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      Neuronal calcium sensor proteins are unable to modulate NFAT activation in mammalian cells

      research-article
      1 , , *
      Biochimica et Biophysica Acta
      Elsevier Pub. Co
      Ca2+, calcium, NFAT, Nuclear Factor of Activated T-cells, NCS, Neuronal Calcium Sensor, CaBP1, Calcium Binding Protein-1, CaM, Calmodulin, CN, Calcineurin, GST, Glutathione-S-transferase, KChIP1, Potassium Channel Interacting Protein-1, GFP, Green Fluorescent Protein, SDS, Sodium Dodecyl Sulphate, PAGE, Polyacrylamide Gel Electrophoresis, HRP, Horse Radish Peroxidase, HEPES, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, EDTA, Ethylenediaminetetraacetic acid, EGTA, Ethylene glycol-bis(2-aminoethylether)-N,N,N′N′-tetra-acetic acid, NTA, N,N-Bis(carboxymethyl)glycine, PMA, Phorbol 12-myristate 13-acetate, NCS family proteins, Calmodulin, Calcineurin activity, NFAT signaling

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          Abstract

          Calcium activated gene transcription through Nuclear Factor of Activated T-cells, (NFAT) proteins, is emerging as a ubiquitous mechanism for the control of important physiological processes. Of the five mammalian NFAT isoforms, transcriptional activities of NFATs 1-4 are stimulated by a calcium driven association between the ubiquitous phosphatase calcineurin and the calcium-sensing protein calmodulin. Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity. In this study we have assessed the ability of NCS proteins to interact directly with calcineurin in vitro and report a specific if weak association between various NCS proteins and the phosphatase. In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity. Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity.

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          Most cited references44

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          A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.

          Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.
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            Calcium oscillations increase the efficiency and specificity of gene expression.

            Cytosolic calcium ([Ca2+]i) oscillations are a nearly universal mode of signalling in excitable and non-excitable cells. Although Ca2+ is known to mediate a diverse array of cell functions, it is not known whether oscillations contribute to the efficiency or specificity of signalling or are merely an inevitable consequence of the feedback control of [Ca2+]i. We have developed a Ca2+ clamp technique to investigate the roles of oscillation amplitude and frequency in regulating gene expression driven by the proinflammatory transcription factors NF-AT, Oct/OAP and NF-kappaB. Here we report that oscillations reduce the effective Ca2+ threshold for activating transcription factors, thereby increasing signal detection at low levels of stimulation. In addition, specificity is encoded by the oscillation frequency: rapid oscillations stimulate all three transcription factors, whereas infrequent oscillations activate only NF-kappaB. The genes encoding the cytokines interleukin (IL)-2 and IL-8 are also frequency-sensitive in a way that reflects their degree of dependence on NF-AT versus NF-kappaB. Our results provide direct evidence that [Ca2+]i oscillations increase both the efficacy and the information content of Ca2+ signals that lead to gene expression and cell differentiation.
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              NFAT signaling: choreographing the social lives of cells.

              Calcium signaling activates the phosphatase calcineurin and induces movement of NFATc proteins into the nucleus, where they cooperate with other proteins to form complexes on DNA. Nuclear import is opposed by kinases such as GSK3, thereby rendering transcription continuously responsive to receptor occupancy. Disruptions of the genes involved in NFAT signaling are implicating this pathway as a regulator of developmental cell-cell interactions.
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                Author and article information

                Journal
                Biochim Biophys Acta
                Biochimica et Biophysica Acta
                Elsevier Pub. Co
                0006-3002
                February 2008
                February 2008
                : 1780
                : 2
                : 240-248
                Affiliations
                The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK
                Author notes
                [* ]Corresponding author. Tel: +44 151 794 5311; fax: +44 151 794 5337. leeh@ 123456liverpool.ac.uk
                [1]

                Current address: Department of Biochemistry, School of Medical Sciences,University of Bristol, University Walk, Bristol, BS8 1TD, UK.

                Article
                BBAGEN26450
                10.1016/j.bbagen.2007.10.011
                2258317
                18005668
                4ed000d5-07fd-4abd-8347-bbe053da9b14
                © 2008 Elsevier B.V.

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 8 August 2007
                : 1 October 2007
                : 18 October 2007
                Categories
                Article

                Biochemistry
                calmodulin,nfat, nuclear factor of activated t-cells,gfp, green fluorescent protein,pma, phorbol 12-myristate 13-acetate,hepes, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid,page, polyacrylamide gel electrophoresis,ncs, neuronal calcium sensor,sds, sodium dodecyl sulphate,egta, ethylene glycol-bis(2-aminoethylether)-n,n,n′n′-tetra-acetic acid,kchip1, potassium channel interacting protein-1,calcineurin activity,edta, ethylenediaminetetraacetic acid,ncs family proteins,gst, glutathione-s-transferase,ca2+, calcium,hrp, horse radish peroxidase,cam, calmodulin,cabp1, calcium binding protein-1,nfat signaling,nta, n,n-bis(carboxymethyl)glycine,cn, calcineurin

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