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)

Ionic channels regulated by extracellular Ca2+ concentration ([Ca2+]o) were examined in freshly isolated rabbit osteoclasts. K+ current was suppressed by intracellular and extracellular Cs+ ions. In this condition, high [Ca2+]o evoked an outwardly rectifying current with a reversal potential of about -25 mV. When the concentration of extracellular Cl ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4',4-diisothiocyanostilbene-2' 2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl- channels. Cd2+ or Ni2+ caused a transient activation of the Cl- current in contrast to the sustained activation elicited by Ca2+. Intracellular 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl- current. Either when the osmolarity of extracellular medium was increased, or when 100 microM cAMP was dissolved in the patch pipette solution, high [Ca2+]o still elicited the Cl- current, indicating that the divalent cation-induced Cl- current was carried through Ca(2+)-activated Cl- channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl- current, indicating that the activation of Cl- current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl- channels under whole cell clamp. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activated the Cl current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl current was mediated through a G protein, and that an increase in [Ca2+]i was critical for the activation of Cl-channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca(2+)-activated Cl- channels.
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PMID:Activation of Cl- channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts. 884 38

Tyrosine kinases (TK) and G proteins act as second, messengers for intracellular signal transduction. TK activates the cascade of protein phosphorylation. G proteins are heterodimer complex with alpha, beta, and gamma subunits. PLC activated by GTP-binding alpha subunit lyses membrane phosphatidyl inositol (PI), releasing diacyl glycerol (DAG) and inositol trisphosphate (IP3). IP3 releases calcium into cytoplasm to activate calcineurin, causing a NF-AT cytoplasmic factor (NF-ATc) to translocate to nucleus. DAG activates protein kinase C (PKC), which synthesizes another nuclear factor NF-ATn. When NF-ATc and NF-ATn assemble to form the complex on the promoter site of DNA, transcription of IL-2 mRNA begins. PKC also induces phosphorylation of I-kappa B to release NF-kappa B. The complex of CsA or FK506 with CyP or FKBP, respectively, inhibits the activation of calcineurin. FKBP-binding rapamycin inhibits cell proliferation and differentiation by inactivation of p70 s6 kinase. RS61443 and mizoribine influence specifically on the de novo pathway of purine biosynthesis. DSG may bind to Hsc 70 and inhibit the translocation of NF-kappa B into nucleus. FTY720 induces lymphocyte-specific apoptosis, independently on Fas-antigen expressions. by modulating bcl-2 genes.
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PMID:[Transplantation immunology and immunosuppressive drug]. 901 Aug 51

This is the first identification of a Ca2+-inhibitable casein kinase (CPK) which we have isolated from the 100000 x g supernatant of Paramecium cell homogenates. The 1000-fold enriched CPK activity depends on millimolar Mg2+ and is inhibited by low concentrations of heparin or by > or = 100 microM Ca2+. Enzyme activity is stimulated by polylysine or polyarginine with either casein or with specific casein kinase-2 (CK-2) peptide substrates (RRRDDDSDDD and RREEETEEE). The enzymic properties are similar with GTP instead of ATP. CPK does not undergo autophosphorylation. In gel kinase assays, enzyme activity is associated with a 36 kDa band. Calmodulin as another characteristic substrate for mammalian CK-2 has not been phosphorylated by this protein kinase. Besides casein, CPK phosphorylates in vitro the catalytic subunit of bovine brain calcineurin (CaN), a typical substrate of type 1 mammalian casein kinase (CK-1) in vitro. Again this phosphorylation is significantly reduced by Ca2+. Thus, CPK combines aspects of different casein kinases, but it is clearly different from any type known by its Ca2+ inhibition. Since CPK also phosphorylates the exocytosis-sensitive phosphoprotein, PP63, in Paramecium, which is known to be dephosphorylated by CaN, an antagonistic Ca2+-effect during phosphorylation/dephosphorylation cycles may be relevant for exocytosis regulation.
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PMID:A novel, calcium-inhibitable casein kinase in Paramecium cells. 903 1

1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel with a conductance of 31 +/- 3 pS. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1. 3. Intracellular ATP is required to prevent run-down of IRK in whole-cell mode. Single channel activity disappeared in inside-out patches exposed to ATP-free solution and in cell-attached patches on cells exposed to metabolic inhibition (KCN, 2-deoxyglucose). 4. The non-hydrolysable ATP analogues, ATP gamma S and adenylyl imidodiphosphate (AMP-PNP), did not prevent run-down. Run-down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A). 5. GTP gamma S and AlF4- inhibited IRK, also in the presence of ATP. GTP beta S antagonized the GTP gamma S effect. Pretreatment of the cells with PTX did not affect the GTP gamma S-induced inhibition. Okadaic acid, however, slowed this inhibition. 6. Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run-down. 7. We conclude that run-down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX-insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein-dependent activation of a phosphatase.
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PMID:Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells. 940 63

Mouse FT210 cells at 39 degreesC cannot enter mitosis but arrest in G2 phase, because they lack Cdc2 kinase activity as a result of a temperature-sensitive lesion in the cdc2 gene. Incubation of arrested cells with the protein phosphatase 1 and 2A inhibitor okadaic acid induces morphologically normal chromosome condensation. We now show that okadaic acid also induces two other landmark events of early mitosis, nuclear lamina depolymerization and centrosome separation, in the absence of Cdc2 kinase activity. Okadaic acid-induced entry into mitosis is accompanied by partial activation of Cdc25C and may be prevented by tyrosine phosphatase inhibitors and by the protein kinase inhibitor staurosporine, suggesting that Cdc25C and kinases distinct from Cdc2 are required for these mitotic events. Using in-gel assays, we show that a 45-kDa protein kinase normally activated at mitosis is also activated by okadaic acid independently of Cdc2 kinase. The 45-kDa kinase can utilize GTP, is stimulated by spermine and is inhibited by heparin. These properties are characteristic of the kinase CK2, but immunoprecipitation studies indicate that it is not CK2. The data underline the importance of a tyrosine phosphatase, possibly Cdc25C, and of kinases other than Cdc2 in the structural changes the cell undergoes at mitosis, and indicate that entry into mitosis involves the activation of multiple kinases working in concert with Cdc2 kinase.
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PMID:Entry into mitosis without Cdc2 kinase activation. 978 81

A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.
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PMID:Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation. 1021 59

In lactating rats, suckling renders mammotropes more responsive to prolactin (PRL)-releasing stimuli and less responsive to PRL-inhibiting secretagogues. We have previously shown that a decrease in the activity of protein phosphatase 2A (PP2A) may be responsible for the decrease in responsiveness to the inhibitory secretagogue dopamine (DA). In our present experiments, we have studied the involvement of the adenylate cyclase (AC), stimulatory and inhibitory GTP-binding proteins and also the role of PP2A in the sensitization phenomenon. Pituitary cells obtained from mother rats separated from their pups for 4 h prior to dispersion (non-suckled), suckled for 10 or 30 min after the separation period (suckled) and without separation (continual suckling) were incubated in the presence of different doses of forskolin to activate AC and DA. In a further study, pituitary cells of non-suckled rats were pretreated with cholera toxin (CTX) or pertussis toxin (PTX) and tested for the stimulatory action of forskolin or TRH on PRL release. Ocadaic acid (OA) pretreatment has been used to investigate the involvement of PP2A. Hormone secretion was measured by the reverse hemolytic plaque assay (RHPA). Our results have shown that cells from non-suckled rats were unresponsive to forskolin. A 10-min suckling stimulus sensitizes pituitary mammotropes to respond with a PRL release to a dose-dependent activation of AC by forskolin. This sensitization of AC becomes a permanent feature of the cells when suckling continues for an additional 20 min. We have also found that pituitary mammotropes from non-suckled dams respond to forskolin or TRH with PRL release when they were preincubated with either PTX or the PP2A inhibitor OA. It clearly indicates that the non-responsive pituitary can be shifted to the responsive stage by uncoupling of inhibitory G-protein from its receptor as well as by inhibition of PP2A. This latter finding, consonant with our previous results, suggests that suckling may cause selective changes in the function of G(i) of mammotropes due to a rapid phosphorylation which can remove tonic, GTP-dependent inhibitory function.
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PMID:Inhibition of protein phosphatase 2A (PP2A) mimics suckling-induced sensitization of mammotropes: involvement of a pertussis toxin (PTX) sensitive G-protein and the adenylate cyclase (AC). 1037 12

The aim of the present study was to investigate whether mechanisms distal to the regulation of Ca2-influx are involved in tolbutamide-induced stimulation and adrenaline- and somatostatin- induced inhibition of insulin secretion in INS-1 cells. Using the patch clamp method, the membrane voltage was either kept constant at -70 mV, or Ca2+-influx was activated by short depolarising pulses to 0 my. These pulses induced an increase in cellular capacitance (Cm) caused by fusion of secretory granules with the plasma membrane. Tolbutamide did not alter, neither Cm under voltage clamp at -70 mV nor increases of Cm due to voltage pulses. The inhibitors of secretion, adrenaline and somatostatin, counteracted the augmentation of [Ca2+]i which was induced by glucose, tolbutamide and forskolin. In the voltage clamp mode, however, where no changes of [Ca2]i. were observed, adrenaline but not somatostatin inhibited the increase of Cm caused by depolarizing voltage pulses. The adrenaline effect on Cm was dependent on the addition of GTP to the pipette solution. When GTP was replaced by GDPbetaS or GTPgammaS, the effect of adrenaline on Cm was abolished. The blockade of calcineurin, by the addition of calcineurin inhibitory peptide (CIP) to the pipette solution, did not affect the adrenaline-induced inhibition of Cm. Moreover. After incubation of the cells with deltamethrin, a calcineurin inhibitor, the stimulation of secretion was attenuated, but the adrenaline-induced inhibition was not affected. Our results suggest that adrenaline-induced inhibition of insulin secretion involves a site of action directly related to the exocytotic membrane fusion. In contrast, the stimulator tolbutamide and the inhibitor somatostatin had no direct effect on exocytosis in INS-1 cells.
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PMID:Effects of adrenaline and tolbutamide on insulin secretion in INS-1 cells under voltage control. 1084 99

1. We used the patch-clamp technique, in conjunction with membrane capacitance measurement, fluorescence measurement of intracellular calcium concentration ([Ca(2+)](i)), and flash photolysis of caged Ca(2+) to study exo- and endocytosis in identified rat corticotrophs. 2. Exocytosis stimulated by depolarization pulses was typically followed by a 'slow' endocytosis that retrieved the membrane with a time constant of approximately 6 s. The efficiency (the endocytosis/exocytosis amplitude ratio) of 'slow' endocytosis was approximately 1.2 at [Ca(2+)](i) < 3 microM and increased to approximately 1.6 at [Ca(2+)](i) > 3 microM. 3. Whole-cell dialysis through a patch pipette did not affect the kinetics and the efficiency of 'slow' endocytosis, but the amplitude of exocytosis was reduced. 4. 'Slow' endocytosis did not require sustained [Ca(2+)](i) elevation and its kinetics was only weakly [Ca(2+)](i) dependent. Our results suggest that 'slow' endocytosis involves a Ca(2+) sensor with a high Ca(2+) affinity (approximately 500 nM). 5. At high [Ca(2+)](i) (> 10 microM), the 'slow' endocytosis was frequently preceded by a 'fast' endocytosis that comprised multiple steps of rapid decrease in membrane capacitance. 6. Neither calmodulin nor calcineurin appeared to be the Ca(2+) sensor for endocytosis because the two forms of endocytosis were not affected by the calmodulin inhibitor calmidazolium (500 microM) or the calcineurin inhibitors cyclosporin A (1 microM) and calcineurin autoinhibitory peptide (1 mg ml(-1)). Ba(2+), a poor activator of calmodulin, could support both forms of endocytosis but slowed the kinetics of 'slow' endocytosis approximately 2-fold. 7. Non-hydrolysable analogues of GTP (GDP-beta-S) and ATP (ATP-gamma-S) also failed to inhibit either form of endocytosis, indicating that neither GTP nor ATP was essential for endocytosis. 8. We suggest that the high Ca(2+) affinity of 'slow' endocytosis may be important for maintaining continuous cycles of exocytosis-endocytosis during sustained adrenocorticotropin secretion in corticotrophs.
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PMID:Endocytosis in identified rat corticotrophs. 1138

The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein.
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PMID:Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors. 1222 6


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