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 Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase.
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PMID:Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance. 761 85

To assess the physiological function of Ca(2+)-dependent protein phosphatase (PP2B) in the yeast Saccharomyces cerevisiae, the phenotypes of PP2B-deficient mutants were investigated. Although PP2B was dispensable for growth under normal conditions, the mutations did, however, cause growth inhibition under certain stress circumstances. The growth of the mutants was inhibited by NaCl and LiCl, but not by KCl, CaCl2, MgCl2 or nonspecific osmotic stresses. Upon shift to high NaCl medium, intracellular Na+ levels of both wild type yeast and the mutants initially increased at a comparable rate. However, internal Na+ in wild type cells started to decline more rapidly than the mutant cells during cultivation in high NaCl medium, indicating that PP2B is important in maintaining a gradient across the membrane. The protection against salt stress was achieved, at least in part, by the stimulation of Na+ export. The maintenance of a high level of internal K+ in high NaCl medium was also PP2B-dependent. In the presence of the immunosuppressant FK506, the growth behaviour and intracellular Na+ and K+ of wild type cells in high NaCl medium became very similar to those of the PP2B-deficient mutant in a manner dependent on the presence of the FK506 binding protein.
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PMID:Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. 769 52

Protein phosphatases PPZ1 and PPZ2 represent a novel form of Ser/Thr phosphatases structurally related to type 1 phosphatases and characterized by an unusual amino-terminal region. We have found that the deletion of PPZ1 gene results in increased tolerance to Na+ and Li+ cations. Simultaneous deletion of PPZ2 gene results in an additional increase in salt tolerance. After exposure to high concentration of Li+, the intracellular content of the cation was markedly decreased in ppz1 delta ppz2 delta mutants when compared to wild type cells. No significant differences were observed between both strains when the Li+ influx was measured, but ppz1 delta ppz2 delta mutants eliminated Li+ more efficiently than wild type cells. This can be explained by the fact that expression of the ENA1 gene, which encodes the major component of the efflux system for these cations, is strongly increased in ppz1 delta ppz2 delta cells. As expected, the disruption of the PPZ genes did not complement the characteristic hypersensitivity for Na+ and Li+ of a ena1 delta strain. The lack of protein phosphatase 2B (calcineurin) has been found to decrease salt resistance by reducing the expression of the ENA1 gene. We have observed that the disruption of the PPZ genes substantially enhances the resistance of the hypersensitive calcineurin-deficient mutants. Since PPZ phosphatases have been found to be functionally related to the protein kinase C/mitogen-activated kinase pathway, we have tested bck1 or mpk1/slt2 deletion mutants and found that they do not display altered salt sensitivity. However, disruption of PPZ1 fails to increase salt resistance in a mpk1/slt2 background. In conclusion, we postulate the existence in yeast of a novel PPZ-mediated pathway involved in salt homeostasis that is opposite to and independent of the recently described calcineurin-mediated pathway.
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PMID:The PPZ protein phosphatases are important determinants of salt tolerance in yeast cells. 776 97

The Na-K-Cl cotransporter of avian salt gland is a membrane-bound 170-kDa protein that is phosphorylated in response to cAMP- and Ca(2+)-dependent secretogogues and is homologous to the Na-K-Cl cotransporter in another Cl-secreting epithelia; the shark rectal gland (Torchia, J., Lytle, C., Pon, D. J., Forbush, B., and Sen, A. K. (1992) J. Biol. Chem. 267, 25444-25450). In the present study we assess the role of Ca2+ and protein kinase C (PKC) activation on the phosphorylation of the Na-K-Cl cotransporter. Although the addition of ionomycin alone did not significantly stimulate cotransporter phosphorylation, concurrent addition of ionomycin plus the tumor promoter phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent increase in phosphorylation. Immunoprecipitation experiments, using a monoclonal antibody which specifically recognizes the cotransporter, suggested that the response to CCh or ionomycin plus PMA was quantitatively similar (5-fold) and was localized exclusively on serine residues. In contrast, when 4 alpha-phorbol was added in the presence of ionomycin, no stimulation was observed. To further assess the involvement of PKC on cotransporter phosphorylation the effects of protein kinase inhibitors were tested. Both staurosporine and calphostin C inhibited phosphorylation of the cotransporter at concentrations known to inhibit PKC, whereas the calmodulin antagonist W-7 had no significant effect. The requirement for Ca2+ was tested further by removing Ca2+ from the incubation medium and stimulating with CCh. Under these conditions, the CCh-stimulated phosphorylation was transient and, furthermore, could be completely inhibited by preloading the cells with the Ca2+ chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid) prior to stimulation. The involvement of protein phosphatases on the phosphorylation of the Na-K-Cl cotransporter was also tested. The addition of okadaic acid stimulated phosphorylation by approximately 3-fold. Taken together these results suggest that the phosphorylation state of the cotransporter involves a dynamic interplay between changes in intracellular Ca2+, PKC, and protein phosphatase activities.
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PMID:Carbachol-stimulated phosphorylation of the Na-K-Cl cotransporter of avian salt gland. Requirement for Ca2+ and PKC Activation. 796 70

The catecholamines dopamine and norepinephrine, play a central role in the regulation of sodium homeostasis and blood pressure. Dopamine inhibits tubular Na+, K(+)-ATPase activity and increases sodium excretion. Norepinephrine stimulates Na+, K(+)-ATPase activity and decreases urinary sodium excretion. The signaling pathway by which these two opposite first messengers regulate Na+, K(+)-ATPase activity involves the dopamine-specific protein phosphatase-1 inhibitor, DARPP-32, and the norepinephrine-activated protein phosphatase-2B, calcineurin. Aberrations in the renal dopamine/norepinephrine system may be the cause of alterations in the regulation of sodium excretion during ontogeny and in salt-sensitive hypertension.
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PMID:Molecular mechanisms involved in catecholamine regulation of sodium transport. 838 80

The roles of casein kinases I and II in the activation of protein phosphatase-1i (PP-1i) by glycogen synthase kinase-3 (GSK-3) were studied using enzyme preparations from porcine heart. PP-1i was activated by GSK-3 and the levels of activation achieved decreased by increasing the ionic strength (0-0.2 M KCl) in the incubation mixtures. At low ionic strength (no KCl added) casein kinase II increased the rate of activation of PP-1i by GSK-3 and the activation proceeded to a slightly greater extent (110-120%) than that obtained by GSK-3 alone. In the presence of 0.14 M KCl only a partial activation of PP-1i by GSK-3 was observed, but when casein kinase II was also added activation was restored to levels observed when PP-1i was activated by GSK-3 in the absence of salt. This effect was shown to be dependent on the concentration of casein kinase II. These results would imply that at low ionic strength casein kinase II and GSK-3 synergistically activate PP-1i as has been previously reported for the rabbit skeletal muscle enzyme (DePaoli-Roach, A. A., J. Biol. Chem. 259, 12144-12152, 1984), whereas, at physiological ionic strength, casein kinase II action may be obligatory for GSK-3 activity. Similar results were obtained when casein kinase I replaced casein kinase II.
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PMID:Hierarchical regulation by casein kinases I and II of the activation of protein phosphatase-1i by glycogen synthase kinase-3 is ionic strength dependent. 838 7

By using a 125I-calmodulin overlay assay, three major high-affinity calmodulin-binding proteins, showing apparent molecular masses of 135, 60, and 50 kDa, have been detected in purified nuclear fractions isolated from rat neurons. It has been shown that after extraction of the nuclei with nucleases and high salt, all these proteins remain strongly associated with the nuclear matrix. The 60- and 50-kDa proteins have been previously identified as subunits of the calmodulin-dependent protein kinase II. We report here the immunoblot identification of the 135-kDa calmodulin-binding protein as myosin light chain kinase. We also show that the calmodulin-dependent protein phosphatase calcineurin is present in the neuronal nuclei and associated with the nuclear matrix. The nuclear localization of both calcineurin and myosin light chain kinase has been confirmed by immunocytochemical studies.
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PMID:Nuclear calmodulin-binding proteins in rat neurons. 838 50

We have recently purified two potent and specific inhibitory polypeptides of protein phosphatase-1 from the particulate fraction of bovine thymus nuclei (Beullens, M., Van Eynde, A., Stalmans, W., and Bollen, M. (1992) J. Biol. Chem. 267, 16538-16544). Here it is reported that these inhibitors, termed NIPP-1a (18 kDa) and NIPP-1b (16 kDa), are excellent substrates (Km = 0.1 microM) for phosphorylation by protein kinase A on both Ser and Thr residues. Phosphorylation was temporally closely related with an activation of NIPP-1. Maximal phosphorylation by protein kinase A (1.5 mol of phosphate/mol of NIPP-1) caused an 8-fold increase in the concentration of NIPP-1 required for half-complete inhibition of the catalytic subunit of protein phosphatase-1, irrespective of the concentration of the phosphatase. Phosphorylation decreased the binding of NIPP-1 to immobilized protein phosphatase-1. NIPP-1 could be efficiently and completely reactivated by incubation with the catalytic subunit of protein phosphatase-2A. The type-1 catalytic subunit was much less effective, however, even when present in a molar excess to NIPP-1. Chromatography of a salt extract of the particulate nuclear fraction of Mono Q revealed three species of PP-1. One of these species, termed PP-1N alpha, contained NIPP-1 as a subunit and could be activated 6-fold by incubation with protein kinase A under phosphorylating conditions. This activation of PP-1N alpha is opposite to the known inhibition of cytoplasmic species of protein phosphatase-1 by protein kinase A.
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PMID:Inactivation of nuclear inhibitory polypeptides of protein phosphatase-1 (NIPP-1) by protein kinase A. 839 Apr 58

Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.
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PMID:Dephosphorylation of catalytic subunit of cAMP-dependent protein kinase at Thr-197 by a cellular protein phosphatase and by purified protein phosphatase-2A. 855 May 70

The yeast PMR2/ENA1 gene encodes an ATPase involved in sodium extrusion and induced by NaCl. At low salt concentrations (0.3 M) induction is mediated by the HOG-MAP kinase pathway, a system activated by non-specific osmotic stress. At high salt concentrations (0.8 M) induction is mediated by the protein phosphatase calcineurin and is specific for sodium. Protein kinase A and Sis2p/Hal3p modulate PMR2/ENA1 expression as negative and positive factors, respectively but Sis2p/Hal3p does not participate in the transduction of the salt signal. Salt stress decreases the level of cAMP and the resulting decrease in protein kinase A activity may contribute to HOG-mediated induction.
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PMID:Multiple transduction pathways regulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast. 861 70


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