DEFINITION
Nuclear factor of activated T-cells (NFAT) is a general name applied to a family of transcription factors shown to be important in immune response.
INTRODUCTION
NFAT is a major stimulation-responsive DNA-binding factor and transcriptional regulator in T cells. NFAT transcription factors induce genes important in cellular processes ranging from the development and activation of lymphocytes. NFAT is expressed in a variety of lymphocytes: T cells, B cells, Natural Killer (NK) cells, monocytes and non-immune related cells (cardiac, muscle and neuronal).
THE GENE
CHEMICAL STRUCTURE AND IMAGINES
Primary structure of the NFAT family :
In humans the NFAT familycomprises five distinct gene products: NFAT1 (also known as NFATc2); NFAT2 (also known as NFATc1); NFAT3 (also known as NFATc4); NFAT4 (also known as NFATc3 and NFATx) and NFAT5 (also known as TonEBP and OREBP).
All isoforms are ubiquitously expressed and each isoform has alternative splice variants that differ in the amino and carboxyl termini. The calcium-regulated isoforms NFAT1–4 share two conserved domains: the Rel-homology region (RHR) and the more moderately conserved NFAT homology region (NHR). NFAT5 has a distinct domain structure and retains only the RHR region of homology to the calcium-regulated isoforms.
Carboxyl-terminal 15-amino acid sequence of NFATx1 is possibly created by tissue-specific splicing and is essential for transactivation activity in T cells. 1998
Alternative polyadenylation events contribute to the induction of NF-ATc in effector T cells. 1999
Secondary Structure :
NFAT secondary structure consist of b sheets and random coils forming three functional domains: a Rel-similarity domain (RSD) for DNA binding and association with AP-1, a NFAT homolog region (NHR) domain, containing localization signalling sequences, and transcriptional activation domains (TAD). NFAT also contains two sequence motifs: a nuclear location signal sequence (NLS) and a nuclear export signal sequence (NES) that permit the import and export of NFAT from the nucleus.
Tertiary Structure :
NFAT tertiary structure has two domains that resemble immunoglobulin (Ig) folds. One domain is a beta barrel comprised of three beta sheets and it binds to DNA; and there are two prominent loop structures within beta barrel: the first loop is more constrained and binds to DNA major grove at 5’ recognition sequence GGAAAA, the second loop binds to the minor grove at the 3’ recognition sequence GGAAAA.
NFAT Signaling: Choreographing the Social Lives of Cells. 2002
Signalling into the T-Cell Nucleus: NFAT regulation. 1998
NFAT Aminoacids Percentage
SYNTHESIS, TURNOVER AND CELLULAR FUNCTIONS
NFAT ACTIVATION
The activation of NFATs by calcium signalling
A key rate-limiting event in the activation of NFATs is a rise in intracellular calcium concentrations. This is initiated by cell surface receptors that stimulate the activation of phospholipase type C (PlC) enzymes such as PlCγ. Efficient activation of NFATs also requires a sustained calcium signal, and this is achieved by the opening of calcium release-activated calcium (CRAC) channels at the plasma membrane. This occurs in response to PlC-initiated emptying of calcium from the endoplasmic reticulum (ER). In turn, calcium release is sensed by the high-affinity ER calcium sensor stromal interaction molecule 1 (STIm1) leading to a conformational change in the CRAC channel protein ORAI1, opening of the channel and an influx of extracellular calcium. Subsequent to the sustained flux through the ER and store-operated calcium channels, calcium binds to calmodulin, which in turn binds to and activates the serine/threonine phosphatase calcineurin. Activation of calcineurin is rate-limiting for NFAT activation in all cells.
Signalling to transcription: store-operated Ca2+ entry and NFAT activation in lymphocytes. 2007
NFATs in the basal state
They are localized to the cytoplasm in an inactive conformation. More than 20 distinct phosphorylation sites are found in multiple distinct serine-rich sequences: the serine-rich region (SRR1) and the SPXX motifs SP1, SP2 and SP3. Dephosphorylation of the SP motifs by calcineurin exposes a nuclear localization sequence and masks a nuclear export sequence, therefore promoting nuclear import leading to transcriptional activation. Calcineurin also maintains NFATs in a dephosphorylated state in the nucleus. Nuclear export of NFATs is crucial as it leads to the termination of transcriptional activity.
NF-AT activation requires suppression of Crm1-dependent export by calcineurin. 1999
Export kinases:
-glycogen synthase kinase 3 (GSK3)
-Akt signalling
-Casein kinase 1 (CK1) (functions as both an export and a maintenance NFAT kinase).
Maintenance and export kinases:
the dual specificity tyrosine phosphorylation- regulated kinases modifiers of the subcellular localization of NFATs.
-DYRK1 functions as an NFAT export kinase.
-DYRK2 phosphorylates NFATs in the cytoplasm and functions as a maintenance kinase.
Glycogen synthase kinase-3 inhibits the DNA binding activity of NFATc. 2000
A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT. 2006
REGULATION
Regulation by calcium
The activation of NFAT proteins is induced by the engagement of receptors that are coupled to the calcium signalling pathway, such as the antigen receptors that are expressed by T and B cells. Receptor ligation leads to the activation of phospholipase C-γ, which hydrolyses phosphatidylinositol-4,5- bisphosphate to produce inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol. InsP3 induces the release of calcium from intracellular stores, which triggers the opening of calcium-release-activated calcium channels in the plasma membrane such that the increased levels of intracellular calcium are maintained. Calcium binds CALMODULIN, which in turn activates the calmodulindependent phosphatase calcineurin.
NFAT proteins are dephosphorylated by activated calcineurin
It leads to their nuclear translocation and the induction of NFAT-mediated gene transcription. Effective phosphate removal by calcineurin requires its docking on NFAT. Interactions between NFAT and calcineurin occur at a specific motif in the N terminus of NFAT, which has the consensus sequence PXIXIT. This motif and constitutes the main docking site for calcineurin on NFAT.
14 phosphorylation sites in NFAT1 are conserved in all calcineurin-regulated NFAT proteins, all but one are dephosphorylated by calcineurin. These sites are located in three different serine-rich motifs:
1. the serine-rich region 1 (SRR1) motif
the SPXX (where X denotes any amino acid) repeat motifs
2. SP2
3. SP3.
Dephosphorylation of the serine residues in these motifs leads to exposure of the NFAT1 nuclear localization signal and nuclear import, and it might also control the DNA-binding affinity of the RHR.
Several kinases phosphorylate NFAT proteins and control their nuclear shuttling:
-glycogen-synthase kinase 3 (GSK3)
-casein kinase 1 (CK1)
-p38
-JUN N-terminal kinase (JNK)..
Autoregulation of NFAT2
NFAT2 is apparently unique in that it is also regulated at the transcriptional level through an autoregulatory loop. This mechanism is isoform specific — only one of the two NFAT2 N-terminal splice variants, NFAT2A, is under the control of an NFAT-dependent inducible promoter. In T cells, NFAT1, which is the main isoform in naive cells, might act together with constitutively expressed NFAT2 and/or NFAT4 to turn on expression of the inducible isoform of NFAT2.
Regulation of the murine Nfatc1 gene by NFATc2. 2002
Regulation by cytokine receptors
Regulation by signalling through the receptors for IL-2 and IL-15 in human peripheral- blood mononuclear cells regulates the binding of NFAT proteins to the promoter of the gene that encodes CX3C-chemokine receptor 1 (CX3CR1). IL-6 signalling through its receptor results in the preferential induction of NFAT1. This leads to increased NFAT1 activity in response to T-cell stimulation.
SUMOYLATION is a new mechanism that regulates NFAT nuclear retention. It is induced by increased intracellular calcium levels.The sumoylation sites of NFAT1 are located in the C terminus.
Dual role of sumoylation in the nuclear localization and transcriptional activation of NFAT1. 2004
Transcriptional partners of NFAT proteins
NFAT proteins can interact with different transcription-factor partners in the nucleus, they are important integrators of calcium signalling with many other signalling pathways in T cells. The structures of monomeric NFAT–DNA complexes emphasize the high flexibility of the linker region that is located between the N-terminal domain of the RHR.
Partners in transcription: NFAT and AP-1
Activator protein 1 (AP1) proteins are the main transcriptional partners of NFAT during T-cell activation. Dimers of FOS and JUN form quaternary complexes with NFAT and DNA on NFAT–AP1 composite sites, which contain two adjacent binding motifs for both transcription factors and are present in many genes that are induced during T-cell activation.Cooperation between NFAT proteins and AP1 integrates two of the main signalling pathways that are induced in response to T-cell stimulation: calcium signalling, which is responsible for the activation of NFAT proteins; and the RAS–MAPK pathway, which induces the expression and activation of FOS and JUN. NFAT–AP1 cooperation during T-cell activation is responsible for a specific pattern of gene expression, which induces the functional changes that characterize an activated T cell.
Partners in transcription: NFAT and AP-1. 2001
DIAGNOSTIC USE
New roles for NFAT
New insights into the role of NFAT proteins in T cell tolerance both in the cell-intrinsic mechanisms of T cell anergy and in the functions of regulatory T cells. Recent studies of the molecular mechanisms of T cell tolerance have the potential to provide new strategies for the treatment of autoimmune disorders in the clinic. Important roles for NFATs are in modulating phenotypes associated with malignancy and tumour progression. NFAT isoforms are overexpressed in human solid tumours and haematological malignancies and they have roles in cancer cell-autonomous functions such as invasive migration, differentiation and the survival of cells in the tumour and its microenvironment. NFATs also have a key role in tumour angiogenesis. Targeting the NFAT pathway should be relevant for the development of anti-cancer therapies.
NFAT, immunity and cancer: a transcription factor comes of age. 2010
NFAT proteins: emerging roles in cancer progression. 2009
