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)

Inhibition of protein tyrosine phosphatase (PTP) activities by vanadate was examined in cultured rat hepatocytes. The incubation of hepatocytes with sodium orthovanadate inhibited PTP activities, measured with labeled polyglutamate tyrosine (4:1) and insulin receptor peptide (1142-1153), in a dose- and time-dependent manner. The PTP activities in cytosolic and particulate fractions were inhibited with the IC50 values of 30-50 and 2-20 microM, respectively. Vanadate-mediated inhibition of protein phosphatase, type 1 (a serine phosphatase) was less pronounced, requiring 50- to 150-fold higher concentrations. Molybdate and tungstate, the other potent inhibitors of PTPs, exerted approximately 70% less inhibition of enzyme activities compared to vanadate in intact liver cells. The cytosolic and particulate PTPs inhibited by vanadate were further resolved by fast protein liquid chromatography on Mono Q and Superose-12 columns. Vanadate exerted stable and differential inhibition of several PTPs. One of them was identified as SHPTP2 (Syp, SHP-2) in cytosolic as well as particulate fractions. Immunoprecipitation of this PTP with Syp-antibody coupled to protein A-agarose confirmed the vanadate-induced decrease in SHPTP2 activity. Vanadate did not alter the expression of SHPTP2 and its distribution between cytosolic and particulate fractions as indicated by the immunoblots. The decrease in the activities of PTPs in vanadate-treated hepatocytes in general was found to be reversed by the reducing agent dithioerythreitol. This study shows that vanadate inhibits many PTPs in intact liver cells, one of them being SHPTP2/SHP-2. The inhibition is stable after chromatography on ion-exchange and gel filtration chromatography. The enzyme inhibition seems to involve the oxidation of the thiol group of PTPs.
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PMID:Inhibition of a Src homology 2 domain containing protein tyrosine phosphatase by vanadate in the primary culture of hepatocytes. 891 24

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.
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PMID:Protein tyrosine phosphorylation in the cyanobacterium Anabaena sp. strain PCC 7120. 907 18

Among the cellular actions of vanadate ions are several that have the potential to be of significance in the regulation of protein phosphorylation. The effects of vanadate on adenosine 3',5' cyclic monophosphate (cAMP)-dependent and independent, alkali-resistant protein phosphorylation in a synaptosomal preparation from rat cortex were examined in this study. Three major vanadate-stimulated, cAMP-independent phosphoproteins (58-, 50-, and 39-kDa) and two cAMP-dependent species (37- and 32-kDa) were detectable. The potentiation between vanadate and cAMP in stimulating the phosphorylation of the latter two proteins is in contrast to the nonadditive combined effect of both on the phosphorylation of other synaptosomal proteins. The two cAMP-dependent, 32P-labeled proteins possess identical or very similar physicochemical properties to two previously cited neuronal phosphoproteins, namely, dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein-32 (DARPP-32) and inhibitor-1 (I-1). Such properties include phosphorylation by cAMP-dependent protein kinase, the presence of an alkali-resistant phosphothreonine residue, comigration on two-dimensional gel electrophoresis, dephosphorylation by type-2B protein phosphatase, and crossreactivity with specific antibodies. Costimulation by cAMP and vanadate of phosphorylation of the latter two proteins on threonine residues, at concentrations of vanadate consistent with the regulation of protein tyrosine phosphatase activity, indicates a unique interaction between these two regulators of protein phosphorylation at the nerve terminus.
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PMID:Adenosine 3',5'-cyclic monophosphate/vanadate-sensitive phosphorylation of DARPP-32- and inhibitor-1-immunoreactive proteins. 928 28

The apical membrane of distal nephron epithelium (A6) has a Ca(2+)-dependent outwardly rectifying Cl- channel with single channel conductances of 3 pS for outward current and 1 pS for inward current under the basal condition. The single channel conductance for inward currents increased as cytosolic Ca2+ concentration ([Ca2+]c) was elevated, while the single channel conductance for outward currents did not change at the range of [Ca2+]c from 10 nM to 1 mM. Insulin (100 nM) increased the single channel conductance for the inward current by increasing the sensitivity to cytosolic Ca2+ by 400-fold, but did not affect the single channel conductance for the outward current. Further, insulin increased the open probability of the channel. These effects of insulin were completely blocked by cyclosporin-A, an inhibitor of protein phosphatase type 2B (PP2B) which dephosphorylates phospho-tyrosine in addition to phosphoserine/threonine, but not by okadaic acid, an inhibitor of protein phosphatase type 1 and 2A. Further, these effects of insulin were also completely blocked by W7, an antagonist of calmodulin which is required for activation of PP2B. Lavendustin A, an inhibitor of protein tyrosine kinase (PTK), mimicked these effects of insulin; this action of lavendustin A required 1 hr after its application, while within 30 min after its application lavendustin A had no significant effects on the single channel conductance. On the other hand, lavendustin A blocked the insulin action for a relatively short time period (i.e., within 30 min after their application). However, H89 (an inhibitor of protein kinase A) or H7 (an inhibitor of protein kinases A, C and G) did not mimic the insulin action. Application of PP2B or protein tyrosine phosphatase to the cytosolic surface of the inside-out patch membrane increased the single channel conductance and the open probability as did insulin in cell-attached patches. The insulin-induced increases in single channel conductance and open probability were reversibly decreased by application of PTK catalytic subunit in the presence of ATP through a decrease in the sensitivity to cytosolic Ca2+, but not by protein kinase A. These observations suggest that as intracellular signalling of insulin action, PP2B-mediated dephosphorylation of phospho-tyrosine of the channel protein (or channel-associated protein) is a novel mechanism for regulation of single channel conductance, and that at least two different types of PTKs regulate the channel characteristics.
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PMID:Protein phosphatase 2B-dependent pathway of insulin action on single Cl- channel conductance in renal epithelium. 949 29

Protein tyrosine phosphatases were analyzed in oocytes of Ascaris suum. Phosphatases dephosphorylating modified acidic lysozyme were present in high-molecular-weight form (M(r) > 600,000) and as a 50- to 55-kDa protein in the soluble fraction. The low-molecular-weight form of the phosphatase cross-reacted with an antiserum raised against human T-cell protein tyrosine phosphatase and was not distinguishable from the 50- to 55-kDa protein tyrosine phosphatase previously described in the muscular layer of the adult worms (B. Schmid et al. 1996, Molecular and Biochemical Parasitology 77, 183-192). The low-molecular-weight form was also present on immunoblots of high-molecular-weight protein tyrosine phosphatase preparations after denaturing electrophoresis. The same or a similar form of the tyrosine phosphatase was also found in detergent extracts from the pelletal fraction. In addition, another tyrosine phosphatase of 180 kDa molecular mass that dephosphorylated myelin basic protein was also found in extracts from the soluble compartment as well as in detergent extracts from the pelletal fraction. It showed no cross-reactivity with antisera raised against soluble mammalian phosphatases and was resistant to inhibition by vanadate. While the activities of the myelin basic protein-dephosphorylating protein phosphatase remained fairly constant during early development of the oocytes, the activity of the enzyme dephosphorylating modified lysozyme in the pelletal fraction decreased to less than 10% of the initial activity between days 3 and 28 of incubation. Immunocytochemical studies of unfertilized and developing Ascaris eggs revealed association of protein tyrosine kinase and protein tyrosine phosphatase with the egg shell, in addition to their presence in the neighborhood of mitochondria. The amount of enzyme changed with the stage of development. In the larval stage (21 days) protein tyrosine kinase had increased in the chitin layer of the shell and in the nuclei while the relative amount of tyrosine phosphatase decreased in accordance with the biochemical data.
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PMID:Ascaris suum: protein phosphotyrosine phosphatases in oocytes and developing stages. 953 68

Run-down of GABA activated Cl- currents was found when rat cerebellar granule cells in culture were studied by the whole-cell patch-clamp technique in the absence of ATP in the pipette medium. This event could be prevented, even in the absence of ATP, by using the perforated-patch technique or by adding to the pipette medium either a blocker of protein tyrosine phosphatase, sodium vanadate, or deltamethrin, a blocker of the protein serine/threonine phosphatase calcineurin. Conversely, run-down could be partially induced, even in the presence of ATP, by blockers of tyrosine kinases. A reduction of GABA(A) receptor activity was also found in outside-out membrane patches when ATP was not on the membrane inside. The run-down phenomenon involved all three conductance levels found in these patches: 11, 20 and 30 pS. In all three cases it was due to a reduction of channels' open probability. The single-channel experiments showed that also in this case run-down was prevented by either sodium vanadate or deltamethrin on the membrane cytoplasmic side. Overall, through relatively unphysiological conditions (cells in culture and patch-clamp techniques), the study of the run-down phenomenon shows that the tyrosine phosphorylation state of GABA(A) receptors is of importance in maintaining it in a proper functional state. The data also show that tyrosine phosphorylation state is controlled by a protein tyrosine phosphatase, whose activity in turn is blocked via serine/threonine phosphorylation.
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PMID:Involvement of phosphatase activities in the run-down of GABA(A) receptor function in rat cerebellar granule cells in culture. 953 23

Protein tyrosine kinases and phosphatases play a vital role in the regulation of cell growth and differentiation in animal systems. However, none of these enzymes has been characterized from higher plants. In this study, we isolated a cDNA encoding a putative protein tyrosine phosphatase (PTPase) from Arabidopsis (referred to as AtPTP1). The expression level of AtPTP1 is highly sensitive to environmental stresses. High-salt conditions increased AtPTP1 mRNA levels, whereas cold treatment rapidly eliminated the AtPTP1 transcript. The recombinant AtPTP1 protein specifically hydrolyzed phosphotyrosine, but not phosphoserine/threonine, in protein substrates. Site-directed mutagenesis defined two highly conserved amino acids, cysteine-265 and aspartate-234, as being essential for the phosphatase activity of the AtPTP1 protein, suggesting a common catalytic mechanism for PTPases from all eukaryotic systems. In summary, we have identified AtPTP1 as a tyrosine-specific protein phosphatase that may function in stress responses of higher plants.
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PMID:Molecular characterization of a tyrosine-specific protein phosphatase encoded by a stress-responsive gene in Arabidopsis. 959 42

In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.
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PMID:Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin. 965 66

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of Aplysia, it recently has been suggested that an intracellular messenger-induced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the "afterdischarge. " In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10. 7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5'-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium/phospholipid-dependent protein kinase C (PKC) inhibitor peptide(19-36). Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.
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PMID:Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities. 972 8

A tight and stable complex with corresponding protein kinases and phosphatases establishes coupling between activators and inactivators. One such example is emerging from the studies of the Ras-dependent MAP kinase cascade signaling pathway. Pervanadate, a potent inhibitor of protein tyrosine phosphatase, stimulates MAP kinase and elicits cell proliferation in cultured mouse fibroblasts which is insensitive to PD 98059, the major inhibitor of upstream MEK, whereas serum- or TPA-triggered proliferation is sensitive to PD 98059. It is suggested that imbalanced coordination between protein kinase and protein phosphatase determines the cellular responses such as cell proliferation. The PD 98059-insensitive cell proliferation upon protein tyrosine phosphatase inhibition is attributed to a MEK bypass pathway.
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PMID:Pervanadate-triggered MAP kinase activation and cell proliferation are not sensitive to PD 98059. Evidence for stimulus-dependent differential PD 98059 inhibition mechanism. 974 31


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