Available online at www.sciencedirect.com

Biochimica et Biophysica Acta 1780 (2008) 240–248 www.elsevier.com/locate/bbagen

Neuronal calcium sensor are unable to modulate NFAT activation in mammalian cells ⁎ Daniel J. Fitzgerald 1, Robert D. Burgoyne, Lee P. Haynes

The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK Received 8 August 2007; received in revised form 1 October 2007; accepted 18 October 2007 Available online 25 October 2007

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 . 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/NFATsignalling 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. © 2007 Elsevier B.V. Open access under CC BY license.

Keywords: NCS family proteins; Calmodulin; Calcineurin activity; NFAT signaling

1. Introduction (NFAT) family of transcription factors. This family of gene regulatory proteins is composed of five members (NFAT1-5) [3,4], Calcineurin (CN) [1] is a serine/threonine specific protein that were first identified as fundamental components of the re- phosphatase present in all eukaryotes that is responsible for the combinatorial immune system of vertebrates [5], but which have modulation of numerous cell signalling pathways. Calcineurin now been implicated in a far broader range of diverse and im- interacts with the ubiquitous EF-hand containing calcium (Ca2+) portant physiological processes. Expression of NFAT proteins is sensor calmodulin (CaM) [2] and this obligate association drives not restricted to cells of the immune system, demonstrated by their calcium dependent phosphatase activity, one of the primary presence and effect on key functions in a selection of other cell and cellular targets of which are the Nuclear Factor of Activated T-cell tissue types including brain and muscle. In a neuronal setting, NFATs have been implicated in axonal remodelling, synaptic Abbreviations: Ca2+, calcium; NFAT, Nuclear Factor of Activated T-cells; plasticity and memory function [6-9]. Transcriptional events me- NCS, Neuronal Calcium Sensor; CaBP1, Calcium Binding Protein-1; CaM, diated by activated NFATs have been linked to aspects of car- Calmodulin; CN, Calcineurin; GST, Glutathione-S-transferase; KChIP1, diovascular development [10,11] and they have also been Potassium Channel Interacting Protein-1; GFP, Green Fluorescent Protein; SDS, Sodium Dodecyl Sulphate; PAGE, Polyacrylamide Gel Electrophoresis; suggested to drive proliferative and apoptotic pathways in epi- HRP, Horse Radish Peroxidase; HEPES, 4-(2-Hydroxyethyl)piperazine-1- thelial cells, fibroblasts, pre-adipocytes, pancreatic β-cells and ethanesulfonic acid; EDTA, Ethylenediaminetetraacetic acid; EGTA, Ethylene osteoblasts [12-16]. The discovery that NFAT proteins can glycol-bis(2-aminoethylether)-N,N,N′N′-tetra-acetic acid; NTA, N,N-Bis(car- influence cell cycle regulation has led to the proposal that they boxymethyl)glycine; PMA, Phorbol 12-myristate 13-acetate may also be involved in certain aspects of tumorigenesis [17]. ⁎ Corresponding author. Tel: +44 151 794 5311; fax: +44 151 794 5337. E-mail address: [email protected] (L.P. Haynes). NFAT proteins in unstimulated cells are heavily phosphory- 1 Current address: Department of Biochemistry, School of Medical Sciences, lated on multiple serine residues by of a number of constitutively University of Bristol, University Walk, Bristol, BS8 1TD, UK. active [18]. This modification acts to mask a nuclear

0304-4165 © 2007 Elsevier B.V. Open access under CC BY license. doi:10.1016/j.bbagen.2007.10.011 D.J. Fitzgerald et al. / Biochimica et Biophysica Acta 1780 (2008) 240–248 241 localisation signal and under such circumstances NFATs remain effect on NFAT dephosphorylation in live HeLa cells in response 2+ predominantly cytosolic and transcriptionally inactive. Stimula- to elevation of [Ca ]i. These data are supported by studies tion of cells with agonists coupled to the phospholipase-C/IP3 indicating that the nuclear translocation of NFAT on elevation of 2+ 2+ generating pathway, depletion of endoplasmic reticulum Ca [Ca ]i to cell nuclei is unaffected in cells expressing NCS-1 or stores and subsequent sustained elevation of intracellular Ca2+ NCS-1 mutants. We therefore suggest that NCS proteins, al- 2+ 2+ concentration ([Ca ]i) due to opening of plasma membrane Ca though exhibiting a detectable in vitro affinity for CN, may not be release activated Ca2+ channels elicits the rapid dephosphoryla- likely to act as physiologically relevant modulators of CN activity tion of NFAT proteins, their nuclear import and upregulation of or NFAT mediated gene transcription in intact cells. target gene expression [3]. Under such circumstances, the Ca2+ signal is integrated upstream of NFAT by CN in conjunction with 2. Materials and methods CaM and it is this protein complex that represents the critical regulatory mechanism for NFAT dephosphorylation and hence 2.1. Recombinant proteins activation of NFAT dependent gene expression. The relationship All recombinant GST fusion proteins used in this study were expressed and between CN/CaM and NFATs is so deeply intertwined that purified as previously described [26]. Purified recombinant calcineurin was nuclear translocation of NFAT proteins has been employed as a obtained from Sigma (Poole, UK) or Biomol (Exeter, UK). Unless otherwise robust assay of CN activity in intact mammalian cells [19-21] and stated all chemicals were of analytical grade and obtained from Sigma. has even found application in large scale screens for proteins involved in calcium induced calcium release [22]. 2.2. Small Scale GST protein binding assays CaM is the primordial member of a superfamily of small EF- 2+ In all binding assays, recombinant GST-fusions proteins (1 μM) were hand containing Ca binding proteins that function to transduce μ 2+ immobilised by incubation with 40 l of glutathione cellulose (GST cellulose) spatial and temporal cellular Ca signals. Other members of the resin (50% bead slurry, Bioline, London, UK) that had been pre-washed in CaM family include the neuronal calcium sensor (NCS) sub- binding buffer (KCl 50 mM, HEPES 20 mM (pH 7.4), EGTA 5 mM, NTA 2+ μ 2+ group of neuronal/neuroendocrine specific proteins [23,24]. 5 mM, CaCl2 4.6 mM (giving a free [Ca ]of1 M)) (+Ca conditions) or 2+ Although only distantly related (21% sequence identity between binding buffer with no added CaCl2 (- Ca conditions) by incubation with constant agitation for 30 min/4 °C. Recombinant calcineurin (1 μM) or bovine CaM and NCS-1) published in vitro studies have identified brain cytosol (∼1 mg, dialysed against the appropriate±Ca2+ binding buffer) potential overlap in target protein interactions between CaM and were then added to immobilised GST proteins in a 100 μl total binding reaction NCS-1 including an apparent positive interaction of both volume and samples incubated with constant agitation for 2 hrs at 4 °C. GST proteins with CN [25]. Since CaM exhibits high and ubiquitous cellulose beads were pelleted by centrifugation (5,000 rpm/1 min/4 °C) and expression these observations led to the speculation that NCS washed with 1 ml of the appropriate binding buffer. This wash step was repeated three times and final bead pellets extracted into 50 μl SDS dissociation buffer proteins may simply represent redundant or alternative mod- (125 mM HEPES pH (6.8), 10% (v/v) sucrose, 10% (v/v) glycerol, 4% (w/v) ulators of classical CaM targets. Evidence from our laboratory SDS, 1% β-mercaptoethanol, 2 mM EDTA) and boiling for 5 min. Samples and others has gone some way to disproving this idea with the were resolved on SDS-PAGE (12.5% gel) and transferred to nitrocellulose filters identification of NCS specific binding proteins that, at present, for western blotting by transverse electrophoresis. Bound calcineurin was de- have no documented interaction with CaM [26]. Further evi- tected using a monoclonal antibody (1:1000, Sigma) followed by incubation with anti-mouse-HRP (1:400, Sigma) and visualisation with ECL reagents. All dence for distinct functionality of the NCS protein family is 2+ western blots were quantitated using ImageJ (National Institutes of Health) apparent from their higher Ca affinity compared to CaM, their densitometry software. differential cellular/tissue distributions and their non-redun- dancy revealed in genetic studies [23,27]. Together these data 2.3. Large scale GST protein binding assays indicate that NCS proteins possess the requisite biochemical properties to allow them respond to unique Ca2+ signals and to This binding assay is essentially as described in [26]. Briefly, each recom- – bind to distinct effector proteins. The possibility exists however binant GST-fusion protein (5 10 mg), including free GST as control, was immobilised onto 3 ml glutathione-Sepharose 4B resin that had been pre-washed that overlap between CaM and NCS target specificities may be extensively with binding buffer by incubation for 2 h at 4 °C with constant of physiological significance and in the case of CN, identifica- agitation. Clarified bovine brain cytosolic extract was applied to the GST affinity tion of a functionally relevant interaction with NCS proteins columns and binding allowed to proceed for 16 h at 4 °C with constant agitation. Each column was washed with a minimum 50 volumes of binding buffer and would be of great interest in view of recent findings implicating 2+ CN activity in aspects of neuronal function. specific Ca -dependent binding proteins eluted using binding buffer containing no added Ca2+(calcium-free or CF buffer). An additional high-salt elution step In order to resolve some of the outstanding questions con- with binding buffer supplemented with 1 M NaCl and no added Ca2+ (High Salt cerning potentially redundant functions of NCS proteins we have or HS buffer) was used to isolate potential Ca2+-independent binding inter- examined whether or not they are able to elicit CN activation in actions. Eluted protein fractions were concentrated by methanol precipitation the physiologically relevant setting of intact mammalian cells and pellets extracted into SDS dissociation buffer by boiling for 5 min. All using NFAT activation as a coupled reporter. We present in vitro western blots were quantitated using ImageJ (National Institutes of Health) densitometry software. biochemical data indicating that various recombinantly expressed NCS family members are able to directly bind CN although to 2.4. Sulfo-SBED cross-linking experiments lower levels compared with CaM. Consistent with these data we have also determined that expression of NCS-1, mutants of this The manufacturers protocol provided by Pierce (Rockford, IL, USA) was protein that have previously been demonstrated to disrupt NCS-1 followed with minor modifications. All steps, except those indicated, were function in vivo, along with other NCS family members have no carried out in the dark. Bait recombinant protein of interest (∼5 mg) was dialysed 242 D.J. Fitzgerald et al. / Biochimica et Biophysica Acta 1780 (2008) 240–248 against binding buffer overnight at 4 °C. Immediately before use, the contents of examine whether the two proteins directly associate. To confirm μ one tube of No-Weigh Sulfo-SBED (Pierce) was dissolved in 22 l of DMSO, a direct interaction we first performed a series of in vitro binding this was then added to 1 ml of dialysed protein. Sulfo-SBED/protein mixes were incubated at room temperature for 30 minutes, centrifuged briefly to remove any assays utilising various recombinantly expressed and purified precipitated, hydrolysed, Sulfo-SBED, and dialysed overnight against binding NCS proteins to act as baits and where either purified recom- buffer. Bovine brain cytosol (500 μl, ∼4 mg protein) dialysed against binding binant CN or bovine brain cytosol were employed as a source of buffer was added to 500μl of the Sulfo-SBED/protein mix. This mixture was prey protein. Initially, purified CN (1 μM) was incubated in the incubated on a rotator at room temperature for 1 hour. After this step, samples μ ∼ presence of various GST-tagged NCS proteins (also at 1 M) in were exposed to long-wave UV illumination (365 nm) at a distance of 5cm μ 2+ from source for 15 minutes. During this step an appropriate volume of Neu- binding buffer containing 1 M free Ca (Fig. 1a, upper panel). travidin beads (Pierce) were washed extensively in binding buffer, 100 μl added CaM was included in this assay as a positive control for CN to the bait/cytosol mixture and samples incubated on a rotator for 1 hour at room binding (Fig. 1a, upper panel, Calmodulin Sepharose). CN was temperature. Bait/cytosol mixes were then centrifuged at 13,000 rpm for 1 minute, observed to bind directly to GST-NCS-1, GST-, supernatants removed and beads washed with 1 ml of binding buffer. This step was GST--δ and GST-CaBP1 but not GST-KChIP1, repeated 6 times. Disulfide bond reduction was then achieved by incubating with 1 ml of 50 mM DTT for 1 hour at room temperature. Beads were subsequently despite equal loading of the proteins (Fig. 1a, lower panel), centrifuged and washed extensively with binding buffer. After the final wash, indicating some degree of interaction specificity. It was also 100 μl of SDS dissociation buffer was added to the beads and samples boiled for apparent from this analysis that, under identical experimental 10 minutes. Samples were separated on SDS PAGE (12.5% gel) and western conditions, seven-fold less CN was bound by GST-tagged NCS blotted with a monoclonal anti-calcineurin antibody (1:1000, Sigma) followed by proteins than by CaM. We next examined if GST-tagged NCS detection with anti-mouse-HRP (1:400, Sigma) and ECL reagents. proteins could efficiently interact with CN present as a com- 2.5. HeLa cell cultures and transfections ponent of a complex protein mixture. GST control protein, GST- NCS-1 and GST-CaBP1 (Calcium Binding Protein-1, a calcium HeLa cells were grown in Dulbecco’s modified Eagle’smedium(DMEM, sensor more closely related to CaM than the NCS family pro- Invitrogen, Paisley, UK) containing 5% foetal bovine serum (Invitrogen), 1% non- teins [28]) were incubated with bovine brain cytosolic protein essential amino acids (Invitrogen), and 1% penicillin/streptomycin (Invitrogen) at extract in a large scale pull-down experiment [26] in the pre- ∼ 2 37 °C in an atmosphere of 5% CO2 and maintained at 1,000,000 cells per 75 cm sence of 1 μM free Ca2+ and Ca2+ dependent binding partners flask. 24 hrs prior to transfection, cells were seeded onto glass cover slips on a 24- 2+ well plate at ∼50,000 cells per well. Transfection mixtures comprised 3 μl eluted by application of Ca free buffer containing EGTA and Fugene™ (Roche, UK) per 1 μg plasmid DNA in a 100 μl total volume of DMEM NTA (Fig. 1b, Calcium Free (CF) lanes). An additional elution and were incubated at room temperature for 30 minutes prior to being added drop- step using 1 M NaCl supplemented buffer was also applied to wise to cells. Cells were assayed 24-48 hrs post-transfection. The NFAT-GFP the samples to elute potential Ca2+ independent binding pro- reporter construct was a kind gift from Professor Anjana Rao (Harvard Medical teins (Fig. 1b, High Salt (HS) lanes). Western blotting for the School, Boston, MA, USA). presence of CN in eluates confirmed a strictly Ca2+ dependent 2.6. Ionomycin induced NFAT translocation interaction with both GST-NCS-1 and GST-CaBP1. No binding was detectable to GST control protein. The signal for CN in the Transfected HeLa cells were washed twice in Krebs buffer (NaCl 145 mM; bovine brain cytosolic extract was compared in the same expe- HEPES 20 mM (pH 7.4); D-(+)-Glucose 10 mM; KCl 5 mM; CaCl2 3 mM; riment (Fig. 1b, Bovine brain cytosol) and it was clear that CN MgCl2 1.3 mM; NaH2PO4 1.2 mM). Cells were subsequently incubated in was not enriched over the brain extract in the Ca2+ free eluates. 500 μl Krebs containing 3 μM ionomycin or 100 μM histamine for required times before either solubilisation in SDS dissociation buffer, to investigate the To provide an independent method to examine the interac- phosphorylation state of NFAT via Western blotting, or fixing in 4% for- tion between NCS proteins and CN, we exploited a recently maldehyde to observe the resulting sub-cellular localisation of NFAT post- developed bi-functional cross-linking reagent [29] which per- treatment via confocal microscopy. For western blotting all samples were re- mits the transfer of a biotin tag from a bait protein of interest to solved on SDS-PAGE (12.5% gel) and proteins transferred to nitrocellulose any specifically interacting prey proteins captured following a filters by transverse electrophoresis. Filters were probed with monoclonal anti- δ GFP (1:1000, JL-8 clone, Invitrogen) followed by incubation with anti-mouse- binding reaction. Samples from a GST-Neurocalcin- binding HRP (1:400, Sigma) and protein visualised with ECL reagents. assay prepared via this method were probed with an anti-CN antibody and the presence of the phosphatase confirmed only in 2.7. Confocal microscopy samples treated with the cross-linker (Fig. 1c, (+) Sulfo) and no CN was detectable in samples devoid of the reagent (Fig. 1c, (-) For confocal laser scanning microscopy, transfected cells were examined Sulfo). Despite use of cross-linking to maintain any low affinity with either a Leica TCS-SP-MP microscope or a Leica TCS-SP2-AOBS mi- interactions, CN was not enriched in the eluate over brain croscope (Leica Microsystems, Heidelberg, Germany) using a 22 μm pinhole and a 63× water immersion objective with a 1.2 numerical aperture. EGFP was cytosol input into each condition (Fig. 1c (+) Sulfo vs. Bovine imaged using excitation at 488 nm and light collection at 500-550 nm. brain cytosol). The data presented in Fig. 1a suggests that the affinity of CN for CaM may be substantially greater than for the NCS proteins 3. Results tested. In order to independently verify these observations we further examined the binding of CN from bovine brain cytosol to 3.1. Direct binding of calcineurin to NCS proteins GST control protein and GST-NCS-1 in direct comparison to CaM (Fig. 1d). We observed Ca2+ dependent binding of CN to A previous in vitro study demonstrated that NCS-1 is cap- CaM (Fig. 1d, Calmodulin Sepharose (+)) that was significantly able of stimulating the phosphatase activity of CN but did not enriched in the eluate compared to bovine brain cytosolic extract, D.J. Fitzgerald et al. / Biochimica et Biophysica Acta 1780 (2008) 240–248 243

in contrast, at this exposure of the western blot, binding to GST- NCS-1 was barely detectable. On longer exposure to film, NCS-1 bound CN was detectable although it should be noted that results obtained from this small scale binding assay are not directly comparable to data presented in Fig. 1b from a large scale binding assay which employed a greater amount of bovine brain cytosolic prey protein. These data are consistent with a high affinity Ca2+ dependent interaction between CaM and CN in contrast to a comparatively weak interaction between NCS-1 and CN.

3.2. Overexpression of NCS proteins has no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2+ elevation in HeLa cells

Although our in vitro studies pointed to CaM as most likely the predominant interacting/activating partner for CN, we speculated that the situation in the complex physiological setting of mam- malian cells might not reflect the results obtained from such a comparatively simple analysis. We therefore attempted to resolve this issue through an examination of a possible interaction bet- ween CN and NCS proteins in intact HeLa cells. For these experiments we obtained a GFP tagged NFAT reporter construct [30], encoding the phosphorylated regulatory domain of the phosphatase, that undergoes a detectable dephosphorylation and nuclear translocation in response to sustained increases in [Ca2+]i. Our rationale was that should there be a functionally relevant NCS protein/CN interaction in cells then there might be discernable alterations in dephosphorylation kinetics/nuclear translocation of the NFAT reporter in cells [30,31] over-expressing NCS proteins as observed for other NCS-1 specific functions [32-35]. In initial studies, we saw no effect on NFAT dephosphoryla- tion or nuclear translocation of over-expression of wild-type NCS-1, hippocalcin or neurocalcin-δ. Since NCS-1 had been identified in a previous report [25] as an in vitro activator of CN Fig. 1. NCS proteins interact with calcineurin in vitro. (a) Upper Panel, Anti- enzymatic activity we decided, however, to focus on this NCS calcineurin western blot of samples from an in vitro binding assay examining family member for this part of the study. A second reason for μ interaction of recombinant CN (1 M) with various GST-tagged NCS proteins examining NCS-1 in further detail also related to the fact that we (all at 1 μM) and to calmodulin sepharose (1 μM) in the presence of buffer containing 1 μM free Ca2+. CN was associated directly with calmodulin had at our disposal several mutant NCS-1 constructs one of sepharose and to a lesser extent with GST-NCS-1, GST-Hippocalcin, GST- which has been previously characterised as a dominant inhibitor Neurocalcin-δ and GST-CaBP1 but not with GST-KChIP1. (a) Lower panel, of NCS-1 function [32]. Such mutants, we reasoned, might assist coomassie blue stained gel highlighting equal loading of GST fusion proteins in unmasking potential functional interactions between NCS-1, input into the binding assay. (b) Anti-calcineurin western blot of samples from CN and NFAT. The first of these mutants, NCS-1E120Q, has been an in vitro binding assay examining the ability of CN derived from bovine brain cytosolic extract to associate with GST control and GST-NCS proteins in both employed in a number of studies due to its ability to interact the absence (High Salt or HS) and presence (Calcium Free or CF) of 1 μM free normally with NCS-1 effectors however, as a consequence of Ca2+. CN was detectable bound to both GST-NCS-1 and GST-CaBP1 only in the impaired Ca2+ binding and conformational change, it can then presence of free Ca2+ and no CN was present in HS samples or GST control 2+ exert a dominant inhibitory phenotype [32-36]. The second protein samples either in the presence or absence of Ca . The amount of CN in NCS-1 mutant we employed in this study, NCS-1G2A, is my- the bovine brain cytosolic extract was shown in a positive control sample of total brain cytosolic lysate resolved on the same gel (Bovine brain cytosol). (c) Anti- ristoylation deficient and, unlike wild type NCS-1 which is calcineurin western blot of samples obtained from a Sulfo-SBED cross-linking constitutively membrane associated through this lipid modifica- experiment (see Materials and Methods for details). CN was detectable in tion, is entirely cytosolic [37,38]. This protein was included in samples from a GST-neurocalcin-δ affinity column treated with the biotin these studies due to the fact that the majority of cellular CN is transfer reagent ((+) Sulfo) but not in its absence ((-) Sulfo). Input bovine brain similarly cytosolic and hence we hypothesised that the G2A cytosolic extract was resolved on the same gel (Bovine brain cytosol). (d) Anti- calcineurin western blot on samples from in vitro binding experiments involving mutation may exaggerate any effects of a potential NCS-1/CN incubation of bovine brain cytosolic protein extract with GST control protein, interaction. calmodulin sepharose and GST-NCS-1 (all at 1 μM) in the absence (-) or In an initial analysis, HeLa cells were co-transfected with 2+ presence (+) of 1 μM free Ca . Total bovine brain cytosolic protein extract was NFAT-GFP along with control empty vector, NCS-1 or NCS-1 separated on the same gel as a positive control for CN. mutant constructs (Fig. 2a). Cells were treated for 7 minutes 244 D.J. Fitzgerald et al. / Biochimica et Biophysica Acta 1780 (2008) 240–248

2+ 2+ 2+ with ionomycin/Ca to elevate [Ca ]i, lysed and NFAT-GFP form of the protein (Fig. 2a). After 7 minutes of Ca /ionomycin detected by western blotting. A band corresponding to the fully treatment NFAT-GFP had redistributed to cell nuclei for all phosphorylated form of NFAT was apparent in 0 time point transfection conditions (Fig. 2b, ionomycin, right hand panels) samples from all transfections (Fig. 2a, P, upper arrow). De- again consistent with the rapid dephosphorylation of the protein phosphorylation of NFAT to two more rapidly migrating species (Fig. 2a). 2+ after sustained [Ca ]i for 7 minutes was observable in all We additionally verified that all NCS-1 expression con- transfected samples and there was no detectable difference in structs used in these studies over-expressed in HeLa cells to the dephosphorylation pattern obtained between control trans- similar extents when co-expressed with NFAT-GFP (Fig. 2c). fected cells and NCS-1 or NCS-1 mutant transfected conditions Western blotting with an anti-NCS-1 antiserum [39] of total cell (Fig. 2a, DeP, lower arrows). These data are fully consistent lystates from HeLa cells transfected identically to those used with results obtained from parallel confocal imaging analyses of in functional assays (Figs. 2a and b) highlighted essentially NFAT-GFP nuclear translocation in HeLa cells transfected and equivalent levels of over-expression of NCS-1, NCS-1G2A and treated in an identical manner but which were then fixed for NCS-1E120Q proteins. There was no detectable NCS-1 protein microscopy (Fig. 2b). For all transfection conditions at time 0 of expression in control, pcDNA transfected, cells (Fig. 2c, pcDNA). Ca2+/ionomycin treatment NFAT-GFP was excluded from cell nuclei and was diffusely cytosolic (Fig. 2b, control, left hand 3.3. Overexpression of NCS-1, NCS-1E120Q and NCS-1G2A has panels), compatible with the presence of the phosphorylated no effect on the dephosphorylation of an NFAT-GFP reporter construct in response to cytosolic Ca2+ elevation in HeLa cells over an acute time course

Our analyses suggested that, over a 7 minute time course, none of the NCS-1 constructs under examination were capable of affecting the dephosphorylation of NFAT-GFP in response to 2+ [Ca ]i elevation and, by inference, nor were they able to influence CN activity in intact mammalian cells. These whole cell data were consistent with our in vitro binding studies however we decided to complete our investigation with two further analyses, firstly examining whether or not NCS-1 might exert a far more subtle effect on the CN/NFAT pathway over an 2+ acute time course of [Ca ]i elevation (Fig. 3a). In these experiments, HeLa cells were again transfected with NFAT-GFP along with control or NCS-1 expression constructs however dephosphorylation was monitored over a short 5 minute time course. For all constructs tested NFAT dephosphorylation was

Fig. 2. Over-expression of NCS-1 and NCS-1 mutant proteins has no detectable effect on NFAT dephosphorylation or NFAT nuclear translocation. (a) Anti-GFP western blot analysis of samples from HeLa cells co-transfected with an NFAT- GFP reporter construct in addition to control empty vector (pcDNA) or the indicated pcDNA based NCS-1 expression constructs. Cells were treated with Ca2+/ionomycin for the indicated times and lysed for SDS-PAGE analysis/ western blotting with anti-GFP antibody. Fully phosphorylated NFAT-GFP was detectable in all transfection conditions at the zero time point (P, upper arrow) and dephosphorylation of this reporter was clearly observable after 7 mins of Ca2+/ ionomycin treatment with a gel shift to two more rapidly migrating species corresponding to dephosphorylated forms of the protein (DeP, lower arrows). (b) Confocal images of NFAT-GFP fluorescence from HeLa cells transfected and treated as for (a) but subsequently fixed and processed for microscopy. NFAT- GFP fluorescence was diffusely cytosolic and excluded from cell nuclei under control, zero time point, conditions for all transfected constructs tested. On elevation of cytosolic Ca2+ for 7 mins (ionomycin) NFAT-GFP fluorescence was observed to translocate from the cytosol to cell nuclei for all transfected con- structs analysed, consistent with dephosphorylation of the protein observed in parallel western blot studies (a). Scale bars 10 μm. (c) Western blot of total HeLa cell lysates with an anti-NCS-1 antiserum. Cells transfected identically to those used in functional assays (a and b) were lysed and subjected to western blotting to determine relative over-expression levels of the NCS-1 proteins analysed in this part of the study. Essentially equivalent levels of expression were detected for NCS-1, NCS-1G2A and NCS-1E120Q constructs. No NCS-1 signal was detectable in control empty vector transfected samples (pcDNA). D.J. Fitzgerald et al. / Biochimica et Biophysica Acta 1780 (2008) 240–248 245

Fig. 3. NCS-1 and NCS-1 mutant proteins do not affect dephosphorylation of NFAT-GFP over an acute time course of intracellular Ca2+ elevation. (a) Anti-GFP western blots on HeLa cell lysates from cells transfected with NFAT-GFP and the indicated pcDNA based expression constructs. Cells were treated with Ca2+/ ionomycin for the indicated times and then lysed directly into SDS dissociation buffer, separated on SDS-PAGE (12.5% gel) and transferred to nitrocellulose for western blotting with an anti-GFP antibody. Fully phosphorylated NFAT-GFP (P, upper arrow) present at time 0 for all transfection conditions was dephosphorylated on Ca2+/ionomycin treatment as monitored by the appearance of more rapidly migrating species by the 5 min time point for all transfection conditions (De-P, lower arrow). (b) Anti-calcineurin western blot on samples derived from an in vitro binding assay where recombinant CN (1 μM) was incubated with GST control protein, GST-NCS-1 or GST-NCS-1E120Q (all at 1 μM) in the absence (-Ca2+) or presence (+Ca2+)of1μM free Ca2+. Samples from duplicate incubations are presented. clearly apparent by the 5 minute time point (a 25 minute time tor expressing cells indicating that the stimulus was below the point was also included to demonstrate complete dephospho- threshold for activation of CN/NFAT-GFP dephosphorylation. rylation of NFAT-GFP) as determined by gel shift of the NFAT- Significantly, cells expressing NCS-1, NCS-1E120Q or NCS-1G2A GFP. Analysis of these earlier time points indicated there was no also failed to exhibit dephosphorylation of NFAT-GFP indicating discernable difference in the pattern of dephosphorylation bet- that over-expression of NCS-1 did not enhance the sensitivity of ween any of the NCS-1 constructs tested and control transfected the CN/NFAT signalling pathway to Ca2+ signals under these cells. conditions (data not shown). In these experiments the calcium induced elevation in cyto- For NCS-1E120Q to be able to exert a dominant negative effect solic free Ca2+ was artificially generated by high concentrations it would have to bind to CN. In order to validate our use of the of ionomycin and external Ca2+. Since NCS-1 has a high NCS-1E120Q mutant as a dominant negative construct in this study affinity for Ca2+ (300 nM) one possibility is that it could we therefore generated a recombinantly purified GST-tagged activate CN under conditions of limited Ca2+ elevation thus fusion construct encoding this protein which was used to confirm increasing the overall cellular sensitivity to a Ca2+ signal. We its ability to bind to purified CN (Fig. 3b). GST-NCS-1E120Q completed, therefore, our analyses with an examination as to retained almost wild-type CN binding activity in this assay whether over-expression of NCS-1 or its mutants could in- although with less strict Ca2+-dependency validating its use in fluence NFAT dephosphorylation in response to a physiological functional assays of NFAT dephosphorylation and nuclear trans- stimulus. Histamine is a well characterised HeLa cell agonist location. For GST-NCS-1 we observed a 13.7-fold increase in that generates oscillatory intracellular Ca2+ signals [40] and binding of CN in the presence of Ca2+ compared to conditions activation of protein C with phorbol esters has been where Ca2+ was absent. In contrast, there was only a 2.2-fold shown to potentiate NFAT driven gene expression elicited by increase in CN binding to GST-NCS-1E120Q in the presence of such Ca2+ oscillations [41-43]. We stimulated HeLa cells trans- Ca2+. fected identically to those in Figs. 2 and 3 with 100 μM his- tamine in both the presence and absence of 50 nM PMA over a 4. Discussion three hour time course and assessed the phosphorylation state of NFAT-GFP by western blotting. No dephosphorylation of Diverse Ca2+ signals are transduced and decoded within cells NFAT-GFP was detectable in these experiments in control vec- by a variety of Ca2+-sensing proteins. Of these Ca2+ responsive 246 D.J. Fitzgerald et al. / Biochimica et Biophysica Acta 1780 (2008) 240–248 proteins the most intensively studied and best understood is conclude that the CaM/CN interaction was likely to be of a calmodulin (CaM) [2]. CaM is the prototypical member of a higher affinity than for interactions between CN and NCS super-family of evolutionarily diverse EF-hand containing Ca2+ proteins. Indeed, published biochemical parameters suggest that sensors to which the NCS sub-family belongs [23]. Whilst the the in vitro Kd for CaM/CN interaction may be ≤0.1 nM [46] NCS proteins are only distantly related to CaM, comparative with that reported for NCS-1/CN being 350 nM [25]. The in studies have identified significant in vitro overlap in their ef- vitro affinity of CaM for CN is therefore three orders of mag- fector specificities [25]. It was proposed that NCS proteins nitude greater than that determined for NCS-1 which would might represent a set of redundant or alternative modulators of agree with the binding data presented in this study. Importantly, bone fide CaM target interactions [25] but the ubiquitous and CaM is expressed at 5-200 fold higher concentrations in cells high level expression of CaM would make this doubtful. than NCS-1 [25] and so CN would be predicted to be bound to 2+ Calcineurin (CN) is a cellular serine/threonine phosphatase CaM rather than NCS-1 at elevated [Ca ]i levels. critical to the activation of NFAT transcription factors and hence To assess whether NCS proteins were capable of modulating in the control of a variety of fundamental physiological pro- CN activity in a cellular environment we employed the de- cesses [3]. CaM has been identified as the essential co-activator phosphorylation and nuclear translocation of a characterised for CN function, however CN has also been identified as a NFAT-GFP reporter construct as a probe of CN phosphatase potential target for NCS-1 [25] and as such represents a can- activity. Co-expression of NCS-1 did not alter the basal phos- didate dual CaM/NCS effector. phorylation state of NFAT-GFP compared with control A series of possibilities exist as a direct consequence of these transfected cells nor did it exert a detectable effect on NFAT- observations. Firstly, NCS proteins could indeed simply re- GFP dephosphorylation/nuclear translocation after sustained 2+ present redundant modulators for CaM targets and as such elevation of [Ca ]i during single endpoint and acute time might be expected to elicit responses and to exhibit biochemical course analyses. Co-expression of the NCS proteins Hippocal- properties closely approximating those observed with CaM. cin, Neurocalcin-δ [26], and the more closely related CaBP1 Secondly, NCS proteins could feasibly interact with CaM (which shares 56% similarity with CaM [47]) were similarly targets in biochemically distinct ways to modulate their ac- without effect in analyses of NFAT-GFP dephosphorylation tivities in specific, CaM independent, manners leading to the (data not shown). From these analyses we cannot formally overall generation of unique physiological endpoints. The latter exclude the possibility that NCS family proteins may activate a has been suggested for dual regulation of voltage-gated Ca2+- pool of cellular CN that does not signal via NFAT depho- channels by both CaBP1 and CaM [44]. With specific regard to sphorylation. This seems unlikely however taking into account possible CN/NFAT activation by NCS-1, this possibility is our biochemical data and the fact that in numerous other studies intriguing in light of data identifying NFAT dependent gene assessing diverse triggers of CN activity, concomitant activation expression as important to aspects of neuronal cell function. of the NFAT pathway is always detected. Thirdly, the possibility also exists that interactions observed In an extension to these studies we also examined the effects between NCS proteins and CaM targets are physiologically of two NCS-1 mutant proteins, NCS-1E120Q and NCS-1G2A,on irrelevant in cells. In this study we have attempted to resolve NFAT-GFP dephosphorylation in HeLa cells. We were able to these issues through an investigation of firstly the biochemical demonstrate that the E120Q mutant of NCS-1 [32] retained characteristics of NCS protein interactions with CN, and approximately wild type binding properties with respect to secondly by examining the potential consequences of NCS interaction with purified CN. However, this protein, which has protein function on activation of the classical CaM-CN-NFAT been routinely observed to exert a dominant negative phenotype pathway in mammalian cells. in various other studies of NCS-1 function [32-35], was unable In a series of in vitro biochemical assays we were able to to generate detectable alterations in NFAT-GFP dephosphoryla- show that there exists a selective and Ca2+ dependent in- tion. In an effort to try and understand if NCS-1 was unable to teraction between various NCS proteins and both purified mediate an in vivo modulation of CN activity due to its res- recombinant CN, and CN derived from bovine brain cytosolic tricted membrane localisation we also examined NFAT-GFP protein extract. We noted during these analyses however that phosphorylation in the presence of co-expressed NCS-1G2A,a significantly more CN bound to immobilized CaM. These data protein that fails to become myristoylated and as a result are consistent with the findings of Schaad et al. indicating that remains cytosolic. Since the bulk of cellular CN is also cytosolic NCS-1 is able to stimulate the phosphatase activity of CN in we reasoned that expression of the G2A mutant might bias any vitro but to a lesser extent than observed for CaM activation. In interaction with CN to generate an observable alteration in experiments utilising bovine brain cytosol as a source of prey dephosphorylation of NFAT-GFP. Our analysis of this mutant protein the difference in binding of the NCS proteins for CN in indicated that it elicited no significant change in the kinetics of comparison to CaM was more greatly magnified, and whilst NFAT-GFP dephosphorylation compared to control transfected considerable quantities of CN could be affinity purified by a cells. In related experiments we tested the ability of NCS-1 and calmodulin sepharose column, the amount of CN associated NCS-1 dominant inhibitory mutants to influence NFAT depho- with NCS family members under identical experimental sphorylation in response to physiological stimuli. We observed conditions was considerably lower. Previous work using a no detectable changes in NFAT dephosphorylation in the pre- biotinylated protein overlay assay also found much less binding sence of over-expressed NCS-1 proteins suggesting that in of CN to NCS-1 compared to CaM [45]. These data led us to addition to sustained elevation of cytosolic free Ca2+, NCS-1 D.J. Fitzgerald et al. / Biochimica et Biophysica Acta 1780 (2008) 240–248 247 proteins are similarly unable to stimulate NFAT dephosphoryla- [13] M. Zayzafoon, Calcium/calmodulin signaling controls osteoblast growth – tion in response to agonist derived Ca2+ signals that were and differentiation, J. Cell Biochem. 97 (2006) 56 70. [14] H. Zhu, W. Gao, H. Jiang, J. Wu, Y.F. Shi, X.J. Zhang, Calcineurin mediates below the threshold for significant activation of CN and NFAT acetylcholinesterase expression during calcium ionophore A23187- dephosphorylation. induced HeLa cell apoptosis, Biochimica et biophysica acta 1773 (2007) Collectively our findings would argue that, although NCS 593–602. proteins are capable of interacting with CN in vitro, they may be [15] J.J. Heit, A.A. Apelqvist, X. Gu, M.M. Winslow, J.R. Neilson, G.R. unable to act as functionally relevant activators of the enzyme in Crabtree, S.K. Kim, Calcineurin/NFAT signalling regulates pancreatic beta-cell growth and function, Nature 443 (2006) 345–349. a cellular context. The possibility remains that there may be [16] C. Mammucari, A. Tommasi di Vignano, A.A. Sharov, J. Neilson, M.C. some level of interplay between NCS proteins and CaM within Havrda, D.R. Roop, V.A. Botchkarev, G.R. Crabtree, G.P. Dotto, Integration protein complexes, exemplified by observations that both CaM of Notch 1 and calcineurin/NFATsignaling pathways in keratinocyte growth and CaBP1 interact with IP3-receptors [40,48,49], that both and differentiation control, Developmental cell 8 (2005) 665–676. NCS-1 and CaM are able to interact with Ca2+ channels [50] [17] M. Buchholz, V. Ellenrieder, An emerging role for Ca2+/calcineurin/NFAT signaling in cancerogenesis, Cell cycle 6 (2007) 16–19 (Georgetown, Tex). and G-protein coupled receptor kinases [34,51,52] and that [18] C.I. Holmberg, S.E. Tran, J.E. Eriksson, L. Sistonen, Multisite phosphor- CaM, NCS-1 and CaBP1 have all been shown to modulate the ylation provides sophisticated regulation of transcription factors, Trends in activity of the transient receptor potential channel, TRPC5 [35]. biochemical sciences 27 (2002) 619–627. It is impossible to formally rule out direct modulation of CN by [19] J. Tothova, B. Blaauw, G. Pallafacchina, R. Rudolf, C. Argentini, C. NCS proteins under all physiological conditions based on the Reggiani, S. Schiaffino, NFATc1 nucleocytoplasmic shuttling is controlled by nerve activity in skeletal muscle, Journal of cell science 119 (2006) data presented in this study however we have provided data to 1604–1611. challenge the idea that CN is a cellular target of the NCS protein [20] S. Puri, B.S. Magenheimer, R.L. Maser, E.M. Ryan, C.A. Zien, D.D. family and instead that CaM appears the more likely regulator Walker, D.P. Wallace, S.J. Hempson, J.P. Calvet, Polycystin-1 activates the of this key signalling phosphatase. calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway, J. Biol. Chem. 279 (2004) 55455–55464. [21] C. Loh, K.T. Shaw, J. Carew, J.P. Viola, C. Luo, B.A. Perrino, A. Rao, Acknowledgements Calcineurin binds the transcription factor NFAT1 and reversibly regulates its activity, J. Biol. Chem. 271 (1996) 10884–10891. This work was supported by grants from The Wellcome Trust. [22] S. Feske, Y. Gwack, M. Prakriya, S. Srikanth, S.H. Puppel, B. Tanasa, P.G. DJF was supported by a Wellcome Trust Prize Studentship. Hogan, R.S. Lewis, M. Daly, A. Rao, A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function, Nature 441 (2006) 179–185. References [23] R.D. Burgoyne, Neuronal calcium sensor proteins: generating diversity in neuronal Ca(2+) signalling, Nat. Rev. Neurosci. 8 (2007) 182–193. [1] F. Rusnak, P. Mertz, Calcineurin: form and function, Physiological reviews [24] R.D. Burgoyne, D.W. O'Callaghan, B. Hasdemir, L.P. Haynes, A.V. 80 (2000) 1483–1521. Tepikin, Neuronal calcium sensor proteins: multitalented regulators of [2] D. Chin, A.R. Means, Calmodulin: a prototypical calcium sensor, Trends neuronal function. Trends Neurosci. 27 (2004) 203–209. Cell Biol. 10 (2000) 322–328. [25] N.C. Schaad, E. De Castro, S. Nef, S. Hegi, R. Hinrichsen, M.E. Martone, [3] G.R. Crabtree, E.N. Olson, NFAT signaling: choreographing the social M.H. Ellisman, R. Sikkink, J. Sygush, P. Nef, Direct modulation of lives of cells, Cell 109 (2002) S67–S79 Suppl. calmodulin targets by the neuronal calcium sensor NCS-1. Proc. Natl. [4] P.G. Hogan, L. Chen, J. Nardone, A. Rao, Transcriptional regulation by Acad. Sci. U. S. A. 93 (1996) 9253–9258. calcium, calcineurin, and NFAT, Genes & development 17 (2003) 2205–2232. [26] L.P. Haynes, D.J. Fitzgerald, B. Wareing, D.W. O'Callaghan, A. Morgan, [5] F. Macian, NFAT proteins: key regulators of T-cell development and R.D. Burgoyne, Analysis of the interacting partners of the neuronal function, Nature reviews 5 (2005) 472–484. calcium-binding proteins L-CaBP1, hippocalcin, NCS-1 and neurocalcin. [6] I.A. Graef, P.G. Mermelstein, K. Stankunas, J.R. Neilson, K. Deisseroth, R.W. Proteomics 6 (2006) 1822–1832. Tsien, G.R. Crabtree, L-type calcium channels and GSK-3 regulate the [27] R.D. Burgoyne, J.L. Weiss, The neuronal calcium sensor family of Ca2+- activity of NF-ATc4 in hippocampal neurons, Nature 401 (1999) 703–708. binding proteins. Biochem. J. 353 (2001) 1–12. [7] T. Yoshida, M. Mishina, Distinct roles of calcineurin-nuclear factor of [28] F. Haeseleer, Y. Imanishi, I. Sokal, S. Filipek, K. Palczewski, Calcium- activated T-cells and protein kinase A-cAMP response element-binding binding proteins: Intracellular sensors from the calmodulin superfamily, protein signaling in presynaptic differentiation, J. Neurosci. 25 (2005) Biochem. Biophys. Res. Comm. 290 (2002) 615–623. 3067–3079. [29] L.C. Trotman, N. Mosberger, M. Fornerod, R.P. Stidwill, U.F. Greber, [8] I.A. Graef, F. Wang, F. Charron, L. Chen, J. Neilson, M. Tessier-Lavigne, Import of adenovirus DNA involves the nuclear pore complex receptor G.R. Crabtree, Neurotrophins and netrins require calcineurin/NFAT CAN/Nup214 and histone H1, Nature cell biology 3 (2001) 1092–1100. signaling to stimulate outgrowth of embryonic axons, Cell 113 (2003) [30] J. Aramburu, F. Garcia-Cozar, A. Raghavan, H. Okamura, A. Rao, P.G. 657–670. Hogan, Selective inhibition of NFAT activation by a peptide spanning the [9] R.D. Groth, P.G. Mermelstein, Brain-derived neurotrophic factor activa- calcineurin targeting site of NFAT, Molecular cell 1 (1998) 627–637. tion of NFAT (nuclear factor of activated T-cells)-dependent transcription: [31] J. Aramburu, M.B. Yaffe, C. Lopez-Rodriguez, L.C. Cantley, P.G. Hogan, a role for the transcription factor NFATc4 in neurotrophin-mediated gene A. Rao, Affinity-driven peptide selection of an NFAT inhibitor more expression, J. Neurosci. 23 (2003) 8125–8134. selective than cyclosporin A, Science 285 (1999) 2129–2133 (New York, [10] R.A. Schulz, K.E. Yutzey, Calcineurin signaling and NFAT activation in N.Y.). cardiovascular and skeletal muscle development, Developmental biology [32] J.L. Weiss, D.A. Archer, R.D. Burgoyne, NCS-1/frequenin functions in an 266 (2004) 1–16. autocrine pathway regulating Ca2+ channels in bovine adrenal chromaffin [11] E.N. Olson, R.S. Williams, Remodeling muscles with calcineurin, cells. J. Biol. Chem. 275 (2000) 40082–40087. Bioessays 22 (2000) 510–519. [33] T.Y. Nakamura, A. Jeromin, G. Smith, H. Kurushima, H. Koga, Y. [12] T.T. Yang, Q. Xiong, H. Enslen, R.J. Davis, C.W. Chow, Phosphorylation Nakabeppu, S. Wakabayashi, J. Nabekura, Novel role of neuronal Ca2+ of NFATc4 by p38 mitogen-activated protein kinases, Molecular and sensor-1 as a survival factor up-regulated in injured neurons, The Journal cellular biology 22 (2002) 3892–3904. of cell biology 172 (2006) 1081–1091. 248 D.J. Fitzgerald et al. / Biochimica et Biophysica Acta 1780 (2008) 240–248

[34] N. Kabbani, L. Negyessy, R. Lin, P. Goldman-Rakic, R. Levenson, [43] R.E. Dolmetsch, K. Xu, R.S. Lewis, Calcium oscillations increase the Interaction with the neuronal calcium sensor NCS-1 mediates desensitiza- efficiency and specificity of gene expression, Nature 392 (1998) 933–936. tion of the D2 dopamine receptor. J. Neurosci. 22 (2002) 8476–8486. [44] A. Lee, R.E. Westenbroek, F. Haeseleer, K. Palczewski, T. Scheuer, W.A. [35] H. Hui, D. McHugh, M. Hannan, F. Zeng, S.Z. Xu, S.U. Khan, R. Catterall, Differential modulation of Cav2.1 channels by calmodulin and Levenson, D.J. Beech, J.L. Weiss, Calcium-sensing mechanism in TRPC5 Ca2+-binding protein 1, Nature Neuroscience 5 (2002) 210–217. channels contributing to retardation of neurite outgrowth, The Journal of [45] B.W. McFerran, J.L. Weiss, R.D. Burgoyne, Neuronal Ca2+-sensor 1: physiology 572 (2006) 165–172. Characterisation of the myristoylated protein, its cellular effects in perme- [36] N. Gamper, V. Reznikov, Y. Yamada, J. Yang, M.S. Shapiro, Phospha- abilised adrenal chromaffin cells, Ca2+-independent membrane-association 2+ tidylinositol 4,5-bisphosphate signals underlie receptor-specific Gq/11- and interaction with binding proteins suggesting a role in rapid Ca signal mediated modulation of N-type Ca2+ channels, J. Neurosci. 24 (2004) transduction, J. Biol. Chem. 274 (1999) 30258–30265. 10980–10992. [46] M.J. Hubbard, C.B. Klee, Calmodulin binding by calcineurin. Ligand- [37] D.W.O'Callaghan, R.D. Burgoyne, Role of in the intracellular induced renaturation of protein immobilized on nitrocellulose, J. Biol. targeting of neuronal calcium sensor (NCS) proteins, Biochem. Soc. Trans. Chem. 262 (1987) 15062–15070. 31 (2003) 963–965. [47] F. Haeseleer, I. Sokal, C.L.M.J. Verlinde, H. Erdjument-Bromage, P. [38] D.W. O'Callaghan, L. Ivings, J.L. Weiss, M.C. Ashby, A.V. Tepikin, R.D. Tempst, A.N. Pronin, J.L. Benovic, R.N. Fariss, K. Palczewski, Five Burgoyne, Differential use of myristoyl groups on neuronal calcium sensor members of a novel Ca2+ binding protein (CABP) subfamily with similarity proteins as a determinant of spatio-temporal aspects of Ca 2+-signal trans- to calmodulin, J. Biol. Chem. 275 (2000) 1247–1260. duction. J. Biol. Chem. 277 (2002) 14227–14237. [48] J. Yang, S. McBride, D.-O.D. Mak, N. Vardi, K. Palczewski, F. Haeseleer, [39] B.W. McFerran, M.E. Graham, R.D. Burgoyne, NCS-1, the mammalian J.K. Foskett, Identification of a family of calcium sensors as protein homologue of frequenin is expressed in chromaffin and PC12 cells and ligands of receptor Ca2+ release channels. Proc. Natl. regulates neurosecretion from dense-core granules, J. Biol. Chem. 273 (1998) Acad. Sci. U. S. A. 99 (2002) 7711–7716. 22768–22772. [49] N.N. Kasri, G. Bultynck, I. Sienaert, G. Callewaert, C. Erneux, L. Missiaen, [40] L.P. Haynes, A.V. Tepikin, R.D. Burgoyne, Calcium Binding Protein 1 is J.B. Parys, H.D. Smedt, The role of calmodulin for inositol 1,4,5- an inhibitor of agonist-evoked, inositol 1,4,5-trisphophate-mediated trisphosphate receptor function, Biochim. Biophys. Acta 1600 (2002) calcium signalling, J. Biol. Chem. 279 (2004) 547–555. 19–31. [41] P.A. Negulescu, N. Shastri, M.D. Cahalan, Intracellular calcium depen- [50] J.L. Weiss, R.D. Burgoyne, Sense and sensibility in the regulation of dence of gene expression in single T lymphocytes, Proceedings of the voltage-gated calcium channels. Trends Neurosci. 25 (2002) 489–491. National Academy of Sciences of the United States of America 91 (1994) [51] A.N. Pronin, J.L. Benovic, Regulation of the G protein-coupled receptor 2873–2877. kinase GRK5 by protein kinase C, J. Biol. Chem. 272 (1997) 3806–3812. [42] R.E. Dolmetsch, R.S. Lewis, C.C. Goodnow, J.I. Healy, Differential [52] T.T. Chuang, L. Paolucci, A. De Blasi, Inhibition of G protein-coupled activation of transcription factors induced by Ca2+ response amplitude and receptor kinase subtypes by Ca2+/calmodulin, J. Biol. Chem. 271 (1996) duration, Nature 386 (1997) 855–858. 28691–28696.