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)

Sucrose-phosphate synthase (SPS) purified from spinach leaves harvested in the dark, was activated by mammalian protein phosphatase 2A (PP2A). Activation of SPS in a fraction from darkened spinach leaves was largely prevented by either okadaic acid or microcystin-LR (specific inhibitors of PPI and PP2A), while inhibitor-2 (a PP1 inhibitor) or Mg2+ (essential for PP2C) were ineffective. In vivo, okadaic acid and microcystin-LR prevented the light-induced activation of SPS and decreased sucrose biosynthesis and CO2 fixation. It is concluded that PP2A is the major SPS phosphatase in spinach. This study is the first to employ microcystin-LR for modulating protein phosphorylation in vivo.
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PMID:Sucrose-phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves. Evidence from the effects of okadaic acid and microcystin. 217 89

Phosphorylation of human erythrocyte ghost membrane proteins was found to be affected by micromolar calcium concentrations. Increasing Ca2+ concentration to 0.2 microM decreased spectrin (band 2) phosphorylation to 30 +/- 6% of control (to which no calcium was added). Decreasing calcium concentration by adding EGTA (0.2mM) to the standard membrane preparation increased spectrin phosphorylation to 575% control. This effect of Ca2+ was more pronounced at higher temperature. At 0 degree C, Ca2+ (0.05mM) had no effect on protein phosphorylation. Sodium fluoride like EGTA caused a four to five fold increase in phosphorylation. Pyrophosphate, a phosphoprotein phosphatase inhibator, had no effect. Once spectrin was phosphorylated in the presence of [gamma-32P]ATP the addition of Ca2+ or EGTA did not decrease or increase its phosphorylation. It is suggested that calcium regulates spectrin phosphorylation either by decreasing kinase activity or by decreasing substrate availability.
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PMID:Calcium regulation of magnesium dependent phosphorylation of human erythrocyte ghost spectrin. 609 25

Bovine thyroid tissue exhibited cAMP-dependent and Ca2+-dependent protein kinase activities as well as a basal (cAMP- and Ca2+-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kinase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent Km values of cAMP and Ca2+ for the membrane-bound protein kinase were 5 . 10(-8) M and 8.3 . 10(-4) M in the presence of 1 Mm EGTA), respectively. The apparent Km values of Mg2+ were 7.10-4M (without (in the cAMP and Ca2+), 5 . 10(-4) M (with cAMP) and 1.3 . 10(-3) M (with Ca2+), and those of ATP were 3.5 . 10(-5)M (with or without cAMP) and 8.5 . 10(-5) M (with Ca2+). The Ca2+-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using 32P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca2+, but was stimulated by a rather broad range (5-25 mM) of Mg2+ and Mn2+. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca2+-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.
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PMID:Properties of enzyme activities involved in protein phosphorylation-dephosphorylation of thyroid plasma membranes. 629 23

The phosphorylation of the Hsp27 complex is rapidly altered in MRC-5 cells when they are exposed to mitogens, cytokines, stress, or serine/threonine protein phosphatase inhibitors. Here we performed experiments to identify which cellular protein phosphatase (PP1, PP2A, or PP2B) is responsible for the in vivo phosphorylation/dephosphorylation of Hsp27. In their purified forms, PP2A dephosphorylates Hsp27 more effectively than PP2B, whereas PP1 is weakly active. Measurements of enzyme activity of lysates derived from inhibitor-treated cells indicated that Hsp27 phosphatase activity is equally sensitive to okadaic acid (PPI/PP2A inhibitor) and cyclosporin (PP2B inhibitor) and that both okadaic acid and cyclosporin treatment inhibited Hsp27 phosphatase activity additively. Together the in vitro data suggest that both PP2A and PP2B can dephosphorylate Hsp27. However, the phosphorylation of Hsp27 in vivo is only affected when cells are treated with PP1 and PP2A inhibitors (okadaic acid, calyculin A) or cantharidin (PP2A inhibitor), but not the PP2B inhibitor, cyclosporin A, suggesting PP2A to be the main enzyme dephosphorylating Hsp27 in the cells. Purification and immunoblotting of Hsp27 phosphatase from MRC-5 cells also suggest it to be PP2A and not PP1 or PP2B. The ability of PP2A to dephosphorylate Hsp27 is shown to be regulated by the phosphorylation state of PP2A itself.
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PMID:Dephosphorylation of the small heat shock protein Hsp27 in vivo by protein phosphatase 2A. 751 Jul 4

The amino acid sequence of a novel mammalian protein phosphatase, termed PPX (and designated PPP4 in the human genome nomenclature), has been deduced from the cDNA and shown to be 65% identical to PP2A alpha and PP2A beta and 45% identical to PPI isoforms, the predicted molecular mass being 35 kDa. PPX was expressed in the baculovirus system. Its substrate specificity and sensitivity to the inhibitors, okadaic acid and microcystin, were similar (but not identical) to the catalytic subunit of PP2A. However, PPX did not bind the 65 kDa regulatory subunit of PP2A. The intracellular localization of PPX was investigated by immunofluorescence using two different antibodies raised against bacterially expressed PPX and a PPX-specific peptide. These showed that although PPX was distributed throughout the cytoplasm and the nucleus, intense staining occurred at centrosomes. The centrosomal staining was apparent in interphase and at all stages of mitosis, except telophase. In contrast, antibodies directed against bacterially expressed PP2A were not specifically localized to centrosomes. The human autoantibody #5051, which stains the pericentriolar material, colocalizes with PPX antibodies, suggesting that PPX may play a role in microtubule nucleation.
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PMID:PPX, a novel protein serine/threonine phosphatase localized to centrosomes. 838 57

New method for purification of phosphatase inhibitor 1 (PPI-1) was developed which avoid the phosphorylation of PPI-1 during the purification and provides a high yield of highly pure preparation. Using this preparation, it was shown that PPI-1 was stoichiometrically phosphorylated by cGMP-dependent protein kinase at Thr-35 and the phosphorylated PPI-1 potently inhibited protein phosphatase 1. Addition of the phosphorylated PPI-1 to beta-escin-skinned single smooth muscle cells resulted in force development of the cells at the submaximal pCa2+. The results suggest that the phosphorylation of PPI-1 can be the mechanism for modifying the Ca2+ sensitivity of smooth muscle contractile response.
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PMID:Enhancement of smooth muscle contraction with protein phosphatase inhibitor 1: activation of inhibitor 1 by cGMP-dependent protein kinase. 860 41

Protein phosphatase inhibitor-1 (PPI-1) has been shown to be present in heart tissue and smooth muscle. Whether PPI-1 is present in cardiomyocytes is not known. The purpose of this study was to determine whether PPI-1 is present and is hormonally regulated in cardiomyocytes. A trichloroacetic acid (TCA) extract enriched in PPI-1 was isolated from guinea pig ventricular cardiomyocytes. The TCA extract inhibited the activity of type 1 protein phosphatase by 20 +/- 4% (n = 3 expts). On phosphorylation by the catalytic subunit of adenosine 3', 5'-cyclic monophosphate-dependent protein kinase, the extent of this inhibition was augmented to 4.5-fold. Dephosphorylation of the phosphorylated TCA extract by type 2 protein phosphatase reduced inhibition to 2 +/- 0.2% (n = 3 expts). To determine whether isoproterenol increases phosphorylation of PPI-1 in cardiomyocytes, the TCA extracts were prepared from cardiomyocytes treated with 1 microM isoproterenol and from untreated cardiomyocytes. The inhibitory activity of the TCA extract in untreated cardiomyocytes was 25 +/- 3% (n = 3 expts) and increased to 75 +/- 2% (n = 3 expts) in isoproterenol-treated cardiomyocytes. With the use of a rabbit skeletal muscle PPI-1 antibody, immunoblots of the TCA extract of cardiomyocytes identified a 28-kDa protein. A 28-kDa protein was also immunoprecipitated from a TCA extract isolated from isoproterenol-treated 32P-labeled cardiomyocytes. The immunoprecipitation was blocked by the addition of excess amounts of purified rabbit skeletal muscle PPI-1. Isoproterenol-treated cardiomyocytes increased the phosphorylation of the 28-kDa protein by 232 +/- 20% (n = 3 expts) compared with untreated cardiomyocytes. We conclude that 1) the 28-kDa protein is PPI-1, 2) PPI-1 is present in ventricular cardiomyocytes, and 3) PPI-1 is hormonally regulated. A decrease in type 1 protein phosphatase activity through phosphorylation of PPI-1 may be an important pathway for augmenting cardiac contractility.
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PMID:Evidence for presence and hormonal regulation of protein phosphatase inhibitor-1 in ventricular cardiomyocyte. 896 52

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

1. Large-conductance Ca(2+)- and voltage-activated potassium (BK) channels are important regulators of cellular excitability. Here, we present a patch-clamp electrophysiological analysis of splice-variant-specific regulation by the synthetic glucocorticoid dexamethasone (DEX) of BK channels consisting of cloned STREX or ZERO alpha-subunit variants expressed in human embryonic kidney (HEK 293) cells. 2. STREX channels in isolated membrane patches were inhibited by protein kinase A (PKA) and this was blocked on pre-treatment of intact cells with DEX (100 nM) for 2 h. 3. The effect of DEX required the synthesis of new mRNA and protein. Furthermore, it required protein phosphatase 2A (PP2A)-like activity intimately associated with the channels, as it was blocked by 10 nM okadaic acid but not by the specific protein phosphatase-1 inhibitor peptide PPI-2. 4. ZERO variant channels that lack the STREX insert were activated by PKA but were not influenced by DEX. ZERO channels containing a mutant STREX domain (S4(STREX)A) were also activated by PKA. Importantly, DEX blocked PKA activation of S4(STREX)A channels in a PP2A-dependent manner. 5. Taken together, the STREX domain is crucial for glucocorticoid regulation of BK channels through a PP2A-type enzyme. Moreover, glucocorticoids appear to induce a generic set of proteins in different types of cells, the actions of which depend on the expression of cell-specific targets.
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PMID:Alternative splicing determines sensitivity of murine calcium-activated potassium channels to glucocorticoids. 1171 61

Since the glucose-lowering effects of vanadium could be related to increased muscle glycogen synthesis, we examined the in vivo effects of vanadium and insulin treatment on glycogen synthase (GS) activation in Zucker fatty rats. The GS fractional activity (GSFA), protein phosphatase-1 (PP1), and glycogen synthase kinase-3 (GSK-3) activity were determined in fatty and lean rats following treatment with bis(maltolato)oxovanadium(IV) (BMOV) for 3 weeks (0.2 mmol/kg/day) administered in drinking water. Skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). In both lean and fatty rats, muscle GSFA was significantly increased at 15 min following insulin stimulation. Vanadium treatment resulted in decreased insulin levels and improved insulin sensitivity in the fatty rats. Interestingly, this treatment stimulated muscle GSFA by 2-fold (p < 0.05) and increased insulin-stimulated PP1 activity by 77% (p < 0.05) in the fatty rats as compared to untreated rats. Insulin resistance, vanadium and insulin in vivo treatment did not affect muscle GSK-3beta activity in either fatty or lean rats. Therefore, an impaired insulin sensitivity in the Zucker fatty rats was improved following vanadium treatment, resulting in an enhanced muscle glucose metabolism through increased GS and insulin-stimulated PPI activity.
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PMID:Oral treatment with vanadium of Zucker fatty rats activates muscle glycogen synthesis and insulin-stimulated protein phosphatase-1 activity. 1219 Jan 10


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