Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.
Mol Cell Biol 1995 Jul
PMID:Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells. 779 92

Characteristics of the cytokine response in resident mouse macrophages to certain Gram-positive and Gram-negative bacteria have been investigated by monitoring the expression of mRNA encoding interleukin-1 alpha and -beta (IL-1 alpha/beta) and tumor necrosis factor-alpha (TNF-alpha). Expression of these cytokine mRNAs occurred within 30-60 min. Both the flavonoid quercetin and phloretin inhibited the expression of IL-1 alpha/beta as well as TNF-alpha mRNA, with quercetin being more potent than phloretin and TNF-alpha expression somewhat more sensitive than that of IL-1 alpha/beta. Expression of all three cytokine mRNAs was also inhibited by prostaglandin E2, with an IC50 of > 1 microM, but not by the phosphodiesterase inhibitor pentoxifylline, although lipopolysaccharide-induced expression of TNF-alpha mRNA was inhibited. Down-regulation of phorbol ester-sensitive isoforms of protein kinase C had virtually no effect on the cytokine response to bacteria, and treatment of resting macrophages with phorbol ester did not cause expression of any of the cytokine mRNAs investigated. Among protein phosphatase inhibitors, cyclosporin A caused extensive inhibition of bacteria-induced expression of both IL-1 alpha/beta and TNF-alpha mRNA, while okadaic acid in itself caused selective induction of TNF-alpha, but not IL-1 alpha/beta mRNA, with a sharp peak at 0.3 microM concentration. At higher concentrations of okadaic acid, at which protein/phosphatase 2B/calcineurin would also be inhibited, the induction was completely reversed. This suggests that critical phosphorylation events, counteracted by one or more okadaic acid-sensitive protein phosphatase(s), and a dephosphorylation event carried out by a cyclosporin-sensitive protein phosphatase are both necessary for transcriptional activation of the TNF-alpha gene.
Mol Immunol 1995 Feb
PMID:Cyclosporin-sensitive expression of cytokine mRNA in mouse macrophages responding to bacteria. 787 67

Molecular cloning of human, mouse and rat brain CaM-stimulated phosphatase has suggested the existence of two genes for the alpha subunit of the enzymes. A alpha and A beta fragments of A alpha and A beta from rat brain library have been expressed in bacteria to produce specific anti-calcineurin A alpha and anti-calcineurin A beta antibodies (Kuno et al., J Neurochem 58: 1643-1651, 1992). Alternative mRNA splicing gives rise to additional calcineurin isozymes with some containing an insertion sequence of ATVEAIEADE. Antibody against synthetic peptide of this insertion sequence has been raised in this study. Three CaM-stimulated phosphatase isozymes previously purified from bovine brain (BPI, BPII, BPIII) (Yokoyama & Wang, J Biol Chem 266: 14822-14829, 1991), along with the bacterially expressed rat A alpha and A beta fragments, were analyzed by two calcineurin alpha subunit monoclonal antibodies VJ6 and VD3, the rat anti-calcineurin A alpha and anti-calcineurin A beta specific polyclonal antibodies, and the insertion peptide antibody. The bovine brain CaM-stimulated phosphatase isozymes BPI and BPIII reacted with both anti-calcineurin A alpha and anti-calcineurin A beta antibodies. While BPII reacted with anti-calcineurin A alpha but not anti-calcineurin A beta antibody, it differed from the expressed A alpha fragment in immunoreactivity towards the monoclonal antibodies. The results show that the bovine brain CaM-stimulated phosphatase isozymes cannot be simply categorized as derived from A alpha or A beta genes products.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1994 Mar 30
PMID:Immunological approach to identify calmodulin-stimulated phosphatase isozymes from bovine brain. 796 92

The X-ray structure of a decameric form of a complex of human cyclophilin A (CypA) with the immunosuppressive drug cyclosporin A (CsA) has been determined. The crystals of space group P43212 with cell dimensions a = b = 95.2 A, c = 280.0 A have five copies of the cyclophilin A/cyclosporin A complex in the asymmetric unit. The structure was solved by molecular replacement techniques, using a known cyclophilin A model. Procedures were developed to construct a self-rotation function using the results of cross-rotation searches. The comparison of experimental and constructed self-rotation maps was an important aid in selecting the correct rotation function solution. The translation functions revealed the presence of a cyclic pentamer. A crystallographic dimer axis passes through the non-crystallographic 5-fold rotation axis of the pentameric asymmetric unit, and generates a decameric "sandwich" of CypA/CsA heterodimers that has 52 symmetry. The five CypA/CsA protomers were refined independently using all data to 2.8 A giving a final crystallographic R-factor of 15.7%. Despite the constraints due to the packing arrangement within the decamer, the CypA and CsA conformations are similar to other CypA/CsA structures determined by X-ray crystallography and NMR spectroscopy. The hydrophobic CsA molecules are embedded in the middle of the decameric sandwich with only 20% of their surface exposed to solvent. The binding loop of CsA (residues 1 to 3 and 9 to 11) comprising 42% of the CsA surface, is buried in the peptidyl-prolyl-cis-trans isomerase active site of the cognate binding partner CypA, while the effector loop (residues 4 to 8) packs in the core of the decamer making hydrogen-bonding and van der Waals contacts with three neighbouring molecules. The environment of CsA in the decamer has been analysed and may provide a mimic for the interactions likely to occur between the CypA/CsA complex and its biological target calcineurin. There is no evidence to suggest that the decameric sandwich itself plays a role in immunosuppression by inhibiting calcineurin. However, the chaperone/foldase activity of CypA could require oligomer formation for its biological function.
J Mol Biol 1994 Dec 09
PMID:The molecular replacement solution and X-ray refinement to 2.8 A of a decameric complex of human cyclophilin A with the immunosuppressive drug cyclosporin A. 799 Jan 29

The solution structure of the periplasmic cyclophilin type cis-trans peptidyl-prolyl isomerase from Escherichia coli (167 residues, MW > 18.200) has been determined using multidimensional heteronuclear NMR spectroscopy and distance geometry calculations. The structure determination is based on a total of 1720 NMR-derived restraints (1566 distance and 101 phi and 53 chi 1 torsion angle restraints). Twelve distance geometry structures were calculated, and the average root-mean-square (rms) deviation about the mean backbone coordinate positions is 0.84 +/- 0.18 A for the backbone atoms of residues 5-165 of the ensemble. The three-dimensional structure of E. coli cyclophilin consists of an eight-stranded antiparallel beta-sheet barrel capped by alpha-helices. The average coordinates of the backbone atoms of the core residues of E. coli cyclophilin have an rms deviation of 1.44 A, with conserved regions in the crystal structure of unligated human T cell cyclophilin [Ke, H. (1992) J. Mol. Biol. 228, 539-550]. Four regions proximal to the active site differ substantially and may determine protein substrate specificity, sensitivity to cyclosporin A, and the composite drug:protein surface required to inhibit calcineurin. A residue essential for isomerase activity in human T cell cyclophilin (His126) is replaced by Tyr122 in E. coli cyclophilin without affecting enzymatic activity.
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PMID:Three-dimensional solution structure of Escherichia coli periplasmic cyclophilin. 813 Jan 88

Calcineurin is a calmodulin-dependent serine-threonine phosphatase found in many cell types but most abundant in neurons. To determine its localization in developing neurons, dissociated cultures from embryonic day 15 rat cerebellum were analyzed immunocytochemically after treatment with cytoskeletal-disrupting drugs. During the initial outgrowth of neurites, calcineurin is enriched in growth cones where its localization depends upon the integrity of both microtubules and actin filaments. Treatment with cytochalasin shifts calcineurin from the growth cone to the neurite shaft, and with nocadozole calcineurin translocates to the cell body. Therefore calcineurin is well positioned to mediate interactions between cytoskeletal systems during neurite elongation. By 14 d in culture, when the neurons have developed extensive neuronal contacts and synapses are present, calcineurin is predominantly in the neurite shaft. Incubation of cultured cells with Cyclosporin A or a specific peptide, both of which selectively inhibit calcineurin's phosphatase activity, prevented axonal elongation. Because the microtubule-associated protein tau appears to play a key role in asymmetric neurite elongation, we examined modifications in its phosphorylation state resulting from calcineurin inhibition. In contrast to the normal development of cerebellar macroneurons in which reactivity with the phosphorylation-dependent antibody, tau-1, progressively increases, there was a persistent inhibition of tau-1 reactivity in cells exposed to Cyclosporin A. These findings suggest a role for calcineurin in regulating tau phosphorylation and possibly modulating other steps required for the determination of polarity.
Mol Biol Cell 1993 Dec
PMID:Calcineurin is associated with the cytoskeleton of cultured neurons and has a role in the acquisition of polarity. 816 6

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation.
Mol Biol Cell 1994 Jan
PMID:Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells: involvement of the conserved lymphokine element 0. 818 61

The crystal structure of the calcium-binding protein calmodulin is used to model the immunologically important calcineurin subunit B. The rough structure is produced by computer-aided homology modeling. Refinement of this using molecular dynamics leads to a suggested structure which appears to satisfy reasonable hydrophilicity and hydrogen-bonding criteria. In the absence of a crystal structure, the model may prove useful in modeling of its interactions with the phosphatase catalytic subunit calcineurin A, and help to explain the calcium modulation of this protein.
J Mol Graph 1993 Mar
PMID:Tertiary structure of calcineurin B by homology modeling. 838 12

Calcineurin is a serine/threonine protein phosphatase which catalyzes the hydrolysis of both phosphoseryl/phosphothreonyl and phosphotyrosyl proteins as well as low molecular weight compounds such as p-nitrophenyl phosphate. It is a hetero-dimeric protein consisting of a 60 kDa A chain and 19 kDa B chain. Calcineurin A is organized into functionally distinct domains such as a catalytic domain, a calcineurin B binding domain, a calmodulin-binding domain, and an inhibitory domain. Calcineurin B has four EF-hand calcium binding domains with a secondary structure that is homologous to calmodulin but its metal binding properties are more similar to troponin-C. The N-terminal myristoyl group of calcineurin B might play a role in the interaction between subunits A and B during phosphorylation/dephosphorylation processes. Crystals of size 0.125 x 0.07 x 0.03 mm and 0.7 x 0.03 x 0.02 mm have been obtained for calcineurin and the A subunit respectively. Crystals of calcineurin show strong diffraction to 5.3 A and weak diffraction to 3.0 A on rotating anode operated at 50 kV and 100 mA. Further work is in progress to improve the X-ray diffraction quality of these crystals and to obtain well diffracting crystals of calcineurin B.
Mol Cell Biochem
PMID:Preliminary crystallization studies of calmodulin-dependent protein phosphatase (calcineurin) from bovine brain. 856 21

The PMC1 gene in Saccharomyces cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work showed that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. We now report that calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter was slightly diminished and a pmc1::lacZ reporter was induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. We propose that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions.
Mol Cell Biol 1996 May
PMID:Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. 862 89


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