5.1. Introduction

In order to respond to changes in the environment, cells need to switch genes on and off. One conserved mechanism that links events at the cell surface to gene expression in the nucleus employs intracellular Ca²⁺. A cytoplasmic Ca²⁺ rise stimulates Ca²⁺-dependent transcription factors, which then regulate gene activity. In neurons, Ca²⁺ entry through voltage-gated Ca²⁺ channels activates Ca²⁺-calmodulin-dependent protein kinases, leading to the phosphorylation of the transcription factor CREB (cAMP response-element binding protein) [1,2]. Phosphorylated CREB is thought to be involved in long-term potentiation [3], a form of learning and memory in the nervous system. In immune cells, transcription factors such as c-fos and the nuclear factor of activated T cells (NFAT) are activated by Ca²⁺ influx through store-operated Ca²⁺- release-activated Ca²⁺ (CRAC) channels [4,5]. These transcription factors often work in tandem to control the expression of chemokines and cytokines that are involved in both the innate and adaptive immune responses [6]. NFAT proteins are a family of five cytosolic proteins that migrate into the nucleus upon activation. For four of the members, translocation is triggered by a rise in cytoplasmic Ca²⁺ concentration [5,7]. NFATs are extensively phosphorylated at rest, resulting in the masking of a nuclear localization sequence. A rise in Ca²⁺ activates the enzyme calcineurin, the target of immunosuppressants cyclosporine A and tacrolimus. Dephosphorylation exposes nuclear localization sequences, enabling NFAT to migrate into the nucleus in a complex with tubulin alpha [8]. Activator protein-1 (AP-1) is a transcription complex composed of members of the fos and jun families that can associate to form a range of homo- and heterodimers [9]. AP-1 activity can be increased by the stimulation of preexisting protein or following enhanced expression and many extracellular signals including G-protein-coupled receptor agonists, cytokines, and growth factors target AP-1 activity. Here, the involvement of cytoplasmic Ca²⁺, particularly localized Ca²⁺ signals or Ca²⁺ microdomains, in activating NFAT and c-fos will be described. The focus is on immune cells, where these processes have been studied in considerable detail.

5.2. Ca²⁺ Entry through CRAC Channels Activates Gene Expression

In immune cells, it has been known for many years that a rise in cytoplasmic Ca²⁺ is essential for expression of various chemokines and cytokines that drive T cell proliferation and the subsequent immune response [10]. Although cells can raise Ca²⁺ through either Ca²⁺ release from internal stores or Ca²⁺ influx across the plasma membrane, Ca²⁺ entry was found to play the major role in immune cell activation. Stimulation in the absence of external Ca²⁺ often failed to evoke any detectable gene expression, whereas robust transcription occurred when external Ca²⁺ was present. For example, studies on the Jurkat human T cell line revealed that antibodies against the T lymphocyte antigen receptor complex evoked Ca²⁺ release from the stores but this failed to induce interleukin 2 (IL-2) expression [11]. In the murine T cell hybridoma B3Z expressing the lacZ reporter gene under the transcriptional control of NFAT, the fraction of lacZ-positive cells fell after stimulation when external Ca²⁺ was reduced [12]. Further evidence for the major role of Ca²⁺ influx in gene expression came from studies on T cells isolated from a 3-month-old immunodeficient boy [13]. Stimulation with anti-CD3 antibody evoked less IL-2 production than in the control and this was pinpointed to impaired Ca²⁺ influx. By contrast, Ca²⁺ release was unaffected. Similar conclusions were reached in rat bone marrow-derived mast cells and RBL-2H3 immortalized mast cell line [14]. Cross-linking of FCεRI receptors led to the activation of NFAT and this was suppressed by the removal of external Ca²⁺. Collectively, these and other studies established the importance of Ca²⁺ influx as a key activator of NFAT and transcription of IL-2.

Cells express a plethora of Ca²⁺ entry channels [15], and each could potentially underlie the Ca²⁺ influx needed for gene expression in immune cells. Several studies, using different approaches, identified the CRAC channel as being the dominant plasmalemmal Ca²⁺ channel that coupled to NFAT activation and gene expression. First, noise analysis of the whole cell Ca²⁺ current in T cells revealed that the chord conductance of the Ca²⁺ channel activated by the T cell receptor was identical to that evoked by thapsigargin [16] (see Chapter 1). Hence, the CRAC channel and the Ca²⁺ channel activated by the T cell receptor, which leads to NFAT activation, are one and the same. Second, the elevation of external K⁺ depolarizes the membrane potential and this was found to reduce Ca²⁺ influx and NFAT reporter gene expression [12] (see Figure 1.1). Because the open probability of voltage-gated Ca²⁺ channels increases as the membrane potential decreases, these results established that the Ca²⁺ influx pathway was not depolarization activated. Third, patch clamp studies on T cells from an immunodeficient infant, in which T cell proliferation was lost, revealed the complete absence of the non-voltage-activated CRAC current following stimulation of either the T cell receptor or after store depletion with thapsigargin [17]. Fourth, mutant cells selected from a population of gamma-irradiated Jurkat NZDipA cells showed defective CRAC channel activity to varying degrees, and the severity of the defect correlated well with the extent of compromised NFAT-dependent reporter gene expression [18]. Collectively, these findings constructed a body of evidence that suggested Ca²⁺ entry through the CRAC channel was required for NFAT activation followed by gene expression. Direct evidence in support of this came when the CRAC channel components were identified at a molecular level.

The first critical component to be identified was the stromal interaction molecule (STIM) family of proteins, STIM1 and STIM2 [19,20]. STIM proteins are found mainly spanning the endoplasmic reticulum, where they function as Ca²⁺ sensors to monitor the Ca²⁺ content of the store [21]. Upon store depletion, they form multimers that migrate to specialized regions of the endoplasmic reticulum located just beneath the plasma membrane [22,23]. Here, they bind to and activate CRAC channels in the plasma membrane.

Gene linkage analysis and siRNA knockdown strategies identified Orai1 as being essential for CRAC channel function [24–26] (see Chapters 2 and 3). Orai1 is a plasma membrane protein with four transmembrane domains. The mutation of the conserved acidic residue E106 in transmembrane domain reduced Ca²⁺ influx and changed ion selectivity, establishing Orai1 as the pore-forming subunit of the CRAC channel [27–29]. A point mutation, R91W, in Orai1 was identified in patients with one form of hereditary severe combined immunodeficiency [25]. This mutant channel was expressed in the plasma membrane but was unable to gate. Importantly, the expression of wild-type Orai1 rescued Ca²⁺ influx in the immunodeficient T cells. Consistent with a fundamental role for Orai1 in T cells, the expression of pore mutant E106Q Orai1 channels in T cells reduced cell proliferation and cytokine secretion [30]. Furthermore, the overexpression of the recombinant Orai1 channel in HEK cells accelerated NFAT activation and reporter gene expression, whereas the knockdown of endogenous Orai1 prevented NFAT activation and gene expression in response to thapsigargin [31]. Moreover, the knockdown of Orai1 suppressed NFAT-driven gene expression following stimulation of either FCεF1 or cysteinyl leukotriene type I G-protein-coupled receptors, demonstrating the physiological relevance of CRAC channels to this form of excitation-transcription coupling [32]. Finally, a relatively specific CRAC channel blocker, Synta66 [33], inhibited NFAT activation following the stimulation of native CRAC channels [31].

In addition to activating NFAT, CRAC channels also induce the expression of the immediate early gene c-fos [34], a component of the AP-1 transcription factor complex. AP-1 and NFAT are considered partners in transcriptional regulation because composite NFAT and AP-1 sites are found on numerous genes that are regulated during the immune response [6].

Stimulation with thapsigargin or leukotriene C₄ (LTC₄), an agonist of G-protein-coupled cysteinyl leukotriene type I receptors, which couple to phospholipase C to generate IP₃ and stimulate protein kinase C, increased c-fos transcription but only when external Ca²⁺ was present [35]. A pharmacological block of CRAC channels with Synta66 also suppressed c-fos induction [35], as did knockdown of Orai1 [36].

5.3. The Importance of Ca²⁺ Microdomains near Open CRAC Channels in the Regulation of Transcription

Although Ca²⁺ entry through CRAC channels is important in activating NFAT and c-fos, several lines of evidence show that Ca²⁺ microdomains near the mouth of open channels are more effective in activating the transcription factors than a rise in bulk cytoplasmic Ca²⁺. First, the Ca²⁺ response to thapsigargin in Ca²⁺-free external solution is transient because plasma membrane Ca²⁺ clearance mechanisms remove cytoplasmic Ca²⁺. In rat basophilic leukemia (RBL) and human embryonic kidney (HEK) cells, the inhibition of the plasma membrane Ca²⁺ ATPase (PMCA) pump with La³⁺ leads to a large and sustained rise in bulk Ca²⁺ following stimulation with thapsigargin in a Ca²⁺-free solution. Although the bulk Ca²⁺ rise was larger than that seen when cells were exposed to thapsigargin in the presence of external Ca²⁺, c-fos transcription was not stimulated [37]. Interestingly, NFAT1 migration into the nucleus did occur upon challenge with thapsigargin in Ca²⁺-free solution supplemented with La³⁺, suggesting a large, nonphysiological bulk cytoplasmic Ca²⁺ rise is sufficient to activate NFAT1 [31]. Second, one important factor that determines the size of Ca²⁺ microdomains is the unitary flux through each channel. This is dictated by the prevailing electrochemical gradient. The K_D for Ca²⁺ permeation through CRAC channels in RBL cells is ∼0.7 mM [38]. Therefore, the unitary flux in 0.5 mM external Ca²⁺ should be considerably smaller than in 2 mM Ca²⁺. When RBL cells were stimulated with thapsigargin in either 0.5 or 2 mM Ca²⁺, bulk Ca²⁺ was similar but the transcription of c-fos only occurred in the higher external Ca²⁺ [37]. Similar findings were obtained for NFAT activation in HEK cells [31]. Conversely, a reduction of the membrane potential by the inhibition of inward rectifier K⁺ channels in RBL cells had little effect on bulk Ca²⁺ but suppressed CRAC channel-driven c-fos expression [37]. Hence, manipulations that reduce the unitary flux and thus the size of the Ca²⁺ microdomain, without compromising the bulk Ca²⁺ rise, impair c-fos and NFAT activation. Third, loading the cytoplasm with the Ca²⁺ chelators EGTA or BAPTA both substantially reduce the rise in bulk cytoplasmic Ca²⁺ following stimulation with thapsigargin. However, EGTA is too slow to prevent the build-up of the Ca²⁺ microdomain and had no inhibitory effect on either c-fos transcription or NFAT activation in RBL or HEK cells [31,37,39]. By contrast, BAPTA has an on-rate for Ca²⁺ ∼500-fold faster than EGTA and therefore reduces the size and lateral extent of the microdomain. In support of a central role for Ca²⁺ microdomains, BAPTA was found to impair both c-fos and NFAT activation following CRAC channel opening [31,37]. Consistent with these earlier studies, BAPTA but not EGTA was also found to suppress NFAT activation by CRAC channels in neural stem cells [40]. Finally, a comparison of the signaling capability of clustered Orai1 channels with dispersed constitutively active V102C mutant Orai1 channels showed that channel clustering was far more effective in activating both c-fos and NFAT than a similar number of active but diffuse ones [36]. Although the Ca²⁺ selectivity of the V102C Orai1 channels is slightly less than for the wild-type channel, the bulk Ca²⁺ was nevertheless increased to a similar extent. Clustering appears favored in signal transduction because the overlapping Ca²⁺ microdomains result in a much higher local Ca²⁺ signal that robustly activates downstream signaling pathways [36].

5.4. How Local Is Local?

An interesting question concerns how local the Ca²⁺ signal near Orai1 needs to be to activate NFAT. Is the Ca²⁺ signal spatially restricted to Orai1 or is a more general smearing of subplasmalemmal Ca²⁺ important? One way of addressing this is to compare the ability of two different plasma membrane Ca²⁺-permeable channels that both raise bulk cytoplasmic Ca²⁺ to similar extent to activate NFAT. The activation of recombinant TRPC3 channels with 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analogue, led to a rise in cytoplasmic Ca²⁺ that was dependent entirely on Ca²⁺ entry [31]. The rise in cytoplasmic Ca²⁺ was similar to that evoked following CRAC channel opening. However, NFAT1 consistently failed to migrate into the nucleus in the presence of OAG [31]. Hence, a general rise in subplasmalemmal Ca²⁺ is not sufficient for NFAT1 activation; rather, it is the Ca²⁺ in the vicinity of CRAC channels that is important.

These findings should not be interpreted to mean that Ca²⁺ entry through TRPC3 is unable to activate NFAT1 in other systems [41]. TRPC3 can form heteromultimers with TRPC1 [42], and TRPC1 channels can be inserted into the plasma membrane following local Ca²⁺ entry through Orai1 [43], suggesting that TRPC and Orai1 channels might colocalize in the membrane. Nevertheless, in mast cells, the direct opening of recombinant TRPC3 channels with OAG is considerably less effective than CRAC channels in activating NFAT1.

5.5. Sensing Local Ca²⁺ near CRAC Channels

For Ca²⁺ microdomains near CRAC channels to activate transcription factors, a Ca²⁺ sensor is required that relays local Ca²⁺ to a downstream signaling pathway. Interestingly, different signal transduction pathways are used to activate c-fos and NFAT [36,37].

In RBL-1 cells, Ca²⁺ microdomains near CRAC channels stimulate c-fos expression through recruitment of the nonreceptor tyrosine kinase Syk [33,37]. A pharmacological block of Syk and a reduction in protein levels using an siRNA-based approach both inhibited c-fos expression in response to thapsigargin [37] or cysteinyl leukotriene type I receptor stimulation [39]. Ca²⁺ entry through the channels was unaffected, suggesting that the involvement of Syk was downstream of CRAC channel activation. Syk is expressed mainly at the cell periphery and remains so after CRAC channel opening [37]. Co-immunoprecipitation studies have found association between Syk and Orai1 under nonstimulated conditions and this increases ∼2-fold after store depletion with thapsigargin [36]. The signal transducers and activators of transcription (STAT) family of transcription factors are widely expressed in the immune system and are activated following phosphorylation by nonreceptor tyrosine kinases [44,45]. Phosphorylated STATs dimerize and then rapidly translocate into the nucleus, where they bind to enhancer elements to regulate gene expression. Following CRAC channel opening, STAT5 was phosphorylated within minutes and this was suppressed by the Syk inhibitor [37]. Hence, Ca²⁺ entry through CRAC channels activates c-fos through the Syk/STAT5 pathway. More recent work using phospho-specific antibodies for the highly related STAT5a and STAT5b proteins found that STAT5a phosphorylation was significantly increased by Ca²⁺ entry through CRAC channels [46]. Basal STAT5b phosphorylation was ∼2-fold higher than for STAT5a, but did not change after stimulation.

NFAT activation is mediated through the Ca²⁺-dependent stimulation of protein phosphatase 2B (calcineurin), the target of immunosuppressants cyclosporine A and tacrolimus. In neurons, the plasmalemmal scaffold protein AKAP79 binds both calcineurin and Ca_v1.2 Ca²⁺ channels, thereby bringing the phosphatase within the realm of the Ca²⁺ microdomain [47,48]. Biochemical pull-down studies revealed that Orai1 was associated with calcineurin after store depletion, and this was prevented following the knockdown of AKAP79 [49]. The knockdown of AKAP79 prevented CRAC channels from activating NFAT, an effect that was rescued by the overexpression of AKAP79 but not by a mutant AKAP protein that was unable to bind calcineurin. In addition to binding calcineurin, AKAP79 was also associated with a fraction of the total NFAT pool [50]. These results show that calcineurin and NFAT are brought close to the CRAC channel microdomain after store depletion, providing a mechanism to activate the enzyme selectively while providing a high local concentration of its target NFAT.

5.6. Parallel Processing of the CRAC Channel Ca²⁺ Microdomain

Pharmacological and siRNA-based approaches reveal that CRAC channel-gated Ca²⁺ microdomains activate Syk and calcineurin through distinct signaling pathways [36]. The inhibition or knockdown of Syk impaired c-fos expression following CRAC channel opening without affecting NFAT activation. Conversely, the inhibition of calcineurin with cyclosporine A suppressed NFAT activation without affecting c-fos expression. Ca²⁺ microdomains therefore recruit two different transcription factors through distinct signaling mechanisms.

In addition to enhancing c-fos expression, Syk also stimulates extracellular signal-regulated kinases (ERK) via protein kinase C [34,51]. This leads to the phosphorylation of both Ca²⁺-dependent phospholipase A₂ (cPLA₂) and 5-lipoxygenase, increasing activities of both enzymes. cPLA₂ hydrolyzes arachidonyl phospholipids to release arachidonic acid, which is then metabolized by 5-lipoxygenase to produce leukotrienes such as LTC₄, a powerful pro-inflammatory signal (see Chapter 11). Although a pharmacological block of Syk suppresses both c-fos transcription and LTC₄ production, ERK inhibition impacted only on LTC₄ production [34]. The parallel processing of the Ca²⁺ microdomain by Syk through two distinct signaling pathways therefore constitutes an effective mechanism to evoke spatially and temporally different cellular responses.

5.7. Caveolin-1 Differentially Regulates NFAT and c-Fos Activities

AP-1 and NFAT often interact cooperatively to control gene expression. For example, genes that encode IL-2-4 and granulocyte-macrophage colony-stimulating factor are transcribed when both NFAT and c-fos are present [52]. By contrast, the transcription of some other genes such as interleukin-13 and tumor necrosis factor-α is induced by NFAT alone [52]. Different gene expression programs can therefore be activated depending on whether NFAT and c-fos operate together or in isolation. This raises an interesting question: If the same signal, namely, Ca²⁺ microdomains near open CRAC channels, activates both NFAT and c-fos, then how can one be activated and not the other? A mechanism must exist that is capable of tunneling the Ca²⁺ microdomain to recruit one transcription factor and not the other. Because Ca²⁺ microdomains activate NFAT and c-fos indirectly, via AKAP79 and Syk, respectively, one possibility is that a membrane scaffolding complex that interacts with numerous signaling pathways determines which transcription factor is activated by the Ca²⁺ microdomain. One candidate for this role is caveolin-1, a protein found in the plasma membrane. Caveolin-1 forms a large oligomeric complex [53], interacts with multiple ion channels [54,55], and helps form signalosomes at the cell periphery [56]. Co-immunoprecipitation studies have found that caveolin-1 associates with Orai1 [57], and two potential interaction sites are on the cytoplasmic N terminus between amino acids 52 and 60 [57] and on transmembrane domain 4, between amino acids 250 and 258 [21].

The overexpression of caveolin-1 in RBL mast cells resulted in enhanced store-operated Ca²⁺ entry [46], which seemed to arise through the stabilization of the STIM1-Orai1 interaction. This action required the scaffolding domain of caveolin-1 because mutations within the critical core motif that is required for association with signaling molecules [58,59] abolished the potentiation of Ca²⁺ entry. Because of the increase in Ca²⁺ flux through CRAC channels, a simple expectation would be that both NFAT and c-fos activities should increase in the presence of caveolin-1. However, this was not the case. Although NFAT reporter gene expression increased after the activation of CRAC channels in cells overexpressing caveolin-1, c-fos expression was actually inhibited [46]. The phosphorylation of STAT5 following Ca²⁺ flux through CRAC channels was prevented by caveolin-1 [46]. Therefore, caveolin-1 differentially regulates the activation of the two transcription factors in response to the same local Ca²⁺ stimulus.

5.8. Modular Regulation by Caveolin-1

One possible explanation for the opposing effects of caveolin-1 on NFAT and c-fos activities relates to the increased Ca²⁺ influx through CRAC channels. c-Fos activity could have a bell-shaped dependence on local Ca²⁺; in this scenario, Ca²⁺ would initially stimulate c-fos but a further rise would then inhibit expression. By contrast, NFAT activity increases quasi-monotonically with local Ca²⁺ until the pathway reaches its maximal capacity. Several lines of evidence argue against this possibility [46]. First, caveolin-1 expression still resulted in the inhibition of c-fos even when stimulus intensity was reduced. Second, reducing the rise in cytoplasmic Ca²⁺ by loading the cytoplasm with EGTA failed to rescue c-fos expression in the presence of caveolin-1. Third, the application of ionomycin after exposure to thapsigargin in wild-type cells raised cytoplasmic Ca²⁺ to high levels, and this led to a further increase in c-fos expression. This Ca²⁺ rise was greater than that seen in cells expressing caveolin-1 and stimulated with thapsigargin, conditions that failed to activate c-fos. In a model where c-fos exhibits a bell-shaped dependence on Ca²⁺ levels, ionomycin stimulation after thapsigargin should have reduced c-fos expression, not increased it. Finally, mutations within the scaffolding domain prevented the increase in Ca²⁺ influx following store depletion but c-fos activity remained suppressed.

Evidence for modular regulation by caveolin-1 of NFAT and c-fos activities has come from experiments with a tyrosine phosphorylation-resistant mutant [46]. Caveolin-1 is phosphorylated by Src family tyrosine kinases on cytosol-facing amino acid residue tyrosine 14 [60], which is thought to tether the Src SH2 domain to phosphorylated caveolin-1 and thus retain Src at specific plasma membrane regions such as focal adhesions [61]. In RBL cells, recombinant caveolin-1 was phosphorylated on tyrosine 14 under resting conditions. Following stimulation with thapsigargin, phosphorylation was only slightly reduced and c-fos expression was suppressed [46]. Expression of a caveolin-1 protein in which tyrosine 14 had been mutated to phenylalanine (caveolin-1-Y14F) fully rescued STAT5a phosphorylation and c-fos expression following CRAC channel opening [46]. This mutant, like wild-type caveolin-1, increased store-operated Ca²⁺ entry as well as NFAT reporter gene expression.

Tyrosine 14 on the cytoplasmic region of caveolin-1 is the locus for the inhibition of c-fos transcription. The phosphorylation of Tyr14 inhibited the ability of Ca²⁺ entry through CRAC channels to activate STAT5a and thus inhibited c-fos expression in RBL cells. By contrast, Tyr 14 phosphorylation of caveolin-1 had no inhibitory effect on NFAT activation, revealing that the phosphorylation status of this site helps determine whether the same local Ca²⁺ signal is capable of simultaneously activating the two transcription factors.

Caveolin-1 is expressed in CD4+ and CD8+ T lymphocytes [62]. CD8+ T cells deficient in caveolin-1 showed normal NFkB activity but an impaired NFAT pathway [62]. Caveolin-1 expression has been reported to inhibit HIV replication through defective NFkB signaling [63]. Hence, caveolin-1 seems to regulate NFAT and NFkB in T cells in a reciprocal manner in certain situations, reminiscent of the regulation of c-fos and NFAT in mast cells.

5.9. Large Bulk Ca²⁺ Rises and c-Fos Gene Expression

In many studies of Ca²⁺-dependent regulation of gene expression, high doses of Ca²⁺ ionophores such as ionomycin have been used to raise bulk Ca²⁺ to high levels in order to activate NFAT or c-fos and thereby gene transcription. At low concentrations (sub μM), ionomycin first releases Ca²⁺ from the stores and this leads to the opening of CRAC channels [64]. However, at higher doses (μM), ionomycin additionally increases Ca²⁺ flux across the plasma membrane directly via its ionophoretic activity. In RBL cells, CRAC channel blockers fail to prevent the rise in bulk Ca²⁺ due to Ca²⁺ entry that is elicited by high concentrations of ionomycin [35]. These large increases in bulk Ca²⁺ may elevate Ca²⁺ sufficiently close to the plasma membrane to match the high local Ca²⁺ within the Ca²⁺ microdomain. However, interpretation is not straightforward. In RBL cells in which both CRAC channels and Ca²⁺ extrusion via the PMCA pumps were inhibited with La³⁺, stimulation with a high dose of ionomycin (5 μM) led to a large increase in cytoplasmic Ca²⁺ and in c-fos transcription [35]. The inhibition of Syk or overexpression of caveolin-1, which both inhibit receptor and thapsigargin-evoked c-fos expression, failed to reduce c-fos activity to ionomycin under these conditions [46]. Hence, a large, grossly nonphysiological rise in bulk Ca²⁺ can activate c-fos gene expression through a pathway not engaged by more physiologically relevant stimuli.

5.10. Conclusion

Ca²⁺ microdomains near open Ca²⁺ channels confer several signaling advantages over a rise in bulk Ca²⁺. These include specificity, speed, and high fidelity in the signal transduction process. Although local Ca²⁺ signals near voltage-operated Ca²⁺ channels have long been known to play a major role in neurotransmitter release at presynaptic active zones [65], the importance of Ca²⁺ signals confined to the vicinity of Ca²⁺ channels in non-excitable cells is now being appreciated. Ca²⁺ microdomains near CRAC channels signal effectively to the nucleus through the recruitment of Ca²⁺-dependent transcription factors including NFATs and c-fos. In mast cells, the scaffolding protein caveolin-1 plays an important role in dictating whether both transcription factors will be activated by the Ca²⁺ microdomain (Figure 5.1). Whereas NFAT signaling is maintained in the presence of caveolin-1, c-fos activity is suppressed. The inhibition is linked to the phosphorylation of tyrosine 14 of caveolin-1, although how this impairs the phosphorylation of STAT5, the transcription factor that couples Ca²⁺ microdomains near CRAC channels to c-fos expression, is currently not known.

Figure 5.1

Cartoon summarizing modular regulation of NFAT and c-fos by caveolin-1 in mast cells. In the absence of caveolin-1, NFAT and c-fos both activate in response to Ca²⁺ microdomains near open CRAC channels. However, when caveolin-1 is phosphorylated on tyrosine (more...)

Modular regulation by the phosphorylated state of a membrane scaffolding protein like caveolin-1 might be a general way to control selectively downstream signaling pathways in response to the same Ca²⁺ microdomain.

References

1.

Wheeler, D.G., Barrett, C.F., Groth, R.D., Safa, P., and Tsien, R.W. CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation-transcription coupling. Journal of Cell Biology. 2008;183:849-863. [PMC free article: PMC2592819] [PubMed: 19047462]

2.

Ma, H., Groth, R.D., Cohen, S.M., Emery, J.F., Li, B., Hoedt, E., Zhang, G., Neubert, T.A., and Tsien, R.W. γCaMKII shuttles Ca²⁺/CaM to the nucleus to trigger CREB phosphorylation and gene expression. Cell. 2014;159:281-294. [PMC free article: PMC4201038] [PubMed: 25303525]

3.

Barco, A., Alarcon, J.M., and Kandel, E.R. Expression of constitutively active CREB protein facilitates the late phase of long-term potentiation by enhancing synaptic capture. Cell. 2002;108:689-703. [PubMed: 11893339]

4.

Parekh, A.B. Store-operated CRAC channels: Function in health and disease. Nature Reviews Drug Discovery. 2010;9:399-410. [PubMed: 20395953]

5.

Hogan, P.G., Chen, L., Nardone, J., and Rao, A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes and Development. 2003;17:2205-2232. [PubMed: 12975316]

6.

Macian, F., Lopez-Rodriguez, C., and Rao, A. Partners in transcription: NFAT and AP-1. Oncogene. 2001;20:2476-2489. [PubMed: 11402342]

7.

Wu, H., Peisley, A., Graef, I.A., and Crabtree, G.R. NFAT signalling and the invention of vertebrates. Trends in Cell Biology. 2007;17:251-260. [PubMed: 17493814]

8.

Ishiguro, K., Ando, T., Maeda, O., Watanabe, O., and Goto, H. Tubulin alpha functions as an adaptor in NFAT-importin beta interaction. Journal of Immunology. 2011;186:2710-2713. [PubMed: 21278340]

9.

Eferl, R. and Wagner, E.F. AP-1: A double-edged sword in tumorigenesis. Nature Reviews Cancer. 2003;3:859-868. [PubMed: 14668816]

10.

Rao, A., Luo, C., and Hogan, P.G. Transcription factors of the NFAT family: Regulation and function. Annual Review of Immunology. 1997;15:707-747. [PubMed: 9143705]

11.

Goldsmith, M.A. and Weiss, A. Early signal transduction by the antigen receptor without commitment to T cell activation. Science. 1988;240:1029-1031. [PubMed: 3259335]

12.

Negulescu, P.A., Shastri, N., and Cahalan, M.D. Intracellular calcium dependence of gene expression in single T lymphocytes. Proceedings of the National Academy of Sciences of the United States of America. 1994;91:2873-2877. [PMC free article: PMC43473] [PubMed: 8146203]

13.

Le Deist, F., Hivroz, C., Partiseti, M., Thomas, C., Buc, H.A., Oleastro, M., Belohradsky, B., Choquet, D., and Fischer, A. A primary T-cell immunodeficiency associated with defective transmembrane calcium influx. Blood. 1995;85:1053-1062. [PubMed: 7531512]

14.

Hutchinson, L.E. and McCloskey, M.A. Fc epsilon RI-mediated induction of nuclear factor of activated T-cells. Journal of Biological Chemistry. 1995;270:16333-16338. [PubMed: 7608202]

15.

Clapham, D.E. Calcium signalling. Cell. 1995;80:259-268. [PubMed: 7834745]

16.

Zweifach, A. and Lewis, R.S. Mitogen-regulated Ca²⁺ current of T lymphocytes is activated by depletion of intracellular Ca²⁺ stores. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:6295-6299. [PMC free article: PMC46915] [PubMed: 8392195]

17.

Partiseti, M., Ledeist, F., Hivroz, C., Fischer, A., Kom, H., and Choquet, D. The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency. Journal of Biological Chemistry. 1994;269:32327-32335. [PubMed: 7798233]

18.

Fanger, C.M., Hoth, M., Crabtree, G.R., and Lewis, R.S. Characterization of T cell mutants with defects in capacitative calcium entry: Genetic evidence for the physiological roles of CRAC channels. Journal of Cell Biology. 1995;131:655-667. [PMC free article: PMC2120614] [PubMed: 7593187]

19.

Liou, J., Kim, M.L., Heo, W.D., Jones, J.T., Myers, J.W., Ferrell, Jr., J.E., and Meyer, T. STIM is a calcium sensor essential for calcium-store-depletion-triggered calcium influx. Current Biology. 2005;15:1235-1241. [PMC free article: PMC3186072] [PubMed: 16005298]

20.

Roos, J., DiGregorio, P.J., Yeromin, A.V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., et al.STIM1, an essential and conserved component of store-operated Ca²⁺ channel function. Journal of Cell Biology. 2005;169:435-445. [PMC free article: PMC2171946] [PubMed: 15866891]

21.

Hogan, P.G., Lewis, R.S., and Rao, A. Molecular basis of calcium signalling in lymphocytes: STIM and ORAI. Annual Review of Immunology. 2010;28:491-533. [PMC free article: PMC2861828] [PubMed: 20307213]

22.

Liou, J., Fivaz, M., Inoue, T., and Meyer, T. Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after calcium store depletion. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:9301-9306. [PMC free article: PMC1890489] [PubMed: 17517596]

23.

Wu, M.M., Buchanan, J., Luik, R.M., and Lewis, R.S. Ca store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. Journal of Cell Biology. 2006;174:803-813. [PMC free article: PMC2064335] [PubMed: 16966422]

24.

Zhang, S.L., Yeromin, A.V., Zhang, X.H.-F., Yu, Y., Safrina, O., Penna, A., Roos, J., Stauderman, K.A., and Cahalan, M.D. Genome-wide RNAi screen of calcium influx identifies genes that regulate Ca release-activated Ca channel activity. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:9357-9362. [PMC free article: PMC1482614] [PubMed: 16751269]

25.

Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S.-V., Tanasa, B., Hogan, P.G., Lewis, R.S., Daly, M., and Rao, A. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006;441:179-185. [PubMed: 16582901]

26.

Vig, M., Peinelt, C., Beck, A., Koomoa, D.L., Rabah, D., Koblan-Huberson, M., Kraft, S., et al.CRACM1 is a plasma membrane protein essential for store-operated calcium entry. Science. 2006;312:1220-1223. [PMC free article: PMC5685805] [PubMed: 16645049]

27.

Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A., and Hogan, P.G. Orai1 is an essential pore subunit of the CRAC channel. Nature. 2006;443:230-233. [PubMed: 16921383]

28.

Yeromin, A.V., Zhang, S.L., Jiang, W., Yu, Y., Safrina, O., and Cahalan, M.D. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature. 2006;443:226-229. [PMC free article: PMC2756048] [PubMed: 16921385]

29.

Vig, M., Beck, A., Billingsley, J.M., Lis, A., Parvez, S., Peinelt, C., Koomoa, D.L., et al.CRACM1 multimers form the ion-selective pore of the CRAC channel. Current Biology. 2006;16:2073-2079. [PMC free article: PMC5685803] [PubMed: 16978865]

30.

Gwack, Y., Srikanth, S., Feske, S., Cruz-Guilloty, F., Oh-hora, M., Neems, D., Hogan, P.G., and Rao, A. Biochemical and functional characterization of Orai proteins. Journal of Biological Chemistry. 2007;282:16232-16243. [PubMed: 17293345]

31.

Kar, P., Nelson, C., and Parekh, A.B. Selective activation of the transcription factor NFAT1 by calcium microdomains near Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Journal of Biological Chemistry. 2011;286:14795-14803. [PMC free article: PMC3083208] [PubMed: 21325277]

32.

Kar, P., Bakowski, D., Di Capite, J., Nelson, C., and Parekh, A.B. Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca²⁺ oscillations and gene expression. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:6969-6974. [PMC free article: PMC3344999] [PubMed: 22509043]

33.

Ng, S.-W., DiCapite, J.L., Singaravelu, K., and Parekh, A.B. Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca²⁺ influx through Ca²⁺ release-activated Ca²⁺ channels. Journal of Biological Chemistry. 2008;283:31348-31355. [PubMed: 18806259]

34.

Chang, W.-C., Nelson, C., and Parekh, A.B. Ca²⁺ influx through CRAC channels activates cytosolic phospholipase A2, leukotriene C4 secretion and expression of c-fos through ERK-dependent and independent pathways in mast cells. FASEB Journal. 2006;20:1681-1693. [PubMed: 17023391]

35.

Di Capite, J., Ng, S.-W., and Parekh, A.B. Decoding of cytoplasmic Ca²⁺ oscillations through the spatial signature drives gene expression. Current Biology. 2009;19:853-858. [PubMed: 19375314]

36.

Samanta, K., Kar, P., Mirams, G.R., and Parekh, A.B. Ca²⁺ channel re-localization to plasma-membrane microdomains strengthens activation of Ca²⁺-dependent nuclear gene expression. Cell Reports. 2015;12:203-216. [PMC free article: PMC4521080] [PubMed: 26146085]

37.

Ng, S.-W., Nelson, C., and Parekh, A.B. Coupling of Ca²⁺ microdomains to spatially and temporally distinct cellular responses by the tyrosine kinase Syk. Journal of Biological Chemistry. 2009;284:24767-24772. [PMC free article: PMC2757180] [PubMed: 19584058]

38.

Fierro, L. and Parekh, A.B. Substantial depletion of the intracellular Ca²⁺ stores is required for macroscopic activation of the Ca²⁺ release-activated Ca²⁺ current in rat basophilic leukaemia cells. Journal of Physiology (London). 2000;522:247-257. [PMC free article: PMC2269755] [PubMed: 10639101]

39.

Ng, S.W., Bakowski, D., Nelson, C., Mehta, R., Almeyda, R., Bates, G., and Parekh, A.B. Cysteinyl leukotriene type I receptor desensitization sustains Ca²⁺-dependent gene expression. Nature. 2012;482:111-115. [PMC free article: PMC3272478] [PubMed: 22230957]

40.

Somasundaram, S., Shum, A.K., McBride, H.J., Kessler, J.A., Feske, S., Miller, R.J., and Prakriya, M. Store-operated CRAC channels regulate gene expression and proliferation in neural progenitor cells. Journal of Neuroscience. 2014;34:9107-9123. [PMC free article: PMC4078087] [PubMed: 24990931]

41.

Poteser, M., Schleifer, H., Lichtenegger, M., Schernthaner, M., Stockner, T., Kappe, C.O., Glasnov, T.N., Romanin, C., and Groschner, K. PKC-dependent coupling of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:10556-10661. [PMC free article: PMC3127924] [PubMed: 21653882]

42.

Yuan, J.P., Zeng, W., Huang, G.N., Worley, P.F., and Muallem, S. STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels. Nature Cell Biology. 2007;9:636-645. [PMC free article: PMC2699187] [PubMed: 17486119]

43.

Cheng, K.T., Liu, X., Ong, H.L., Swaim, W., and Ambudkar, I.S. Local Ca²⁺ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²⁺ signals required for specific functions. PLoS Biology. 2011;9:e10011025. [PMC free article: PMC3050638] [PubMed: 21408196]

44.

Darnell, J.E.J. STATs and gene regulation. Science. 1997;277:1630-1635. [PubMed: 9287210]

45.

Bromberg, J.F. Activation of STAT proteins and growth control. Bioessays. 2001;23:161-169. [PubMed: 11169589]

46.

Yeh, Y.-C. and Parekh, A.B. Distinct structural domains of caveolin-1 independently regulate Ca²⁺ release-activated Ca²⁺ channels and Ca²⁺ microdomain-dependent gene expression. Molecular and Cellular Biology. 2015;35:1341-1349. [PMC free article: PMC4372695] [PubMed: 25645930]

47.

Oliveria, S.F., Dell’Acqua, M.L., and Sather, W.A. AKAP79/150 anchoring of calcineurin controls neuronal L-type Ca²⁺ channel activity and nuclear signalling. Neuron. 2007;55:261-275. [PMC free article: PMC2698451] [PubMed: 17640527]

48.

Li, H., Pink, M.D., Murphy, J.G., Stein, A., Dell’Acqua, M.L., and Hogan, P.G. Balanced interactions of calcineurin with AKAP79 regulate Ca²⁺-calcineurin-NFAT signaling. Nature Structural & Molecular Biology. 2012;19:337-345. [PMC free article: PMC3294036] [PubMed: 22343722]

49.

Kar, P., Samanta, K., Kramer, H., Morris, O., Bakowski, D., and Parekh, A.B. Dynamic assembly of a membrane signaling complex enables selective activation of NFAT by orai1. Current Biology. 2014;24:1361-1368. [PMC free article: PMC4062936] [PubMed: 24909327]

50.

Kar, P. and Parekh, A.B. Distinct spatial Ca²⁺ signatures selectively activate different NFAT transcription factor isoforms. Molecular Cell. 2015;58:232-243. [PMC free article: PMC4405353] [PubMed: 25818645]

51.

Chang, W.C., Di Capite, J., Singaravelu, K., Nelson, C., Halse, V., and Parekh, A.B. Local calcium influx through calcium release-activated calcium (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal. Journal of Biological Chemistry. 2008;283:4622-4631. [PubMed: 18156181]

52.

Macian, F., Garcia-Rodriguez, C., and Rao, A. Gene expression elicited by NFAT in the presence or absence of cooperative recruitment of Fos and Jun. EMBO Journal. 2000;19:4783-4795. [PMC free article: PMC302068] [PubMed: 10970869]

53.

Sargiacomo, M., Scherer, P.E., Tang, Z., Kubler, E., Song, K.S., Sanders, M.C., and Lisanti, M.P. Oligomeric structure of caveolin: Implications for caveolae membrane organization. Proceedings of the National Academy of Sciences of the United States of America. 1995;92:9407-9911. [PMC free article: PMC40994] [PubMed: 7568142]

54.

Alioua, A., Lu, R., Kumar, Y., Eghbali, M., Kundu, P., Toro, L., and Stefani, E. Slo1 caveolin-binding motif, a mechanism of caveolin-1-Slo1 interaction regulating Slo1 surface expression. Journal of Biological Chemistry. 2008;283:4808-4817. [PubMed: 18079116]

55.

Pani, B., Ong, H.L., Brazer, S.W., Liu, X., Rauser, K., Singh, B.J., and Ambudkar, I.S. Activation of TRPC1 by STIM1in ER-PM microdomains involves release of the channel from its scaffold caveolin-1. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:20087-20092. [PMC free article: PMC2785296] [PubMed: 19897728]

56.

Ludwig, A., Howard, G., Mendoza-Topaz, C., Deerinck, T., Mackey, M., Sandin, S., Ellisman, M.H., and Nichols, B.J. Molecular composition and ultrastructure of the caveolar coat complex. PLoS Biology. 2013;11:e1001640. [PMC free article: PMC3754886] [PubMed: 24013648]

57.

Yu, F., Sun, L., and Machaca, K. Constitutive recycling of the store-operated Ca²⁺ channel Orai1 and its internalization during meiosis. Journal of Cell Biology. 2010;191:523-535. [PMC free article: PMC3003315] [PubMed: 21041445]

58.

Couet, J., Li, S., Okamoto, T., Ikezu, T., and Lisanti, M.P. Identification of peptide and protein ligands for the caveolin scaffolding domain. Journal of Biological Chemistry. 1997;272:6525-6533. [PubMed: 9045678]

59.

Li, S., Okamoto, T., Chun, M., Sargiacomo, M., Casanova, J.E., Hansen, S.H., Nishimoto, I., and Lisanti, M.P. Evidence for a regulated interaction between heterotrimeric G proteins and caveolin. Journal of Biological Chemistry. 1995;270:15693-15701. [PubMed: 7797570]

60.

Lee, H., Volonte, D., Galbiati, F., Iyengar, P., Lublin, D.M., Bregman, D.B., Wilson, M.T., et al.Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: Identification of a c-src/Ca_v-1/Grb7 signaling cassette. Molecular Endocrinology. 2000;14:1750-1775. [PubMed: 11075810]

61.

Gottlieb-Abraham, E., Shvartsman, D.E., Donaldson, J.C., Ehrlich, M., Gutman, O., Martin, G.S., and Henis, Y.I. Src-mediated caveolin-1 phosphorylation affects the targeting of active Src to specific membrane sites. Molecular Biology of the Cell. 2013;24:3881-3895. [PMC free article: PMC3861084] [PubMed: 24131997]

62.

Tomassian, T., Humphries, L.A., Liu, S.D., Silva, O., Brooks, D.G., and Miceli, M.C. Caveolin-1 orchestrates TCR synaptic polarity, signal specificity, and function in CD8 T cells. Journal of Immunology. 2011;187:2992-3002. [PMC free article: PMC3881976] [PubMed: 21849673]

63.

Wang, X.M., Nadeau, P.E., Lin, S., Abbott, J.R., and Mergia, A. Caveolin 1 inhibits HIV replication by transcriptional repression mediated through NF-kB. Journal of Virology. 2011;85:5483-5493. [PMC free article: PMC3094981] [PubMed: 21430048]

64.

Morgan, A.J. and Jacob, R. Ionomycin enhances Ca²⁺ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane. Biochemical Journal. 1994;300:665-672. [PMC free article: PMC1138219] [PubMed: 8010948]

65.

Neher, E. Vesicle pools and Ca²⁺ microdomains: New tools for understanding their roles in neurotransmitter release. Neuron. 1998;20:389-399. [PubMed: 9539117]

Yi-Chun Yeh

Department of Physiology, Anatomy and Genetics

University of Oxford

Oxford, United Kingdom

Anant B. Parekh

Department of Physiology, Anatomy and Genetics

University of Oxford

Oxford, United Kingdom