Ca2+/Calmodulin-independent Activation of Calcineurin from Dictyostelium by Unsaturated Long Chain Fatty Acids

Kessen, Ursula; Schaloske, Ralph; Aichem, Annette; Mutzel, Rupert

doi:10.1074/jbc.274.53.37821

Cited by 32 publications

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“…Screening of the ZAP cDNA Expression Library Using 35 S-VU1-CaM-Titration of bacteriophage and screening of the cDNA library were essentially carried out as described previously (39). Variations in protocol were implemented with respect to the use of 35 S-radiolabeled recombinant CaM as a probe.…”

Section: Methodsmentioning

confidence: 99%

“…Variations in protocol were implemented with respect to the use of 35 S-radiolabeled recombinant CaM as a probe. An overnight culture of an XL1-Blue strain of Escherichia coli in LB media containing 0.2% maltose was pelleted and resuspended in 10 mM MgSO 4 .…”

Section: Methodsmentioning

confidence: 99%

“…Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using 35 S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca 2؉ -dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum.…”

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confidence: 99%

“…Dictyostelium CN has been extensively characterized (31)(32)(33). FK506 and cyclosporin A do not inhibit growth or aggregation but do affect cell differentiation, yet the regulation and roles of Dictyostelium CN are still under analysis (34,35). In addition to acting as CaM's effectors, CaMBPs bind different targets based upon cytosolic Ca 2ϩ levels; they localize CaMs to subcellular locales and act as capacitators in CaM function (36).…”

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Nucleomorphin

Myre

O’Day

2002

Journal of Biological Chemistry

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Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using 35 . The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca 2؉ -dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Nucleomorphin

Myre

O’Day

2002

Journal of Biological Chemistry

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Ganglioside contained in the neuronal tissue‐enriched acidic protein of 22 kDa (NAP‐22) fraction prepared from the detergent‐resistant membrane microdomain of rat brain inhibits the phosphatase activity of calcineurin

Kobayashi

Silva

Kumanogoh

et al. 2015

J of Neuroscience Research

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Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). Neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) is one of the major protein components of neuronal DRM. To determine the cellular function of NAP-22, interacting proteins were screened with an immunoprecipitation assay, and calcineurin (CaN) was detected. Further studies with NAP-22 prepared from DRM and CaN expressed in bacteria showed the binding of these proteins and a dose-dependent inhibitory effect of the NAP-22 fraction on the phosphatase activity of CaN. On the other hand, NAP-22 expressed in bacteria showed low binding to CaN and a weak inhibitory effect on phosphatase activity. To solve this discrepancy, identification of a nonprotein component that modulates CaN activity in the DRM-derived NAP-22 fraction was attempted. After lyophilization, a lipid fraction was extracted with chloroform/methanol. The lipid fraction showed an inhibitory effect on CaN without NAP-22, and further fractionation of the extract with thin-layer chromatography showed the presence of several lipid bands having an inhibitory effect on CaN. The mobility of these bands coincided with that of authentic ganglioside (GM1a, GD1a, GD1b, and GT1b), and authentic ganglioside showed an inhibitory effect on CaN. Treatment of lipid with endoglycoceramidase, which degrades ganglioside to glycochain and ceramide, caused a diminution of the inhibitory effect. These results show that DRM-derived NAP-22 binds several lipids, including ganglioside, and that ganglioside inhibits the phosphatase activity of CaN.

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Functional and phenotypic relevance of differentially expressed proteins in calcineurin mutants of Caenorhabditis elegans

et al. 2006

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Calcineurin is a heterodimeric serine/threonine protein phosphatase, important for many cellular processes such as T-cell regulation, cardiac hypertrophy and kidney development. We previously reported the characterization of Caenorhabditis elegans calcineurin mutants as providing a simple but excellent genetic model system for studying in vivo functions of calcineurin. Calcineurin loss-of-function mutants, cnb-1(lf), and gain-of-function mutants, tax-6(gf), show certain opposite phenotypes as well as some similar phenotypes. In order to explain the phenotypic similarity observed in both loss-of-function and gain-of-function mutants, we examined the proteins that followed similar trends in both mutants relative to wild-type worms by using 2-DE. Interestingly, VHA-13, HSP-6 and phosphoenolpyruvate carboxykinase are down-regulated in both mutants. A total of 96 differentially regulated proteins were identified by MALDI-TOF/MS. Among these, 42 proteins are up-regulated and 54 proteins are down-regulated in calcineurin mutants. Furthermore, knock-down of about 30% of the genes, which are down-regulated in calcineurin mutants, showed some of the phenotypes of calcineurin-null mutants. This analysis suggests the functional relevance of these proteins to calcineurin activity in C. elegans.

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Ca2+/Calmodulin-independent Activation of Calcineurin from Dictyostelium by Unsaturated Long Chain Fatty Acids

Cited by 32 publications

References 40 publications

Nucleomorphin

Nucleomorphin

Ganglioside contained in the neuronal tissue‐enriched acidic protein of 22 kDa (NAP‐22) fraction prepared from the detergent‐resistant membrane microdomain of rat brain inhibits the phosphatase activity of calcineurin

Functional and phenotypic relevance of differentially expressed proteins in calcineurin mutants of Caenorhabditis elegans

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