SnapShot: Ca²⁺-Calcineurin-NFAT Signaling

Ca²⁺-calcineurin-NFAT signaling transmits signals to the nucleus from a wide variety of receptors and is required for developmental events as diverse as axon outgrowth, cardiac morphogenesis, lung morphogenesis, neural crest diversification, epithelial stem cell maintenance, and immune responses. The pathway appears to be particularly dedicated to vertebrate-specific morphogenic events, perhaps reflecting its first evolutionary appearance in vertebrates. Calcineurin-NFAT signaling is the target of the drugs cyclosporin A and FK506 and several viral immune modulators. In addition, its malfunction is implicated in Down’s syndrome, diabetes, and cardiac hypertrophy.

Receptors that induce the entry of Ca²⁺ into the cell resulting in the activation of calcineurin phosphatase are shown in the lower left. Ca²⁺ binds to the regulatory subunit of calcineurin as well as to calmodulin to activate the phosphatase activity of calcineurin (Stemmer and Klee, 1994). The immunosuppressive drugs FK506 and cyclosporin A inhibit calcineurin by binding first to the abundant intracellular proteins, FKBP and cyclophilin, respectively. These protein complexes then bind to calcineurin, preventing substrate access (Liu et al., 1991). The specificity of these calcineurin inhibitors is due to the large composite surface arising from FKBP bound to FK506 (see inset structure) (Griffith et al., 1995) or cyclosporin A bound to cyclophilin. FK506 and cyclosporin A are highly specific probes of the NFAT signaling pathway in tissues where the concentration of calcineurin is less than the concentration of FKBP or cyclophilin (Graef et al., 2001). Calcineurin removes several phosphate residues from the N terminus of NFATc proteins, the Ca²⁺-calcineurin-sensitive subunits of NFAT transcription complexes. Removal of the phosphates exposes nuclear localization sequences in NFATc proteins leading to their rapid entry into the nucleus (Beals et al., 1997a). Once in the nucleus, the NFATc proteins assemble on DNA with partner proteins generically termed NFATn (lower right) that are often the endpoints of other signaling pathways (Crabtree and Olson, 2002). In most cases, NFAT-dependent transcription requires that the Ca²⁺ signaling be coincident with MAP kinase signaling, providing a mechanism for signal integration and coincidence detection.

The targets of NFAT signaling (panel, upper right) are largely cytokines, growth factors and their receptors, proteins involved in cell-cell interactions, as well as many microRNAs. Of particular importance are positive feedback loops initiated by direct binding of NFATc1 and NFATc4 to their promoters as well as the regulation of Ca²⁺ channels, such as the IP3 receptor, responsible for the release of intracellular Ca²⁺ stores (Genazzani et al., 1999). This positive feedback loop appears to be important in committing cells to specific fates. A negative feedback loop mediated by the NFAT-dependent activation of RCAN (DSCR1) gene expression appears to constrain the pathway (Arron et al., 2006).

NFATc proteins are actively removed from the nucleus by first priming with Dyrk1a or protein kinase A (PKA) followed by phosphorylation by GSK3 (Arron et al., 2006; Beals et al., 1997b; Gwack et al., 2006). The inhibition of GSK3 by AKT and PI3 kinase (PI3K) signaling increases the level of NFATc proteins in the nucleus and hence provides a second means of coincidence detection and signal integration. Many of the phenotypes of Down’s syndrome are thought to be due to the duplication of the DSCR and Dyrk1a genes on chromosome 21. Increased gene dosage both reduces NFATc import into the nucleus and also facilitates export leading to a reduction in NFATc function and compromised positive feedback (Arron et al., 2006).

Abbreviations

AKT

the cellular homolog of the acute transforming oncogene v-AKT

BCR

B cell receptor

CRAC

Ca²⁺ release-activated Ca²⁺ channel

DSCR1/RCAN

Down’s syndrome critical region 1, now called regulator of calcineurin

Dyrk1a

dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A

FKBP

FK506-binding protein

GSK3

glycogen synthease kinase 3

IP3R1

inositol 1,4,5-triphosphate receptor 1

NFAT

nuclear factor of activated T cells

NMDA

N-methyl-D-aspartate receptor

NFATc

Ca²⁺-calcineurin-dependent subunits of NFAT complexes, also cyclosporin-sensitive subunit of NFAT complexes

NFATn

generic name for nuclear subunits of NFAT-transcription complexes

NF-κB

nuclear factor binding to the immunoglobulin kappa locus in B cells

PI3K

phosphatidylinositol 3-kinase

IP3

inositol 1,4,5-triphosphate

PLCγ

phospholipase gamma

STIM

stromal interaction molecule

Sox2

sex determination-box containing gene

TRP

transient receptor potential channel

TCR

T cell receptor

VEGF

vascular endothelial growth factor