cells, and we suggest that PTEN may play a role in the regulation of T cell survival and TCR signaling by directly opposing phosphatidylinositol 3-kinase.
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PMID:The tumor suppressor PTEN regulates T cell survival and antigen receptor signaling by acting as a phosphatidylinositol 3-phosphatase. 1065 43

H/K-ATPase preparations (the G1 membrane) from pig stomach contain both kinases and phosphatases and show reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) residues of the alpha-chain of H/K-ATPase. The Tyr-kinase is sensitive to genistein and quercetin and recognized by anti-c-Src antibody. The Ser-kinase is dependent on Ca(2)(+) (K(0.5) = 0.9 microM), sensitive to a PKC inhibitor, and recognized by antibodies against PKCalpha and PKCbetaII. The addition of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS) caused a dramatic increase in the phosphorylation of added synthetic copolymer substrates and permitted the phosphorylation of maltose-binding proteins fused with the N-terminal domain of alpha-chains. The phosphotyrosine phosphatase was inhibited by vanadate. The phosphoserine phosphatase was inhibited by okadaic acid and by inhibitor-2. The presence of protein phosphatase-1 was immunologically detected. Column chromatographic separation of CHAPS-solubilized G1 membrane and others indicate the apparent molecular weight of the Src-kinase to be approximately 60 kDa, the PKCalpha and/or PKCbII to be approximately 80 kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be approximately 35 kDa. These data show that these membrane-bound enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role of which is unknown.
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PMID:Membrane enzyme systems responsible for the Ca(2+)-dependent phosphorylation of Ser(27), the independent phosphorylation of Tyr(10) and Tyr(7), and the dephosphorylation of these phosphorylated residues in the alpha-chain of H/K-ATPase. 1078 91

A phosphoprotein phosphatase (PPase M-I) that dephosphorylates serine and threonine residues of histones was isolated from the goat cauda-epididymal sperm plasma membrane and partially characterized. The PPase was solubilized from the sperm membrane by treating it with 0.1 N NaOH at pH 11.4 and the solubilized enzyme was partially purified by concanavalin A-sepharose affinity chromatography and high-performance liquid chromatography (HPLC), revealing it to be a 520-kDa protein. The PPase gave a single protein band in native polyacrylamide gel electrophoresis (PAGE), but in the presence of SDS it resolved into multiple proteins (35-170 kDa) showing that the isolated enzyme contained a few contaminating proteins. The enzyme is a glycoprotein because it binds with high affinity to concanavalin A. It was maximally active at pH 8.0 and its activity was not dependent on bivalent metal ions. The enzyme is a specific phosphatase as it displayed higher affinity for dephosphorylation of large molecular weight phosphate esters. The PPase showed broad substrate specificity for the dephosphorylation of a variety of proteins. The membrane-associated PPase was strongly (70-80%) inhibited by detergents (0.5%) such as Nonidet P-40, Lubrol PX, Triton X-100 and Tween-20. Pyrophosphate (5 mm) and orthovanadate (400 microM) had no significant effect on the activity of the isolated PPase whereas polyamines such as spermine (10 mM) and spermidine (10 mM) slightly inhibited (20%) the enzymatic activity. Inorganic phosphate (10 mM) and NaF (10 mM), the well-known inhibitors of the cytosolic PPases, had no appreciable effect on the activity of PPase M-I, indicating that the membrane-bound PPase is distinct from the cytosolic PPases. The enzyme was radiolabelled when the intact spermatozoa were subjected to lactoperoxidase-mediated radioiodination reaction. The results show that the PPase M-I is an ecto-enzyme that may play an important role in sperm physiology by causing the dephosphorylation of the sperm outer surface phosphoproteins.
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PMID:Partial purification and characterization of a phosphoprotein phosphatase from sperm plasma membrane. 1097 6

Gastric vesicles purified from acid-secreting rabbit stomach display K(+) permeability manifested by the valinomycin-independent proton pumping of H(+)-K(+)-ATPase as monitored by acridine orange quenching. This apparent K(+) permeability is attenuated by the treatment of the membrane with 5 mM Mg(2+), and this phenomenon has been attributed to membrane-bound phosphoprotein phosphatase. However, with the exception of the nonspecific inhibitor pyrophosphate, protein phosphatase inhibitors failed to inhibit the loss of K(+) permeability. Preincubation of the membrane with neomycin, a phospholipase C inhibitor, surrogated the effect of Mg(2+), whereas another inhibitor, U-73122, did not. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) restored the attenuated K(+) permeability by treatment with either Mg(2+) or neomycin. Furthermore, either phosphatidylinositol bound to phosphatidylinositol transfer protein or phosphatidylinositol 4,5,6-trisphosphate (PIP(3)) surrogated the effect of PIP(2). Mg(2+) and neomycin reduced K(+) permeability in the membrane as determined by Rb(+) influx and K(+)-dependent H(+) diffusion. Treatment with Mg(2+) reduced the contents of PIP(2) and PIP(3) in the membrane. These results suggest that PIP(2) and/or PIP(3) maintain K(+) permeability, which is essential for proton pumping in the apical membrane of the secreting parietal cell.
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PMID:Phosphatidylinositol is essential determinant for K+ permeability involved in gastric proton pumping. 1151 91

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are regulated tightly by protein kinases and phosphatases. The regulatory domain of CFTR has about 20 potential sites for phosphorylation by protein kinases A (PKA) and C (PKC). The reason for this large number of sites is not known, however their conservation from fish to humans implies that they play important roles in vivo. PKA is an important activator, and its stimulation of CFTR is enhanced by PKC via mechanisms which are not fully understood. The physiological stimuli of CFTR are not known for some epithelia, and it appears likely that other serine/threonine and even tyrosine kinases also regulate CFTR in particular tissues. Phosphatases that deactivate CFTR have yet to be identified definitively at the molecular level, however CFTR is regulated by a membrane-bound form of protein phosphatase-2C (PP2C) in several cell types. Patch-clamp studies of channel rundown, co-immunoprecipitation, chemical cross-linking studies, and pull-down assays all indicate that CFTR and PP2C are closely associated within a stable regulatory complex. Understanding the regulation of CFTR by PP2C is a priority due to its potential as a target for pharmacotherapies in the treatment of cystic fibrosis.
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PMID:Regulation of the CFTR channel by phosphorylation. 1184 11

The presence of cAMP-dependent protein kinase (PKA) in the plasma membrane compartment and its association with an A-kinase anchoring protein (AKAP150) is implicated in mediating cAMP regulatory events in the rat myometrium. The association of PKA with purified myometrial plasma membrane declined gradually between Day 16 and Day 21 of gestation, with a decrease of 53% +/- 11% of the catalytic subunit and of 61% +/- 7% of the regulatory subunit at Day 21 compared with Day 19. To determine the role of progesterone in this association, pregnancy was prolonged by administration of progesterone or shortened by administration of the antiprogestin RU486. Progesterone treatment maintained PKA association with plasma membrane at Day 21 at 123% +/- 23% (catalytic subunit) and 92% +/- 4% (regulatory subunit) of Day 19 levels. In contrast, protein phosphatase 1, protein phosphatase 2B, phospholipase Cbeta(3), and AKAP150 concentrations in the plasma membrane did not change over this interval or with progesterone treatment. Changes in PKA coimmunoprecipitated with membrane-associated AKAP150 paralleled those in total plasma membrane on Days 19 and 21 and on Day 21 following progesterone treatment. In contrast, plasma membrane PKA catalytic and regulatory subunits decreased by 20 h after RU486 injection on Day 15 of pregnancy to levels resembling those on Day 21. These data indicate that progesterone prevents the decline in PKA associated with myometrial plasma membrane and with AKAP150 in the pregnant rat. The decrease in membrane-bound PKA between Days 19 and 21 and after RU486 treatment precedes the onset of parturition in both experimental paradigms. The loss of plasma membrane PKA may be critical for the decrease in the inhibitory effect of cAMP on oxytocin-induced phosphatidylinositide turnover that occurs near the end of pregnancy and may contribute to enhanced myometrial contractile responsiveness near term.
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PMID:Progesterone prevents the pregnancy-related decline in protein kinase A association with rat myometrial plasma membrane and A-kinase anchoring protein. 1213 3

In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B' subunit, B56 epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B' subunit (sequenced to be B56 alpha) or with the B alpha regulatory subunit have no phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56 epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (approximately 20 ng/mouse retina) and 9 times more of the C subunit (approximately 4 ng/mouse retina) than of the B56 epsilon subunit (approximately 0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56 epsilon subunit, greatly prefers phosphorylated phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic phosducin and membrane-bound rhodopsin, by the same enzyme.
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PMID:Light-driven translocation of the protein phosphatase 2A complex regulates light/dark dephosphorylation of phosducin and rhodopsin. 1242 13


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