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Cell Calcium 42 (2007) 145156
Signalling to transcription: Store-operated Ca2+
entry and NFAT activation in lymphocytes
Yousang Gwack, Stefan Feske, Sonal Srikanth, Patrick G. Hogan, Anjana Rao
Department of Pathology, Harvard Medical School, The CBR Institute for Biomedical Research, 200 Longwood Avenue, Bos ton, MA 02115, USA
Received 3 March 2007; received in revised form 20 March 2007; accepted 21 March 2007
Available online 18 June 2007
Abstract
In cells of the immune system that are stimulated by antigen or antigenantibody complexes, Ca 2+ entry from the extracellular medium
is driven by depletion of endoplasmic reticulum Ca2+ stores and occurs through specialized store-operated Ca2+ channels known as Ca2+-
release-activated Ca2+ (CRAC) channels. The process of store-operated Ca2+ influx is essential for short-term as well as long-term responses
by immune-system cells. Short-term responses include mast cell degranulation and killing of target cells by effector cytolytic T cells, whereas
long-term responses typically involve changes in gene transcription and include T and B cell proliferation and differentiation. Transcription
downstream of Ca2+ influx is in large part funneled through the transcription factor nuclear factor of activated T cells (NFAT), a heavily
phosphorylated protein that is cytoplasmic in resting cells, but that enters the nucleus when dephosphorylated by the calmodulin-dependent
serine/threonine phosphatase calcineurin. The importance of the Ca2+/calcineurin/NFAT signalling pathway for lymphocyte activation is
underscored by the finding that the underlying defect in a family with a hereditary severe combined immune deficiency (SCID) syndrome is
a defect in CRAC channel function, store-operated Ca2+ entry, NFAT activation and transcription of cytokines, chemokines and many other
NFAT target genes whose transcription is essential for productive immune defence.
We recently used a two-pronged genetic approach to identify Orai1 as the pore subunit of the CRAC channel. On the one hand, we initiated a
positional cloning approach in which we utilised genome-wide single nucleotide polymorphism (SNP) mapping to identify the genomic region
linked to the mutant gene in the SCID family described above. In parallel, we used a genome-wide RNAi screen in Drosophila to identifycritical regulators of NFAT nuclear translocation and store-operated Ca2+ entry. These approaches, together with subsequent mutational and
electrophysiological analyses, converged to identify human Orai1 as a pore subunit of the CRAC channel and as the gene product mutated in
the SCID patients.
2007 Elsevier Ltd. All rights reserved.
Keywords: Nuclear factor of activated T cells; CRAC channels; Calcineurin; T cell activation; Cytokine expression
Ca2+ is a universal regulator of intracellular signalling
[14]. As described in other reviews in this volume, Ca2+
is utilised as a second messenger by essentially all cells in
multicellular organisms, where it regulates diverse aspects ofcellular function.Increasesin intracellular free Ca2+ ([Ca2+]i)
levels modulate many intracellular processes by activat-
ing ubiquitous Ca2+ sensors such as calmodulin (CaM); in
Corresponding author at: Department of Pathology, Harvard Medical
School, The CBR Institutefor Biomedical Research,Rm 152,Warren Alpert
Bldg, 200 Longwood Avenue, Boston, MA 02115, USA.
Tel.: +1 617 278 3260; fax: +1 617 278 3280.
E-mail address: [email protected] (A. Rao).
turn, calmodulin activates a large number of calmodulin-
dependent proteins including the kinases CaMKII and
CaMKIV and the phosphatase calcineurin which together
shape both the early and late phases of the subsequent cellularresponse [5].
The importance of Ca2+ as an intracellular second mes-
senger is emphasised by the many different mechanisms,
which work together to maintain Ca2+ homeostasis within
intracellular compartments (reviewed in [1,2]). The endo-
plasmic reticulum (ER) is a substantial reservoir of stored
Ca2+, and is the principal Ca2+ store mobilised for signalling.
The free Ca2+ concentration in the ER is estimated to be
in the 100700M range, and about an order of magnitude
0143-4160/$ see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2007.03.007
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146 Y. Gwack et al. / Cell Calcium 42 (2007) 145156
more Ca2+ is bound to low-affinity sites in the ER lumen and
is therefore available for eventual mobilisation. Cytoplasmic
Ca2+ ([Ca2+]i) levels aretypically 70100 nM,whereas extra-
cellular Ca2+ levels ([Ca2+]o)are104-fold higher,12 mM.
Ca2+ entersthe cytoplasm through channels located in ER and
plasma membranes, and is extruded across the plasma mem-
brane and into ER Ca2+
stores by Ca2+
ATPases localisedin the ER and the plasma membrane (SERCA and PMCA
pumps, respectively). In some cells, Na+/Ca2+ exchangers in
the plasma membrane (NCX) contribute to maintaining the
low resting levels of [Ca2+]i.
Cells have several mechanisms for regulated Ca2+ entry;
the predominant mechanism utilised depends on the cell
type and manner of stimulation involved [14]. Four general
classes of Ca2+ entry channels have been described: voltage-
gated Ca2+ channels (Cav) such as the L-type Ca2+ channel
(LTCC); channels gated by physical parameters (tempera-
ture, mechanical forces, etc.), which are often members of
the TRP (transient receptor potential) family; channels gated
by ligand/receptor interaction, some of which are also TRP
family members; and store-operated Ca2+ channels gated by
depletion of intracellular Ca2+ stores (reviewed in [14,69].
Of these, voltage-gated Ca2+ channels, and the subtype of
store-operated Ca2+ channels found in immune cells and
termed Ca2+-release-activated Ca2+ (CRAC) channels, are
markedly Ca2+-selective, showing a preference for Ca2+ over
Na+ in physiological solutions that is estimated at 1000:1
[3,4,9].
Store-operated Ca2+ channels open upon stimulation of
receptors coupled to phospholipase C (reviewed in [14]).
Receptor tyrosine kinases (RTKs) and immunoreceptors
(antigen and Fc receptors on immune cells) activate phos-pholipase C gamma (PLC), while certain G protein-coupled
receptors (GPCR) activate PLC. Activated PLC hydrolyses
phosphatidylinositol-4,5-bisphosphate (PIP2), thereby gen-
erating the second messengers inositol-1,4,5-trisphosphate
(InsP3) and diacylglycerol. InsP3 releases Ca2+ from the ER
by binding to InsP3 receptors (InsP3R). The resulting deple-
tion of ER Ca2+ stores promotes a transient elevation of
[Ca2+]i, and opens store-operated Ca2+ channels through a
process whose molecular mechanism is not yet fully under-
stood (see below).
1. CRAC channels and the regulation of Ca2+ entry
in lymphocytes and mast cells
In this review, we focus on cells of the immune system, in
which the functional consequences of Ca2+ signalling have
been exhaustively studied [3,10]. Binding of antigen to T
and B cell antigen receptors (TCR, BCR), and binding of
antigenantibody complexes to Fc receptors on mast cells,
monocytes, macrophages and natural killer cells, results in
a transient rise in intracellular free Ca2+ ([Ca2+]i) levels
as a result of the release of Ca2+ from ER stores triggered
by InsP3. However, the volume of the ER is estimated at
only 1% of cytoplasmic volume in T lymphocytes and
3% in the RBL (rat basophilic leukemia) mast cell line
(reviewed in [11]). Since cytoplasmic Ca2+ is extruded by
PMCA pumps, release of Ca2+ from ER stores cannot by
itself support a sustained elevation of [Ca2+]i. Rather, as
mentioned above, store depletion triggers the opening of
a specific class of store-operated Ca2+
channels known asCRAC channels [3,4,9]. Under normal physiological con-
ditions, CRAC channels remain open as long as antigen is
present andstores remaindepleted, thus leading to a sustained
increase in [Ca2+]i that lasts until the antigenic stimulus dies
away.
Unlike TRP channels [7], the CRAC channels of mast
cells and lymphocytes have been unambiguously established
as store-operated Ca2+ channels [3,4,9]. The gating mecha-
nism for these channels involves depletion of Ca2+ stores per
se, rather than a response to Ca2+ released into the cytoplasm
or to second messengers such as diacylglycerol (reviewed
in [3,4,9,11]). The receptor-proximal events PLC acti-
vation, InsP3 generation that lead to store depletion canbe bypassed and store depletion can be achieved directly by
treating cells with a variety of agents: thapsigargin, which
blocks the SERCA pump responsible for maintaining the
stores; intracellular application of Ca2+ chelators such as
BAPTA, which promote passive store depletion by acting
as a cytoplasmic Ca2+ sink; or calcium ionophores such as
ionomycin, which, at the concentrations commonly used to
activate immune cells, operate primarily by providing an
additional pathway for Ca2+ efflux from stores (reviewed in
[3,4,9,11]).
The basic biophysical and electrophysiological features of
CRAC channels have beenwell established through studies inmany laboratories (reviewed in [3,4,9,11]). Fig. 1 illustrates
one of our own experiments, performed in collaboration with
Drs. Murali Prakriya and Richard Lewis at Stanford [12].
CRAC channels show a characteristic IV relationship with
pronounced inward rectification and a very high selectivity
for Ca2+ over monovalent cations. The ratio of the perme-
ability coefficients for Ca2+ and Na+ has been estimated at
1000:1, a degree of selectivity otherwise documented only
in voltage-gated (Cav) Ca2+ channels. Another hallmark of
the CRAC channel is its susceptibilty to potentiation by low
concentrations (35M) of 2-aminoethoxydiphenyl borate
(2-APB) [4,9] (Fig. 1).
T cells also express voltage-gated and Ca2+-activated
K+ channels in a pattern that depends on the activa-
tion/differentiationstatus of the cells[13]. These K+ channels
modulate the rate of Ca2+ entry through CRAC channels by
altering membrane potential: hyperpolarisation increases the
driving force for Ca2+ influx while depolarisation decreases
it. Kv1.3 channels maintain membrane potential and there-
fore the driving force on Ca2+: in turn Ca2+ entry and
the resulting increased [Ca2+]i levels are thought to acti-
vate the opening of Ca2+-activated IKCa1 channels. Toxins
and compounds that block Kv1.3 channels cause membrane
depolarisation, thus reducing thedriving force on Ca2+, atten-
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Y. Gwack et al. / Cell Calcium 42 (2007) 145156 147
Fig. 1. Electrophysiological characteristics of CRAC channels in human T cells. (A) Ca2+ and Na+ currents through CRAC channels in a control T cell (whole-
cell configuration). Thecell waspretreated with thapsigarginto activate CRACchannels, then exposedsequentially to 20mM Ca2+ or to a divalent-free solution
(DVF) extracellularly to record Ca2+ and Na+ currents, respectively. Peak currents during steps to 100 mV are shown. (B) Currentvoltage relationship of the
Ca2+ current recorded in A (average of 10 traces recorded at the time points indicated by the blue bar). Note the pronounced inward rectification characteristic
of ICRAC . (C) 2-APB (5M) strongly potentiates ICRAC. Reproduced with permission from ref. [12] (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article).
uating Ca2+ influx and [Ca2+]i increase, and inhibiting T cell
activation [13].
2. NFAT is a major target of Ca2+ signalling in many
cell types
Ca2+ signalling activates nuclear factor of activated T
cells (NFAT), a family of four transcription factors (NFAT1-
4, also known as NFATc1-c4) (reviewed in [10,1417])
(Fig. 2). NFAT regulates gene transcription during T cell
activation and differentiation, osteoclast differentiation, car-
diac valve development and differentiation of slow-twitch
skeletal muscle fibers, among others; it is also implicated
in many pathological processes, among them transplant
rejection, osteoporosis, myocardial hypertrophy, allergy and
autoimmune disease [10,1417]. NFAT proteins are heav-ily phosphorylated and reside in the cytoplasm; when cells
are stimulated, they are dephosphorylated by calcineurin,
a calmodulin-dependent serine/threonine phosphatase, and
translocate to the nucleus [10,1417]. The phosphorylated
serine residues of NFAT are located primarily within four
conserved sequence motifs in a conserved regulatory domain
[10,18]. Phosphorylated NFAT exposes a nuclear exportsequence (NES), which binds the exportin Crm1; dephos-
phorylation results in a conformational change that masks
the NES and exposes a nuclear localization sequence (NLS)
which binds importins [18]. The most N-terminal phosphory-
lated motif, SRR1, controls NLS exposure and accessibility
of the remaining phosphorylated residues to calcineurin [18],
while the two conserved motifs flanking the NLS (SP-2
and SP-3) control DNA-binding affinity, NES exposure and
nuclear export (reviewed in [10]).
The different serine-rich motifs of NFAT proteins are tar-
geted by different kinases, allowing for fine-tuned regulation
of NFAT activation [1820]. The kinases can be classified as
maintenance kinases which act in the cytoplasm of restingcells to keep NFAT in its phosphorylated state, and export
kinases which re-phosphorylate NFAT in the nucleus. Three
families of constitutively-active kinases DYRK, CK1 and
Fig. 2. Schematic view of the NFAT activation cycle.
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GSK3 act concertedly to phosphorylate NFAT [20]. CK1-
family kinases are both maintenance and export kinases,
which phosphorylate the SRR-1 region of NFAT1 and other
NFAT proteins [19]. DYRK-familykinases phosphorylate the
SP-3 motif of NFAT1, thereby priming for GSK3-mediated
phosphorylation at the SP-2 motif; DYRK2, which is cyto-
plasmic, serves as the maintenance kinase while DYRK1A,which resides in the nucleus, is the export kinase [20]. In
addition, other intracellular signalling pathways influence the
phosphorylation state of NFAT. For instance, MAP kinases
facilitate the nuclear export of NFAT proteins by potentiating
the ability of CK1 to phosphorylate the SRR1 motif; con-
versely, the activity of GSK3 is suppressed by Akt, a kinase
activated in response to diverse signalling pathways in dif-
ferent cell types (reviewed in [10]). Thus the level of active
nuclear NFAT depends both on the parameters of Ca2+ influx
as described below, and on which inducible kinases are active
under the particular stimulation conditions imposed.
3. Ca2+ signalling and transcriptional activation in
cells of the immune system
Uponstimulationthrough immunoreceptors, cells of the
immune system B cells which bind antigen through the
BCR, T cells which bind MHC/peptide complexes through
the TCR, mast cells which bind antigen-IgE complexes
through the Fc receptor, and natural killer (NK) cells which
bind antigen-IgG complexes through Fc receptors activate
similar downstream signalling pathways and transcription
factors (reviewed in [2124]). Each of these immunorecep-
tors is coupled to tyrosine kinases of the Src and ZAP70/Sykfamilies, whose activation results in tyrosine phosphoryla-
tion andactivation of PLC, andconsequent generation of the
second messengers InsP3 and diacylglycerol. InsP3-mediated
depletion of ER Ca2+ storesresults in CRAC channel opening
and Ca2+ influx across the plasma membrane, thus driving
activation of the transcription factor NFAT. Diacylglycerol
binds to two distinct classes of signalling enzymes, Ras-
GRP and protein kinase C, thus activating MAP kinase and
IKK (IB kinase) pathways, which lead to activation of the
AP-1 (Fos-Jun) and NFB transcription factors respectively
[2527]. MAP kinases are responsible for activation of the
AP-1 transcription factor, which consists of homo- and het-
erodimers of Jun family proteins, as well as heterodimers
of Fos and Jun [25,26]. In response to stimulation through
immunoreceptors, Fos and Jun proteins are synthesised and
also activated posttranslationally by site-specific phosphory-
lation; for instance, Jun is modified by members of the family
of Jun N-terminal kinases (JNK) [26]. Together, NFAT, AP-1
and NFB act in concert with secondary transcription factors
to drive the transcription of a large number of genes that reg-
ulate lymphocyte proliferation and differentiation (reviewed
in [10,2527]).
NFAT, AP-1 and NFB were shown to be optimally acti-
vated in response to different patterns of Ca2+ signalling in
JurkatT cells [2830]. Transient highCa2+ spikes evokedsus-
tained activation of JNK and NFB, but not NFAT, whereas
prolongedlow increasesin [Ca2+]i, which were insufficientto
activate JNK or NFB, sufficed to activate NFAT [28]. Acti-
vation of NFAT and NFB was also sensitive to the frequency
of [Ca2+]i oscillations: low frequency oscillations activated
NFB, whereas high frequencies activated both NFAT andNFB [29,30]. Moreover, oscillations enhanced signalling
efficiency specifically at low levels of stimulation [29,30].
4. Biological consequences of Ca2+ entry in
immune-system cells
[Ca2+]i increases in lymphocytes and mast cells are
coupled to a variety of antigen-dependent responses, both
rapid and long-term. (i) The rapid responses are indepen-
dent of new gene transcription: they include degranulation
of allergen-exposed mast cells, which occurs in minutes,
and target cell killing by cytolytic T cells, which is com-plete within a few hours. Mast cells that are coated with
immunoglobulin E (IgE) degranulate and release proteases,
prostaglandins,leukotrienes,histamineand manyother medi-
ators when exposed to appropriate allergens [23]; similarly,
cytolytic T cells attack and lyse infected, malignant or trans-
planted cells by secreting the pore-forming protein perforin
and specialised proteases known as granzymes [31]. (ii) In
contrast, the long-term responses involve transcriptional pro-
grammes initiated by sustained Ca2+ signalling (reviewed
in [10,15,25]): they include proliferation, differentiation and
acquisition of effector function by nave T and B lympho-
cytes following their first encounter with antigen, as well astranscription of cytokine, chemokine and other activation-
associated genes by differentiated effector T cells upon
secondary exposure to antigen. The productive interaction of
T cells with antigen-presenting cells is accompanied by sus-
tained activation of Ca2+, phosphatidylinositol (PI) 3-kinase,
NFAT and other signalling pathways, as shown by monitor-
ing [Ca2+]i elevation and production of PI-3,4,5-phosphate
(PIP3) in individual T cells for many hours [32]. Moreover,
sustainedactivationis required to maintain ongoing transcrip-
tional responses, as shown by adding inhibitors (e.g. the PI
3-kinase inhibitor wortmannin or the calcineurin inhibitors
cyclosporin A (CsA) or FK506) to T cells after initiation
of T cell activation [3234]. (iii) A third and very inter-
esting Ca2+-dependent programme is activated in B cells
exposed to self-antigens while circulating in a healthy host:
these cells show only a modest basal elevation of [Ca2+]i(150200 nM), which nevertheless causes low-level activa-
tion of multiple signalling pathways, and sends a substantial
fraction of the total NFAT in the cell (30%) to the nucleus
[3537]. Continuous occupancy of the BCR activates potent
negative feedback mechanisms that cause the B cells to enter
an anergic or unresponsive state, such that they become
unable to respond to strong antigen stimulation with an ele-
vation of [Ca2+]i. In vivo, anergic B cells are very short-lived
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Y. Gwack et al. / Cell Calcium 42 (2007) 145156 149
and thus are unable to provoke autoimmune destruction by
producing antibodies that react to self [3638]. The anergic
state decays rapidly upon dissociation of bound antigen from
the B cell surface [36]. Anergy is a transcriptional state that
is strongly dependent on NFAT, as shown by analysis of B
cell anergy in mice lacking NFAT1 [39].
The existence of a similar negative regulatory programmeis well established in T cells [40], and many of the relevant
target genes (anergy-associated genes) have been identified
[41,42]. A substantial number of these are known signalling
proteins or transcription factors thatfunction in negative feed-
back loops to attenuate T cell responses. Established negative
regulators that are transcriptional targets of the anergy pro-
gramme mediated by Ca2+, calcineurin and NFAT include:
(i) the diacylglycerol kinases DGK and DGK [43]; (ii) the
transcriptional regulators Egr2 and Ikaros [44,45]; (iii) the E3
ligases Itch, Cbl-b and Grail [46,47]; and (iv) an unidentified
palmitoyltransferase that anchors the transmembrane adap-
tor LAT in cholesterol-rich lipid microdomains in the plasma
membrane, thereby facilitating T cell signalling [48].
5. Severe combined immunodeficiencies resulting
from defects in CRAC channel function
Theimportance of Ca2+ influxthrough CRAC channels for
normal immune defence against pathogens is highlighted by
the existence of at least three families of patients with severe
combined immunodeficiency (SCID) secondary to a lack of
Ca2+ influx and ICRAC [4951]. The children with SCID
were originally identified by their susceptibility to recur-
rent infections, and later shown to be completely deficientin store-operated Ca2+ entry as measured by Ca2+ imaging
in single cells. We have shown that T cells from the two
affected patients in one of these families show marked atten-
uation of NFAT dephosphorylation and nuclear translocation
in response to TCR stimulation or treatment with pharma-
cological agents such as ionomycin or thapsigargin, with no
obvious defect in activation of the transcription factors AP-1
and NFB [49,52,53]. As a result, the T cells are defective in
transcription of multiple cytokine and chemokine genes, fully
explaining the patients severe immune deficiency [52,53].
The cells are completely deficient in CRAC channel func-
tion as judged by electrophysiological measurements [12].
The molecular defect in these patients has been identified as
a point mutation in the CRAC channel pore subunit, Orai1
([54,55]; see below). One patient, whose older sibling suc-
cumbed to a severe infection, was rescued by administration
of recombinant IL-2 and bone marrow transplantation, and
he is now essentially normal except for a slight degree of
non-progressive muscle hypotonia and a mild case of a syn-
drome termed ectodermal dysplasia with anhydrosis [52].
This finding emphasises that although CRAC currents are
broadly apparent in non-excitable cells(includingDrosophila
S2 cells, [56]), there is clearly some lymphocyte selectivity
that could be exploited therapeutically.
Even brief nuclear entry of NFAT can have significant
biological consequences, as shown by using T cells from the
SCID patients mentioned above. Despite the fact that they
have almost imperceptible store-operated Ca2+ influx and
CRAC channel activation, release of Ca2+ from ER stores
is normal in these cells, and leads to transient NFAT dephos-
phorylation and nuclear import [12,52,53]. Surprisingly, thistransient activation permits almost normal induction of a
small number of NFAT-dependent genes [52]. Tomakeamore
quantitative assessment of the number of genes controlled by
Ca2+ signalling, cDNA microarrays were used to compare the
transcriptional profiles of normal T cells and SCID T cells
underboth resting and activated conditions [53]. As expected,
loss of CRAC channel function was linked to pronounced
changes in the expression of activation-associatedgenes. Sur-
prisingly, almost half of the genes whose expression was
altered upon activation showed increased expression in the
SCID patients cells, implying that calcium signalling acti-
vates a complex transcriptional programme in which nearly
as many genes are repressed (40%) as activated (60%).Use of the calcineurin inhibitor CsA showed that a majority
of Ca2+-dependent gene expression in T cells is funneled
through the calcineurin (and presumably the NFAT) sig-
nalling pathway [53].
6. Identification of CRAC channel components and
regulators: a two-pronged genetic approach
We used two different but complementary genetic screens
to identify upstream regulators of NFAT [20,54]. The
first was a positional cloning approach aimed at identi-fying the gene defective in the SCID patients mentioned
above (Fig. 3). The second was a genome-wide Drosophila
RNAi screen (Fig. 4), made possible (i) by the fact
that Drosophila cells have a store-operated Ca2+ channel
with electrophysiological characteristics very reminiscent
of CRAC channels [56], and (ii) by the establishment of
the Drosophila RNAi Screening Centre by Norbert Perri-
mon and Bernard Mathey-Prevot at Harvard Medical School
[5760].
At the time that we initiated our efforts, the molecular
identity of the CRAC channel was not known, but there was
much discussion of thepossibilitythat theCRAC channel was
formed by one or more TRP family members (reviewed in
[4,7]). We therefore examined the SCID patients T cells and
fibroblasts for TRP family members known to be expressed in
these cell types, and sequencedthe relevant TRP genes. How-
ever, we did not observe altered expression of TRP mRNA
and/or protein, nor did we find any mutations in coding and
junctional sequences of the TRP genes that we analysed (S.F.,
A.R., unpublished).
In addition to identifying CRAC channel components and
regulators, we were interested in identifying the kinase that
phosphorylated the SP3 motif of NFAT1, since it had eluded
biochemical identification for many years [18,19]. Also, it
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150 Y. Gwack et al. / Cell Calcium 42 (2007) 145156
Fig. 3. Identification by genome-wide SNP analysis of the region linked to the mutation in the SCID patients cells. (a) Pedigree of the SCID family. Black
squares, affected patients. Yellow and double-coloured symbols, unaffected and presumed heterozygous carriers of the disease gene based on experimentally
measured Ca2+ influx. (b) Map of the genomic region linked to the SCID mutation. The ORAI1 gene is indicated. Adapted with permission from ref. [54] (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
was clear that the positional cloning approach would yield
one component of the pathway leading from store depletionto CRAC channel opening, but identifying other components
obviously called for a more global approach. Because our
objective was to find all potential regulators of NFAT, we
decided to use NFAT nuclear translocation as the readout of
the global RNAi screen (Fig. 4), rather than thapsigargin-
evoked changes in [Ca2+]i as preferred by other groups that
performed similar screens [61,62]. This choice turned out to
be ideal in our screen of 21,800 gene products, we obtained
only 16 robust candidates whose depletion prevented NFAT
nuclear translocation, and as expected the candidates
included nuclear transport proteins and calcineurin sub-
units [20,63]. In contrast, two fluorescence-based screens
for candidates whose depletion abolished store-operated
Ca2+ influx, monitored by use of a Ca2+ indicator dye,
yielded1500 candidates and 75 filtered hits, respectively
[61,62].
7. Positional cloning of the mutation in the SCID
patients cells
The SCID patient family that we were investigating was
initially very small, and not amenable to standard positional
cloning approaches. We had access to T cells and DNA
from two parents who were first cousins and so were pre-
sumed to be heterozygous for the mutant gene (assumingan autosomal recessive mode of inheritance) and their two
affected children. The parents were clinically normal, and
initial testing of their T cells showed no obvious defect in
store-operated Ca2+ influx when 2 mM extracellular Ca2+
was used. However, when Ca2+ influx was monitored at
lower concentrations of [Ca2+]o (0.2 to 0.5 mM), a signif-
icant deficit became apparent, with lower peak [Ca2+]i as
well as a lower apparent rate of [Ca2+]i increase observed
in the parents cells [54]. This modification of the assay
allowed us to test a panel of additional family members,
including the grandparents and an unaffected sibling, bring-
ing the total number of family members analysed to 23, of
whom 13 appeared to be heterozygous carriers of the mutant
gene (Fig. 3a). DNA samples from all family members were
then used for genome-wide SNP (single nucleotide polymor-
phism) mapping using microarrays, permitting simultaneous
genotyping of more than 10,000 SNPs in each individuals
genome [54].
SNP data were evaluated using two independent link-
age analyses. The first (standard homozygosity mapping)
assumed an autosomal recessive mode of inheritance based
on the clinical phenotype, and evaluated SNP data just from
the two patients, their parents, their unaffected brother and
their grandparents (grey shaded area in Fig. 3a); the sec-
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Y. Gwack et al. / Cell Calcium 42 (2007) 145156 151
Fig. 4. Genome-wide Drosophila RNAi screen to identify upstream regulators of NFAT. (Left) Candidates whose RNAi-mediated depletion ( ) would be
expected to prevent nuclear translocation of a GFP-NFAT fusion protein in thapsigargin-treated cells. (Right) Candidates whose RNAi-mediated depletion ( )
would be expected to cause aberrant nuclear localisation of NFAT in unstimulated cells.
ond utilized the remainder of the pedigree (green box in
Fig. 3a), and assumed an autosomal dominantmode of inher-
itance based on our identification of heterozygous carriers
of the mutant gene by phenotypic analysis of store-operated
Ca2+
influx. The dominant analysis identified a unique regionon chromosome 12q24 with a LOD score of3.8, clearly
overlapping with one of six regions (on six different chromo-
somes, LOD scores 1.51.9) identified in the homozygosity
mapping analysis. Because the two analyses were run on
different sets of individuals and were fully independent, it
was possible to add the parametric LOD scores to obtain
a statistically robust combined LOD score of 5.7 (odds of
500,000: one in favour of linkage), defining an 9.8 Mb
candidate region that was overwhelmingly likely to contain
the true gene (Fig. 3b). Genomic sequencing of six known
genes in this region with a potential role in Ca2+ signalling
or Ca2+ binding (shown in blue in Fig. 3b) did not reveal any
mutations in exons or immediately adjacent genomic regions,
but did allow us to narrow down the candidate homozygous
region from 9.8 to an 6.5 Mb interval which contained
74 genes [54].
8. Genome-wide Drosophila RNAi screen to identify
upstream regulators of NFAT
In parallel with genome-wide SNP mapping, we con-
ducted a genome-wide RNAi screen for NFAT regulators
in Drosophila. We chose to perform the RNAi screen in
Drosophila rather than in mammalian cells, partly because
the mammalian RNAi screens that were being set up at
the same time at Harvard Medical School did not cover
the entire human genome, and more importantly, because
RNAi in Drosophila cell cultures is much more efficientthan in mammalian cells [64]. This is primarily because
long double-stranded RNAs (300700 basepairs) are used,
which yield multiple 21-nt siRNAs after processing by Dicer.
Long double-stranded nucleic acids cannot be used in mam-
malian cells since they elicit an interferon response. Another
advantage ofDrosophila RNAi screens is that there are typ-
ically fewer members of each protein family in Drosophila
than in mammals, thereby reducing redundancy; for instance,
Drosophila has one member each of the Stim and Orai pro-
tein families,whereas mammals have two and threemembers,
respectively.
Our decision to use NFAT in the Drosophila RNAi screen
was unusual because the four calcium-regulated NFAT pro-
teins emerged only in vertebrates and are not represented
in Drosophila. Drosophila does possess one protein termed
NFAT (Drosophila NFAT, dNFAT); however, this protein
is more closely related to mammalian NFAT5 (also termed
TonEBP or OREBP) than to the calcium-regulated vertebrate
NFATs [6567].
Only 2 of the 16 candidates, dOrai and dStim ( Fig. 5a,
b), were unambiguously identified as regulators of store-
operated Ca2+ influx in a secondary flow cytometry-based
screen [54,63]. Stromal interaction molecule (STIM) had
been previously identified as an essential regulator of
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Fig. 5. STIM1, Orai1, and CRAC channel activation. (a) The luminal portion of human STIM1 contains an EF-hand paired with a second vestigial EF-hand
sequence that does not bind Ca2+ (not shown), followed by a SAM domain. The cytoplasmic segment contains coiled-coil regions and other regions whose
structure is not known (shown as blobs). The cytoplasmic region of STIM1 is long enough to span the distance between the ER and a closely apposed plasma
membrane. (b) Human Orai1 is an intrinsic plasma membrane protein with four transmembrane segments. The black rectangles in TM1 and TM3 show the
approximate locations of E106 and E190, glutamate residues with a key role in ion selectivity. The red rectangle shows the location of the R91W mutation in
SCID patients [54]. The approximate positions of the N-linked carbohydrate moiety and the HA epitope tag inserted into the TM3-TM4 loop are shown. (c)
The current model of STIM1-Orai1 signalling. The ER Ca2+ sensor STIM1 is distributed throughout the ER in resting cells, with its EF-hand occupied by Ca2+
(grey). On depletion of luminal Ca2+, bound Ca2+ dissociates from the EF-hand (red), eliciting a conformational change in STIM1 and causing it to localise
into puncta. At sites of ER-plasma membrane apposition, signals from STIM1 (red arrows) activate Ca2+ influx through the CRAC channel, a subunit of which
is Orai 1. Adapted with permission from ref. [11] (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article).
store-operated Ca2+ influx in RNAi screens performed inde-
pendently in Drosophila and in mammalian cells [68,69].
The protein product of the Drosophila gene olf186-F was
named Drosophila Orai by one of us (Y.G.); the cor-
responding names for the human proteins are Orai1, 2
and 3 [54] with an alternate proposed nomenclature being
CRACM1, M2 and M3 [61,70]. The genes encoding the
human proteins have been assigned thesymbols TMEM142A,
TMEM142B and TMEM142C (HUGO Gene Nomenclature
Committee).
A successful byproduct of the RNAi screen was the iden-
tification of the elusive SP-3 kinase for NFAT1 [20]. NFATkinases, and a multiude of other kinases, were identified in
a screen for proteins that sent NFAT-GFP to the nucleus
even under resting conditionsthe converse of the screen
used to identify Stim and Orai (Fig. 4). Analysis of the
mammalian homologues of several of these kinases showed
unambiguously that the SP-3 motif of NFAT1 is a target
for phosphorylation by members of the DYRK family of
kinases [20]. The same screen yielded diverse regulators of
calcium homeostasis, including SERCA pumps, Na+/Ca2+
exchangers, K+ channels, and various cation/Ca2+ channels
[20].
9. The ER Ca2+ sensor STIM
RNAi-mediated depletion of Drosophila Stim in S2R
cells, or depletion of either of its two human homologues,
STIM1 and STIM2, in HeLa cells, led to a markeddecreasein
store-operated Ca2+ influx [68,69]. In contrast, in Jurkat cells
STIM1 depletion suppressed thapsigargin-mediated Ca2+
influx whereas STIM2 depletion had little or no effect [68].
The question of the relative roles of STIM1 and STIM2 is
discussed in many of the other reviews in this volume, and
will not be addressed here. Analysis of gene-disrupted mice,
especially those bearing conditional alleles of the Stim1 andStim2 genes, will likely resolve the controversies.
STIM1 is a single-pass transmembrane protein (Fig. 5a)
localized both in the ER and in the plasma membrane.
There is considerable controversy about the role of plasma
membrane STIM1; this topic is also exhaustively reviewed
elsewhere in this volume and will not be discussed here. It
is clear, however, that ER STIM1 is sufficient for activation
of store-operated Ca2+ influx, and that there is no absolute
requirement for STIM1 in the plasma membrane, at least
in the initial activation of Ca2+ influx [71,72]. STIM pro-
teins possess conserved N-terminal Ca2+-binding EF hands
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Y. Gwack et al. / Cell Calcium 42 (2007) 145156 153
localisedeither extracellularly, wherethey would be expected
to bind Ca2+ constitutively, or within the ER lumen, where
they would be expected to sense the luminal Ca2+ concentra-
tion. The EF hand of STIM1 has a Kd for Ca2+ binding that is
estimated at 200600M [73], closely matched to the Ca2+
concentration in the ER lumen (100700M) [74].
The mechanism by which STIM1 couples ER store deple-tion to CRAC channel opening is not clear at present. Store
depletion causes STIM1, which is normally diffusely dis-
tributed in the ER, to relocalise into regions of ER-plasma
membrane apposition, which are visible in the light micro-
scope as small clusters or aggregates that have been termed
puncta (Fig. 5c). EF hand mutants of STIM1 that do not
bind Ca2+ effectively are constitutively localised in puncta
[69,75]. The propensity of STIM1 to aggregate in conditions
of low Ca2+ concentration is recapitulated by a small frag-
ment containing only the functional EF hand, an adjacent
vestigial EF hand (P.G.H., unpublished), and the adjacent
SAM domain (sterile- motif, a domain that in many cases
mediates protein-protein interactions) [73]. The minimummean distance between the ER and the plasma membrane
at sites of punctum formation has been estimated by elec-
tron microscopy as 17 10 nm, close enough for a potential
direct interaction between STIM1 in the ER membrane and
Orai1 in the plasma membrane [71]. Elegant experiments
from Rich Lewis laboratory have shown that STIM1 aggre-
gation precedes the onset of store-operated Ca2+ entry and
CRAC channel opening, suggesting a causal role [76], and
there is evidence that overexpressed Orai1 coaggregates with
overexpressed STIM1 after store depletion [76,77]. In Jurkat
T cells treated with cytochalasin D, large STIM1 aggregates
were shown to correspond to sites of localised Ca2+
influx,and roughly, to sites of Orai1 aggregation as well [76].
Stim and Orai are clearly key components of the path-
way leading from from ER store depletion to CRAC channel
opening. Overexpression of dStim and dOrai in Drosophila
S2 cells, or Orai1 and STIM1 in HEK293 cells or RBL cells,
results in a striking increase in store-operated Ca2+ entry,
and more dramatically, in CRAC currents [62,72,78,79],
suggesting that these two proteins are the only limiting
components of the pathway. Other components may yet be
discovered: potential players include proteins involved in
Stim trafficking, proteins that recruit STIM1 to sites of ER-
plasmamembraneapposition,proteins that arepart of a larger
CRAC channel complex at the plasma membrane, and pro-
teins involved in organising Orai1 within this putative larger
complex. These presumed additional players may be abun-
dant and stable proteins that are difficult to deplete by RNAi.
Stim and Orai were the only ones found in common in the
three Drosophila genome-wide RNAi screens performed by
three independent groups [54,6163]; indeed Stim served as a
positive control based on previous reports [68,69]. Although
the splicing factor Noi emerged as a potential candidate in
two of three screens (our own screen and one of the Ca2+-
based screens [61]), our secondary screens suggested that it
was not a relevant player in the Ca2+ entry pathway [63].
Syntaxin 5, a SNARE protein involved in vesicle fusion, was
identified in only one of the screens, but was suggested to
have a role in the pathway based on the fact that its depletion
inhibited store-operated Ca2+ influx by 23-fold [62].
As an ER Ca2+ sensor, it is likely that STIM1 has roles in
a variety of pathways unrelated to CRAC. Indeed there have
been several reports that STIM1 couples to TRP channelsand is involved in their gating. Again, this point is thoroughly
covered elsewhere in this volume and we refer the reader to
these other reviews.
10. Biochemical properties of Orai1, Orai2 and
Orai3
All three Orai proteins can localise to the plasma mem-
brane, as unambiguously demonstrated by showing that
epitope-tagged Orai, in which theHA epitope tagwas inserted
into the predicted TM3-TM4 loop, was detected by surface
staining of unpermeabilised cells [54,63]. Orai1 has a con-sensus N-glycosylation sequence (NVS) and is glycosylated
when expressed in HEK293 cells, but mutation of this residue
does not impair either surface localisation or function [63]
(Fig.5b). In contrast Orai2 andOrai3do notpossess a consen-
sus sequence for N-glycosylation and are not glycosylated.
Orai1 is a stable dimer in non-ionic detergent solutions,
as shown by glycerol gradient centrifugation [63]. Moreover,
treatment of cells with a cell-permeant crosslinking agent
showed that the protein existed as dimers and possibly as
higher-order multimers (tetramers?) in cells, both before and
after treatment with thapsigargin. Co-immunoprecipitation,
a technique that provides no information about stoichiom-etry, also showed that Orai1 molecules bearing different
epitope tags could associate with one another in detergent
solution [63,70]. Further, Orai1 could co-immunoprecipitate
with Orai2 and Orai3 under the same conditions [63]. Further
experiments are needed to confirm these latter associations
and determine whether mixed dimers or multimers of Orai
proteins exist in cells, and whether they are functional ion
channels.
11. Orai1 is a pore subunit of the CRAC channel
Mutational analyses by several groups have confirmed that
Orai1 is a pore subunit of the CRAC channel [55,70,80]. All
three groups focused on conserved acidic residues, which
are essential for Ca2+ permeation through all Ca2+ channels
that have been analysed so far [6]. In our own experiments
[55], we first showed by RNAi-mediated knockdown of
endogenous Orai in Drosophila cells and reconstitution with
RNAi-resistant mutants in which the conserved glutamates
had been substituted with the sterically similar glutamine
that two conserved glutamates in transmembrane segments
1 and 3 were essential for store-operated Ca2+ entry. We
then replaced the corresponding glutamates (E106 and E190)
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154 Y. Gwack et al. / Cell Calcium 42 (2007) 145156
in human Orai1 with alanine (A), which truncates the side-
chain to a methyl group; aspartate (D), which decreases the
length of the side-chain by a single methylene but preserves
the negative charge; or glutamine (Q), in which the nega-
tively charged carboxylate (COO) has been replaced with
the uncharged but polar amide (CONH2). The mutant pro-
teins were introduced into SCID T cells, and store-operatedCa2+ influx was analysed. The E190Q substitution and all
three substitutions at residue 106 decreased store-operated
Ca2+ influxconsiderably. We usedthe whole-cellpatchclamp
technique to analyse SCID T cells reconstituted with E106D
and E190Q, two mutants whose function was substantially
impaired but which carried sufficient current for reproducible
electrophysiological measurements. Both mutants showed a
decrease in selectivity for Ca2+, measured as an increase in
the relative permeability for Na+ and Cs+ over Ca2+ [55].
In divalent-free solutions, Na+ can permeate through wild-
type CRAC channels but influx is half-maximally blocked by
20M Ca2+ [4,9]; howeverthe E106D mutantshows an10-
fold shift in the dose-response curve, requiring 200MCa2+ to block Na+ influx to the same extent [55]. Similar
results were obtained by Yeromin et al. using Drosophila
Orai, in a context in which it was overexpressed together with
Drosophila Stim in S2 cells [80]. Yeromin et al. also found
that when either of two specific aspartate and asparagine
residues in theextracellular loop ofDrosophila Orai,between
transmembrane segments 1 and 2, was replaced with alanine,
the ability of Gd3+ to block the CRAC current was decreased,
suggesting that these residues were located at the extracellu-
lar mouth of the pore [80]. Finally, Vig et al. overexpressed
human Orai1 and STIM1 in HEK293 cells, and showed again
that E106 and E190 were critical for CRAC channel func-tion [70]. They also confirmed that simultaneous mutation of
D110 and D112 in the extracellular TM1-TM2 loop to ala-
nine decreased the ability of micromolar Ca2+ concentrations
to block Na+ currents in divalent-free medium [70]. These
results are consistent, and show that Orai1 is a pore subunit
of the CRAC channel.
12. Conclusion and perspectives
The stage is now set for detailed analysis of the mecha-
nism of store-operated Ca2+ entry through CRAC channels.
Previously proposed models were: (i) direct conformational
coupling of proteins which sense store depletion in the
ER (previously thought to be InsP3 receptors, now most
likely STIM1) to components of the CRAC channel com-
plex (now, Orai1 and associated proteins); (ii) an insertional
model in which CRAC channels located in intracellular vesi-
cles traffic to the plasma membrane in response to store
depletion (potentially consistent with the identification of
Syntaxin 5 as a verified candidate in a Drosophila screen
[62], but not consistent with the fact that Orai1 is primarily
located in the plasma membrane [63]); and (iii) a diffusible
messenger model in which CRAC channels are gated by
a small, diffusible Ca2+ influx factor that is released from
depleted stores (possibly as a result of STIM1 aggrega-
tion into puncta). Each of these models was supported by
some circumstantial evidence, but conflicting opinions and
reports abounded (reviewed in [81]). With the molecular
players in hand, however, the controversies should soon be
resolved.Several questions remain. (i) The first is the structure of
the CRAC channel itself: is it composed of homomultimers
or heteromultimers of the Orai, and does it contain other
channel subunits or intracellular components that serve to
organise the complex? How do residues in the pore determine
the characteristic properties of the CRAC channel: its very
low single-channel conductance, its high selectivity for Ca2+
over Na+, its sensitivity to removal of all divalent cations, its
calcium-dependent inactivation, its dualsensitivity to 2-APB,
etc.? (ii) A second outstanding question is how CRAC chan-
nels are gated; the mechanism clearly involves STIM1 but
it remains to be determined how STIM1 and Orai1 interact,
how their interaction is regulated by store depletion, and how
STIM1 reaches sites of ER-plasma membrane apposition.
In T cells, both store-operated Ca2+ entry and NFAT-driven
reporter activity were substantially diminished by RNAi-
mediated depletion of WAVE2, an effector protein which
regulates actin polymerisation downstream of Rac1 [82]. Itis
now feasible to ask whether WAVE2 depletion interferes with
the function of known components in the Orai-Stim pathway,
for instance by impairing STIM1 aggregation in response to
store depletion. (iii) A third major question involves the bio-
logical roles of STIM1, STIM2, Orai1, Orai2 and Orai3 in
different cell types and tissues: are the interactions among
these proteins promiscuous or selective, do they interact withother types of proteins (e.g. TRPs), and do they regulate func-
tions other than store-operated Ca2+ entry? (iv) Finally, the
surviving SCID patient has only mild extra-immunological
phenotypes [52], suggesting that blocking the Orai1 channel
will be valuable therapeutically. This possibility remains to
be tested. Given the existence of Orai2 and Orai3, it will be
necessary to identify inhibitors selective for channels con-
taining Orai1, especially if functional channels are formed
that contain heteromultimers of Orai1 with Orai2, Orai3 or
other unrelated proteins.
Acknowledgements
This work was supported by NIH grants GM075256 and
AI40127 to A.R., and NIH grant AI066128 and grants from
the March of Dimes and Charles H. Hood Foundations to S.F.
References
[1] M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality
of calcium signalling, Nat. Rev. Mol. Biol. 1 (2000) 1121.
[2] E. Carafoli, The calcium-signalling saga: tap water and protein crys-
talis, Nat. Rev. Mol. Cell. Biol. 4 (2003) 326332.
-
7/31/2019 Signaling nFAT
11/12
Y. Gwack et al. / Cell Calcium 42 (2007) 145156 155
[3] R.S. Lewis, Calcium signalling mechanisms n T lymphocytes, Annu.
Rev. Immnol. 19 (2001) 497521.
[4] A.B.Parekh,J.W.PutneyJr., Store-operatedcalciumchannels, Physiol.
Rev. 85 (2005) 757810.
[5] P. James, T. Vorherr, E. Carafoli, Calmodulin-binding domains: just
two faced or multi-faceted? Trends Biochem. Sci. 20 (1995) 3842.
[6] W.A. Sather, E.W. McCleskey, Permeation and selectivity in calcium
channels, Annu. Rev. Physiol. 65 (2003) 133159.
[7] D.E. Clapham, TRP channels as cellular sensors, Nature 426 (2003)
517524.
[8] C. Montell, L. Bimbaumer, V. Flockerzi, The TRP channels, a remark-
ably functional family, Cell 108 (2002) 595598.
[9] M. Prakriya, R.S. Lewis, CRAC channels: activation, permeation, and
the search for a molecular identity, Cell Calc. 33 (2003) 311321.
[10] P.G. Hogan, L. Chen, J. Nardone, A. Rao, Transcriptional regulation
by calcium, calcineurin, and NFAT, Genes Dev. 17 (2003) 22052232.
[11] P.G.Hogan,A. Rao,DissectingICRAC, a store-operatedcalcium current,
Trends Biochem. Sci. 32 (2007) 235245.
[12] S. Feske, M. Prakriya, A. Rao, R.S. Lewis, A severe defect in CRAC
Ca2+ channel activation and altered K+ channel gating in T cells from
immunodeficient patients, J. Exp. Med. 202 (2005) 651662.
[13] K.G. Chand, H. Wulff, C. Beeton, M. Pennington, G.A. Gutman, M.D.
Cahalan,K+ channelsas targetsfor specific immunomodulation, Trends
Pharmacol. Sci. 25 (2004) 280289.
[14] G.R. Crabtree, E.N. Olson, NFAT signalling: choreographingthe social
lives of cells, Cell 109 (Suppl.) (2002) S67.
[15] F. Macian, NFAT proteins: key regulators of T-cell development and
function, Nat. Rev. Immunol. 5 (2005) 472484.
[16] J. Heineke, J.D. Molkentin, Regulation of cardiac hypertrophy by
intracellular signalling pathways, Nat. Rev. Mol. Cell Biol. 8 (2006)
589600.
[17] V. Horsley, G.K. Pavlath, NFAT: ubiquitous regulator of cell differen-
tiation and adaption, J. Cell Biol. 156 (2002) 771774.
[18] H. Okamura, J. Aramburu, C. Garcia-Rodriguez, J.P. Viola, A. Ragha-
van, M. Tahiliani, X. Zhang, J. Qin, P.G. Hogan, A. Rao, Concerted
dephosphorylation of the transcription factor NFAT1 induces a con-
formational switch that regulates transcriptional activity, Mol. Cell 6
(2006) 539550.[19] H. Okamura, C. Garcia-Rodriguez, H. Martinson, J. Qin, D.M. Vir-
shup, A. Rao, A conserved docking motif for CK1 binding controls the
nuclear localization of NFAT1, Mol. Cell Biol. 24 (2004) 41844195.
[20] Y. Gwack, S. Sharma, J. Nardone, B. Tanasa, A. Iuga, S. Srkanth, H.
Okamura, D. Bolton, S. Feske, P.G. Hogan, A. Rao, A genome-wide
Drosophila RNAi screen identifies DYRK-family kinases as regulators
of NFAT, Nature 441 (2006) 646650.
[21] R.T. Abraham, A. Weiss, Jurkat T cells and development of the T-cell
receptor signalling paradigm, Nat. Rev. Immunol. 4 (2004) 301308.
[22] S.B. Gauld, J.M. Dal Porto, J.C. Cambier, B cell antigen receptor sig-
nalling: roles in cell development and disease, Science 296 (2002)
16411642.
[23] H. Turner, J.P. Kinet, Signalling through the high-affinity IgE receptor
Fc epsilonRI, Nature 402 (1999) 6760 Suppl. B24-B30.
[24] L.L. Lanier, NK cell recognition, Annu. Rev. Immunol. 23 (2005)225274.
[25] F. Macian, C. Lopez-Rodriguez, A. Rao, Partners in transcription:
NFAT and AP-1, Oncogene 20 (2001) 24762489.
[26] M. Karin, E. Gallagher, From JNK to pay dirt: jun kinases, their bio-
chemistry, physiology and clinical importance, IUBMB Life 57 (2005)
283295.
[27] J. Schulze-Luehrmann, S. Ghosh, Antigen-receptor signalling to
nuclear factor kappa B, Immunity 5 (2006) 701715.
[28] R.E. Dolmetsch, R.S. Lewis, C.C. Goodnow, J.I. Healy, Differential
activation of transcription factors induced by Ca2+ response amplitude
and duration, Nature 386 (1997) 855858.
[29] R.E. Dolmetsch, K. Xu, R.S. Lewis, Calcium oscillations increase
the efficiency and specificity of gene expression, Nature 392 (1998)
933936.
[30] W. Li, J. Llopis, M. Whitney, G. Zlokamik, R.Y. Tsien, Cell-permeant
caged InsP3 ester shows that Ca2+ spike frequency can optimize gene
expression, Nature 392 (1998) 936941.
[31] C. Nagler-Anderson, N.L. Allbriton, C.R. Verret, H.N. Eisen, A com-
parison of the cytolytic properties of murine primary CD8 + cytotoxic
T lymphocytes and cloned cytotoxic T cell lines, Immunol. Rev. 103
(1988) 111125.
[32] J.B. Huppa, M. Gleimer, C. Sumen, M.M. Davis, Continuous T cell
receptor signalling required for synapse maintenance and full effector
potential, Nat. Immunol. 4 (2003) 749755.
[33] C. Loh, K.T.Y. Shaw, J.A. Carew, J.P.B. Viola, B.A. Perrino, A. Rao,
Calcineurin binds the transcription factor NFAT1 and reversibly regu-
lates its activity, J. Biol. Chem. 271 (1996) 1088410891.
[34] L.A. Timmerman, N.A. Clipstone, S.N. Ho, J.P. Northrop, G.R. Crab-
tree, Rapid shuttling of NF-AT in discrimination of Ca2+ signals and
immunosuppression, Nature 383 (1996) 837840.
[35] J.I. Healy, R.E. Dolmetsch, L.A. Timmerman, J.G. Cyster, M.L.
Thomas, G.R. Crabtree, R.S. Lewis, C.C. Goodnow, Different nuclear
signals are activated by the B cell receptor during positive versus neg-
ative signalling, Immunity 4 (1997) 419428.
[36] S.B. Gauld, R.J. Benschop, K.T. Merrell, J.C. Cambier, Maintenance
of B cell anergy requires constant antigen receptor occupancy and
signalling, Nat. Immunol. 6 (11) (2005 Nov.) 11601167.
[37] K.T. Merrell, R.J. Benschop, S.B. Gauld, K. Aviszus, D. Decote-
Ricardo, L.J. Wysocki, J.C. Cambier, Identification of anergic B cells
within a wild-type repertoire, Immunity 6 (2006) 953962.
[38] D.A. Fulcher, A. Basten, Reduced life span of anergic self-reactive B
cells in a double-transgenic model, J. Exp. Med. 179 (1994) 125134.
[39] R. Barrington, M. Borde, A. Rao, M. Carroll, NFAT1 involvement in
B cell self-tolerance, J. Immunol. 177 (2006) 15101515.
[40] R.H. Schwartz,T cellanergy, Annu. Rev. Immunol.21 (2003) 305334.
[41] F. Macian, F.J.G. Cozar, S.-H. Im, H.F. Horton, M.C. Bryne, A. Rao,
Transcriptional mechanisms underlying lymphocyte tolerance, Cell
109 (2002) 719731.
[42] V. Heissmeyer, A. Rao, E3 ligases in T cell anergy: turning immune
responses into tolerance, Science STKE 241 (2004) 15.
[43] B.A. Olenchock, R. Guo, J.H. Carpenter, M. Jordan, M.K. Topham,
G.A. Koretzky, X.P. Zhong, Disruption of diacylglycerol metabolismimpairs the induction of T cell anergy, Nat. Immunol. 7 (2006)
11741181.
[44] M. Safford, S. Collins, M.A. Lutz, A. Allen, C.T. Huang, J. Kowalski,
A. Blackford, M.R. Horton, C. Drake,R.H. Schwartz,J.D. Powell, Egr-
2 and Egr-3 are negative regulators of T cell activation, Nat. Immunol.
6 (2005) 472480.
[45] S. Bandyopadhyay, M. Dure, M. Paroder, N. Soto-Nieves, I. Puga, F.
Macian, Interleukin 2 gene transcription is regulatedby Ikaros-induced
changes in histone acetylation in anergic T cells, Blood (2006) [Epub
ahead of print].
[46] V. Heissmeyer,F. Macian,S.-H. Im, R. Varma,S. Feske, K. Venuprasad,
M.-S.Jeon, H. Gu, Y.-C.Liu, M.L. Dustin, A. Rao,Calcineurin imposes
T cell unresponsiveness through targeted proteolysis of signalling pro-
teins, Nat. Immunol. 5 (2004) 255265.
[47] D.L. Mueller, E3 ubiquitin ligases as T cell anergy factors, Nat.Immunol. 5 (2004) 883890.
[48] M. Hundt, H. Tabatha, M.S. Jeon, K. Hayashi, Y. Tanaka, R. Krishna,
L. De Giorgio,Y.C. Liu, M. Fukata, A. Altman, Impairedactivationand
localization of LAT in anergic T cells as a consequence of a selective
palmitoylation defect, Immunity 24 (2006) 513522.
[49] S. Feske, J.M. Muller, D. Graf, R.A. Kroczek, R. Drager, C. Niemeyer,
P.A. Baeuerle, H.H. Peter, M. Schlesier, Severe combined immunod-
eficiency due to defective binding of the nuclear factor of activated T
cells in T lymphocytes of twomalesiblings, Eur. J. Immunol.26 (1996)
21192126.
[50] M. Partiseti, F. Le Deist, C. Hivroz, A. Fischer, H. Korn, D. Choquet,
The calcium current activated by T cell receptor and store depletion in
human lymphocytes is absent in a primary immunodeficiency, J. Biol.
Chem. 269 (1994) 3232732335.
-
7/31/2019 Signaling nFAT
12/12
156 Y. Gwack et al. / Cell Calcium 42 (2007) 145156
[51] F. Le Deist,C. Hivroz, M. Partiseti, C. Thomas, H.A. Buc, M. Oleastro,
B. Belohradsky, D. Choquet, A. Fischer, A primary T-cell immunodefi-
ciency associated with defective transmembrane calcium influx, Blood
85 (1995) 10531062.
[52] S. Feske, R. Draeger, H.H. Peter, K. Eichmann, A. Rao, The dura-
tion of nuclear residence of NFAT determines the pattern of cytokine
expression in human SCID T cells, J. Immunol. 165 (2000) 297305.
[53] S. Feske, G. Giltnane,R. Dolmetsch, L. Standt, A. Rao,Gene regulation
mediated by calcium signals in T lymphocytes, Nat. Immunol.2 (2001)
316324.
[54] S. Feske, Y. Gwack, M. Prakriya, S. Srikanth, S.-H. Puppel, B. Tanasa,
P.G. Hogan, R.S. Lewis, M. Daly, A. Rao, A mutation in Orai1 causes
immune deficiency by abrogating store-operated Ca2+entry and CRAC
channel function, Nature 441 (2006) 179185.
[55] M. Prakriya, S. Feske, Y. Gwack, S. Srikanth, A. Rao, P. Hogan, Orai1
is an essential pore subunit of the CRAC channel, Nature 443 (2006)
230233.
[56] A.V. Yeromin, J. Roos, K.A. Stauderman, M.D. Cahalan, A store-
operated calcium channel in Drosophila S2 cells, J. Gen. Physiol. 123
(2004) 167182.
[57] N. Perrimon, B. Mathey-Prevot, Applications of high-throughput RNA
interference screens to problems in cell and developmental biology,
Genetics 175 (2007) 716.
[58] C.J. Echeverri, N. Perrimon, High-throughput RNAi screening in cul-
tured cells: a users guide, Nat. Rev. Genet. 7 (2006) 373384.
[59] I. Flockhart, M. Booker, A. Kiger, M. Boutros, S. Armknecht, N.
Ramadan, K. Richardson, A. Xu, N. Perrimon, B. Mathey-Prevot, Fly-
RNAi: the Drosophila RNAi screening center database, Nucl. Acids
Res. 34 (2006) D489D494 (Database issue).
[60] S. Armknecht, M. Boutros, A. Kiger, K. Nybakken, B. Mathey-Prevot,
N. Perrimon, High-throughput RNA interference screens in Drosophila
tissue culture cells, Meth. Enzymol. 392 (2005) 5573.
[61] M. Vig, C. Peinelt, A. Beck, D.L. Koomoa, D. Rabah, M. Koblan-
Huberson, S. Kraft, H. Turner, A. Fleig, R. Penner, J.P. Kineet,
CRACM1 is a plasma membrane protein essential for store-operated
Ca2+ entry, Science 312 (2006) 12201223.
[62] S.L. Zhang, A.V. Yeromin, X.H. Zhang, Y. Yu, O. Safrina, A. Penna, J.
Roos, K.A. Stauderman, M.D. Cahalan, Genome-wide RNAi screenof Ca2+ influx identifies genes that regulate Ca2+ release-activated
Ca2+ channel activity, Proc. Natl. Acad. Sci. U S A 103 (2006) 9357
9362.
[63] Y. Gwack, S. Srikanth, S. Feske, F. Cruz-Guilloty, M. Oh-hora, D.
Neems, P.G. Hogan, A. Rao, Biochemical and functional characteriza-
tion of Orai-family proteins, J. Biol. Chem. 282 (2007) 1623216243.
[64] J.C. Clemens, C.A. Worby, N. Simonson-Leff, M. Muda, T. Maehama,
B.A. Hemmings, J.E. Dixon, Use of double-stranded RNAinterference
in Drosophila cell lines to dissect signal transduction pathways, Proc.
Natl. Acad. Sci. U S A 97 (2000) 64996503.
[65] C. Lopez-Rodriguez, C.L. Antos, J.M. Shelton, J.A. Richardson, F. Lin,
T.I. Novobrantseva,R.T. Bronson,P.Igarashi,A. Rao,E.N. Olson, Loss
of NFAT5 results in renal atrophy and lack of tonicity-responsive gene
expression, Proc. Natl. Acad. Sci. U S A 101 (2004) 23922397.
[66] J. Aramburu, K. Drews Elger, A. Estrada-Gelonch, J. Minguillon,B. Morancho, V. Santiago, C. Lopez-Rodriguez, Regulation of the
hypertonic stress response and other cellular functions by the Rel-
like transcription factor NFAT5, Biochem. Pharmacol. 72 (2006)
15971607.
[67] I.A. Graef, J.M. Gastier, U. Francke, G.R. Crabtree, Evolutionary
relationships among Rel domains indicate functional diversification
by recombination, Proc. Natl. Acad. Sci. U S A 98 (2001) 5740
5745.
[68] J. Roos, P.J. DiGregorio, A.V. Yeromin, K. Ohlsen, M. Lioudyno,
S. Zhang, O. Safrina, J.A. Kozak, S.L. Wagner, M.D. Cahalan, G.
Velicelebi, K.A. Stauderman, STIM1, an essential and conserved com-
ponent of store-operatedCa2+ channelfunction, J. CellBiol. 169(2005)
435445.
[69] J. Liou, M.L. Kim, W.D. Heo, J.T. Jones, J.W. Myers, J.E. Ferrell Jr.,
T. Meyer, STIM is a Ca2+ sensor essential for Ca2+-store-depletion-
triggered Ca2+ influx, Curr. Biol. 15 (2005) 12351241.
[70] M. Vig, A. Beck, J.M. Billingsley, A. Lis, S. Parvez, C. Peinelt,
D.L. Koomoa, J. Soboloff, D.L. Gill, A. Fleig, J.P. Kinet, R. Penner,
CRACM1 multimersform the ion-selective pore of the CRAC channel,
Curr. Biol. 16 (2006) 17.
[71] M.M. Wu, J. Buchanan, R.M. Luik, R.S. Lewis, Ca2+ store depletion
causes STIM1 to accumulate in ER regions closely associated with the
plasma membrane, J. Cell Biol. 174 (2006) 803813.
[72] J.C. Mercer, W.I. Dehaven, J.T. Smyth, B. Wedel, R.R. Boyles, G.S.
Bird, J.W. Putney Jr., Large store-operated calcium selective currents
due to co-expression of Orai1 and Orai2 with the intracellular calcium
sensor, Stim1, J. Biol. Chem. 281 (2006) 2497924990.
[73] P.B.Stathopulos,G.Y. Li, M.J.Plevin, J.B.Ames, M. Ikura, Stored Ca2+
depletion-induced oligomerization of stromal interaction molecule 1
(STIM1) via the EF-SAM region. An initiation mechanism for capaci-
tive Ca2+ entry, J. Biol. Chem. 281 (2006) 3585535862.
[74] J. Meldolesi, T. Pozzan, The endoplasmic reticulum Ca2+ store: a view
from the lumen, Trends Biochem. Sci. 23 (1998) 1014.
[75] S.L. Zhang, Y. Yu, J. Roos, J.A. Kozak, T.J. Deerinck, M.H. Ellisman,
K.A. Stauderman, M.D. Cahalan, STIM1 is a Ca2+ sensor that acti-
vates CRAC channels and migrates from the Ca2+ store to the plasma
membrane, Nature 437 (2005) 902905.
[76] R.M. Luik, M.M. Wu, J. Buchanan, R.S. Lewis, The elementary
unit of store-operated Ca2+ entry: local activation of CRAC chan-
nels by STIM1 and CRACM1 (Orai1), Nat. Cell Biol. 8 (2006) 815
825.
[77] P. Xu, J. Lu, Z. Li, X. Yu, L. Chen, T. Xu, Aggregation of STIM1underneath the plasma membrane induces of Orai1, Biochem.Biophys.
Res. Commun. 350 (2006) 969976.
[78] C. Peinelt, M. Vig, D.L. Koomoa, A. Beck, M.J. Nadler, M. Koblan-
Huberson, A. Lis, A. Fleig, R. Penner, J.P. Kinet, Amplification of
CRAC current by STIM1 and CRACM1 (Orai1), Nat. Cell Biol. 8
(2006) 771773.
[79] J. Soboloff, M.A. Spassova, X.D. Tang, T. Hewavitharana, W. Xu,
D.L. Gill, Orai1 and STIM reconstitute store-operatedcalcium channel
function, J. Biol. Chem. 281 (2006) 2066120665.
[80] A.V. Yeromin, S.L. Zhang, W. Jiang, Y. Yu, O. Safrina, M.D. Cahalan,
Molecular identification of the CRAC channel by altered selectivity in
a mutant of Orai, Nature 443 (2006) 226229.
[81] J.T. Smyth, W.I. Dehaven, B.F. Jones, J.C. Mercer, M. Trebak, G.
Vazquz, J.W. Putney Jr., Emerging perspectives in store-operated Ca 2+
entry: roles of Orai, Stim, and TRP.[82] J.C. Nolz, T.S. Gomez, P. Zhu, S. Li, R.B. Medeiros, Y. Shumizu, J.K.
Burkhardt, B.D. Freedman, D.D. Billadeau, The WAVE2 complex reg-
ulates actin cytoskeletal reorganization and CRAC-mediated calcium
entry during T cell activation, Curr. Biol. 16 (2006) 2434.