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Key Event Title
Interference, nuclear localization of NFAT
Key Event Components
|genetic interference||NFAT protein||increased|
Key Event Overview
AOPs Including This Key Event
|All life stages||High|
Key Event Description
The nuclear factor of activated T cells (NFAT) is a substrate of calcineurin (CN) (Rao et al. 1997). A NFAT has an N-terminal with a plurality of SP motifs rich in serine and proline, which are controlled by means of phosphorylation and dephosphorylation. There is a nuclear localization signal (NLS) held between these SP regions as well as a nuclear export signal (NES) in the N-terminal adjacent to the SP motifs (Beals et al. 1997, Zhu and McKeon 1999, Serfling et al. 2000). SP motifs ordinarily are phosphorylated, which covers the NLS and leaves the NES exposed, so that NFAT localizes in cytoplasm. When SP motifs are dephosphorylated by activated CN, the NLS is exposed and the NES is covered, thereby promoting nuclear localization of NFAT (Matsuda and Koyasu 2000, Zhu and McKeon 1999). When T-cell activation takes place, T-cell–receptor- mediated stimulus increases the intracellular concentration of calcium and activates a regulatory subunit (CnB), which subsequently induces a catalytic subunit (CnA) phosphatase activation, leading to dephosphorylation of NFAT thereby promoting nuclear localization of NFAT. CNI-immunophilin complexes inhibit CN phosphatase activation, thereby interfering with NFAT nuclear localization (Bhattacharyya et al.2011).
Concentration-dependent reduction of in vitro nuclear localization of NFAT was evident at concentrations from 0.1 nM (Jurkat T cells) or 10nM (human CD4+ T cells) and up to 1 μM (1000 nM) under the conditions of 2 hours treatment (Maguire et al. 2013).
How It Is Measured or Detected
Nuclear translocation of NFAT can be tested by imaging flowcytometer, in which lymphocytes are treated with fluorescence-labeled anti-NFAT antibody and DAPI (nuclear stain) and intracellular distribution of NFAT is analyzed by imaging flowcytometry with image analysis (Maguire O et al. 2013).
Interference with translocation of NFAT to the nucleus can be detected using gel mobility shift assays of nuclear or cytoplasmic extracts electrophoresed with end-labeled NFAT-binding site from human IL-2 enhancer (Flanagan et al. 1991).
Domain of Applicability
NFAT expresses in B cells, mast cells, neutrophils, granulocytes, dendritic cells, macrophages, and natural killer cells as well as T cells from humans, rodents and other mammalian species (Rao et al. 1997).
Evidence for Perturbation by Stressor
- Rao, A., Luo, C., and Hogan, PG. (1997). Transcription factors of the NFAT family: regulation and function. Annual Review of Immunology 15: 707-47.
- Beals, C.R., Clipstone, N.A., Ho, S.N. and Crabtree, G.R. (1997). Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction. Genes & development 11 (7): 824-34.
- Zhu, J. and McKeon, F. (1999). NF-AT activation requires suppression of Crm1-dependent export by calcineurin. Nature. 398(6724): 256-60.
- Serfling, E., Berberich-Siebelt, F., Chuvpilo, S., Jankevics, E., Klein-Hessling, S., Twardzik, T., and Avots, A., (2000). The role of NF-AT transcription factors in T cell activation and differentiation. Biochimica et Biophysica Act 1498 (1): 1-18.
- Matsuda, S., Koyasu, S. (2000). A second target of cyclosporin A and FK506. Tanpakushitsu kakusan koso. 45(11): 1823-1831.
- Bhattacharyya, S., Deb, J., Patra, A.K., Thuy Pham, D.A., Chen, W., Vaeth, M., Berberich-Siebelt, F., Klein-Hessling, S., Lamperti, E.D., Reifenberg, K., Jellusova, J., Schweizer, A., Nitschke, L., Leich, E., Rosenwald, A., Brunner, C., Engelmann, S., Bommhardt, U., Avots, A., Müller, M.R., Kondo, E. and Serfling, E. (2011). NFATc1 affects mouse splenic B cell function by controlling the calcineurin-NFAT signaling network. The Journal of experimental medicine 208 (4): 823-39.
- Flanagan, W.M., Corthésy, B., Bram, R.J. and Crabtree, G.R. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352 (6338): 803-7.
- Maguire O., Tornatore K.M., O'Loughlin K.L., Venuto R.C., Minderman H.(2013). Nuclear translocation of nuclear factor of activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus.