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)

The bumetanide-sensitive component of pHi recovery from an NH4Cl-induced acute alkaline load was used as a measure of Na(+)-K(+)-2Cl- cotransport activity in rat parotid acini. Acinar treatment with NaF/AlCl3 (15 mM NaF plus 10 microM AlCl3) induced a 5-fold stimulation in the initial rate of bumetanide-sensitive pHi recovery. This effect was dependent on NaF concentration (K1/2 approximately 7 mM) and was blunted in the presence of the Al3+ chelator desferal mesylate suggesting that it might be due to the aluminofluoride ion, AlF-4. NaF/AlCl3 treatment did not increase acinar intracellular cAMP levels but did result in an increase in intracellular calcium concentration (from 87 +/- 5 to 181 +/- 2 nM) and in acinar cell shrinkage (12 +/- 1%). But the stimulation of the Na(+)-K(+)-2Cl- cotransporter by NaF/AlCl3 persisted in acini which had been depleted of their intracellular Ca2+ stores. In these acini no effect of NaF/AlCl3 on intracellular calcium or cell volume was observed, indicating that stimulation of the cotransporter was not secondary to either of these phenomena. The effect of NaF/AlCl3 on the cotransporter was blocked by the protein kinase inhibitor K252a indicating the involvement of a protein phosphorylation event. This result is consistent with either NaF/AlCl3-dependent protein kinase activation or phosphatase inhibition. The stimulation of the cotransporter by NaF/AlCl3 was mimicked by the protein phosphatase inhibitor calyculin A; however, this effect was not blocked by K252a suggesting that a different protein kinase from that associated with NaF/AlCl3 may be involved. The data indicate that the Na(+)-K(+)-2Cl- cotransporter in this tissue is under tight regulatory control, in all likelihood via multiple protein kinase/phosphatase systems. The physiological roles of these regulatory events in modulating acinar fluid secretion driven by the Na(+)-K(+)-2Cl- cotransporter remain to be elucidated.
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PMID:Activation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors. 138 25

The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12.FK-506.calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807-815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506-binding protein. Our characterization of the FKBP12.FK-506.CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12.FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12.FK-506, FKBP51.FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.
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PMID:Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex. 138 26

Migration of human polymorphonuclear neutrophils on vitronectin is dependent on repeated transient increases in the concentration of intracellular free calcium ([Ca2+]i). A specific peptide inhibitor of the Ca(2+)-calmodulin-dependent phosphatase calcineurin was introduced into the cytoplasm of neutrophils. The peptide inhibited neutrophil migration on vitronectin by interfering with the release of the cells from sites of attachment. A similar reduction in motility on vitronectin occurred when cells were treated with the immunosuppressant FK506, which also inhibits calcineurin when bound to its binding protein, FKBP. These results indicate that a rise in [Ca2+]i reduces integrin-mediated adhesion to vitronectin by a mechanism that requires calcineurin activity.
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PMID:Inhibition of neutrophil chemokinesis on vitronectin by inhibitors of calcineurin. 138 29

Cyclosporin A and FK-506 are important therapeutic agents that have found widespread use in preventing graft rejection during tissue transplantation. Research efforts aimed at elucidating the molecular mechanism of action of these drugs have, in addition to defining their immunosuppressive functions, led to the identification of two new gene families whose products may function as components of several diverse signal transduction pathways. In the presence of the immunosuppressive drugs, some members of the receptor families interact with the Ca2+/calmodulin-dependent protein phosphatase 2B, also known as calcineurin. Inhibition of phosphatase activity may effect several downstream biochemical processes. In this way, cyclosporin A and FK-506 have proved to be useful probes of signaling events in both lymphocytic and other cell types.
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PMID:FK-506 and cyclosporin A: immunosuppressive mechanism of action and beyond. 138 51

This work describes the relationship between the cytoplasmic free calcium concentration ([Ca2+]i) measured by the fluorescent Ca(2+)-indicator fura-2, the phosphorylation of the myosin light chain and the force development in the phasic longitudinal smooth muscle layer of guinea-pig ileum and the tonic rabbit pulmonary artery. The close temporal relationship between the rise in cytoplasmic Ca2+ and the initiation of force development as well as the rather good correlation between cytoplasmic Ca2+ and force maintenance leaves little doubt about cytoplasmic Ca2+ being the primary regulator of force. However the present experimental evidence indicate that [Ca2+]i and force are not invariably tightly coupled in smooth muscle. A dissociation between the time course of [Ca2+]i and force was found in the tonic rabbit pulmonary artery but not in the phasic ileum of the guinea-pig. In contrast, there was a pronounced decline in the Ca(2+)-sensitivity of the contractile apparatus (desensitization to Ca2+) in the guinea-pig ileum during prolonged depolarization, an observation not found in the pulmonary artery. Such desensitization could reflect the activation of highly active myosin light chain phosphatase(s) and the different Ca(2+)-sensitivities of tonic and phasic smooth muscles can, at least in part, be due to differences in myosin light chain kinase/phosphatase activity ratios. The sensitivity of the regulatory/contractile apparatus to Ca2+ was increased by agonists in intact and in permeabilized preparations. Furthermore a different sensitizing potentiation between different agonists was observed. The mechanism of the "sensitization" of the contractile response to Ca2+ could act through the activation of the phosphorylation of a protein phosphatase inhibitor, thereby inhibiting the myosin light chain phosphatase. The experiments therefore show that different levels of tension may be present at the same [Ca2+]i and indicate that the Ca(2+)-sensitivity can be modulated in smooth muscle.
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PMID:Modulation of the Ca(2+)-sensitivity in phasic and tonic smooth muscle. 141 85

Okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, profoundly influenced the activity of the NADPH oxidase of human neutrophils. It strongly inhibited stimulation of superoxide generation by phorbol 12-myristate 13-acetate (PMA) and impaired translocation of protein kinase activity and of the two cytosolic components p47-phox and p67-phox to the plasma membrane. The increase in the phosphorylation of the cytochrome b-245 subunits p22-phox and gp91-phox after stimulation was also blocked. Inhibition of activity was associated with a decrease in cytosolic free Ca2+ and was reversed by the Ca2+ ionophore A23187, which also restored protein translocation and phosphorylation of the cytochrome. This effect of A23187 was itself blocked by preincubation with cyclosporin A, suggesting that calcineurin might be involved in the re-activation process. In contrast with PMA, the response to the bacterial peptide fMet-Leu-Phe was greatly prolonged after an initial decrease in the rate of onset of NADPH oxidase activity.
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PMID:Okadaic acid produces changes in phosphorylation and translocation of proteins and in intracellular calcium in human neutrophils. Relationship with the activation of the NADPH oxidase by different stimuli. 141 26

A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to immunoblot with anti-tyrosine phosphate antibodies. Taken together, these results indicate that insulin activates this novel cytosolic protein kinase by a mechanism that causes its phosphorylation on serine or threonine residues.
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PMID:Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver. 153 38

The delta-subspecies of protein kinase C (delta PKC) was purified to near homogeneity from the Triton X-100 extract of the rat brain particulate fraction by successive chromatographies on S-Sepharose fast flow, phenyl 5PW, heparin 5PW, hydroxyapatite, and Mono Q columns. The purified enzyme was a doublet with molecular masses of 78 and 76 kDa on SDS/PAGE. The doublet proteins were separated partially by Mono Q column chromatography; both were recognized by the antibodies raised against synthetic oligopeptides, parts of the deduced amino acid sequence of the rat delta PKC. Protein phosphatase 2A treatment suggested that the 78-kDa protein was a phosphorylated form of the 76-kDa protein. To confirm the structural and genetic identity of the doublet proteins, delta PKC was expressed in COS 7 cells by transfecting its cDNA-constructed plasmid and was purified for comparison. This recombinant enzyme was also a doublet. The enzymes isolated from the brain and COS 7 cells showed identical reactivities with delta PKC-specific antibodies, chromatographic behaviors, and V8 protease peptide mappings. In addition, these two enzyme preparations were indistinguishable from each other in their responses to phosphatidylserine, diacylglycerol, phorbol esters, free fatty acids, Ca2+, and enzyme inhibitors. Comparison was also made between the enzymologic properties of delta PKC and alpha PKC, which were distinctly different from each other.
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PMID:Isolation and characterization of delta-subspecies of protein kinase C from rat brain. 154 50

We have reviewed the literature, which supports an important role for dopamine withdrawal in the regulation of PRL secretion. Concentrations of dopamine in the hypophyseal portal circulation are sufficient to occupy the majority of dopamine receptors (1) and tonically suppress PRL secretion (20-26). Brief escapes from dopaminergic regulation associated with the secretion of PRL have been observed (37-41). Therefore, dopamine regulates secretion of PRL both by occupancy of, as well as dissociation from, specific D2 dopamine receptors. The rapid off rate from its receptor (2) is consistent with signals transmitted through brief decreases in dopamine concentration. The removal of dopamine for 10 min results in increases in intracellular cAMP and presumably activation of protein kinase A (39, 138) as well as activation of phospholipase C (137, 138) and protein kinase C (136). The removal of dopamine results directly in the release of PRL (37-41). Furthermore, the brief removal of dopamine results in the long-term potentiation of the PRL-releasing action of TRH (38-40). The potentiating action of dopamine withdrawal appears to be mediated by the activation of protein kinase A since pretreatment with VIP, a hormone that signals via protein kinase A, also potentiates the action of TRH (39). TRH stimulates PRL release via Ca2+/protein kinase C (177-184). The potentiating action of dopamine removal is selective for the Ca2+/protein kinase C pathway since dopamine removal does not potentiate the PRL-secreting action of VIP (38, 87, 92). The action of TRH is potentiated up to 30 min after the return of dopamine and the suppression of PRL to basal levels (38). In Fig. 10, dopamine dissociation from its receptor or VIP association to its receptor are shown separated by a broken line to indicate that by the time the potentiation of the action of TRH is tested, either dopamine is again occupying its receptor or VIP is no longer present. Therefore, the effect of protein kinase A activation is remembered by the lactotroph. We hypothesize that the responsiveness of the cell to TRH is potentiated by the phosphorylation of proteins by protein kinase A. Two potential substrates for protein kinase A are voltage-dependent Ca2+ channels and protein phosphatase inhibitors that would prolong the action of protein kinase C. When TRH occupies its receptor, intracellular Ca2+ levels are increased first from intracellular stores and subsequently by extracellular Ca2+ influx (187-189). Intracellular Ca2+ is mobilized by increased levels of IP3(128). Extracellular Ca2+ enters the lactotroph via voltage-dependent Ca2+ channels (189, 190).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Dissociation of dopamine from its receptor as a signal in the pleiotropic hypothalamic regulation of prolactin secretion. 161 63

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.
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PMID:Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A. 164 16


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