Abstract
Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.
Highlights
The structural complexity ofNFAT was first appreciated by Flanagan et al (1991), who proposed a two-component structure consisting of a PMA-inducible nuclear component resistant to Cyclosporin A (CsA) and FK-506 and a T cell-specific cytosolic component present in unstimulated T cells that translocated to the nucleus upon ionomycin or PHA stimulation
NFATp is present in the cytoplasm of unstimulated murine T cells, it translocates to the nucleus upon Ca2 + -dependent stimulation, and its translocation is blocked by CsA. 2 Purified NFATp is able to directly bind to the murine IL-2 NFAT motif and to form an NFAT complex in association with Fos and Jun proteins (Jain et al, 1993a; McCaffrey et al, 1993b)
We show that the constitutive factor found in nuclear extracts of unstimulated Band T cell lines is a human homologue of murine NFATp and that its ability to reconstitute an NFAT complex in cooperation with Fos and Jun is inhibited by CsA treatment
Summary
Vol 270, No 35, Issue of September 1, pp. 20653-20659, 1995 Printed in U.S.A. Phosphorylation of the Transcription Factor NFATp Inhibits Its DNA Binding Activity in Cyclosporin A-treated Human B and T Cells*. We show that the constitutive factor found in nuclear extracts of unstimulated Band T cell lines is a human homologue of murine NFATp and that its ability to reconstitute an NFAT complex in cooperation with Fos and Jun is inhibited by CsA treatment. This inhibition is due to loss of DNA-binding activity of human NFATp resulting from an increase in its phosphorylation level as in vitro phosphatase treatment completely restores DNA-binding activity of the factor. These observations demonstrate an additional mechanism for CsA-mediated inhibition of NFAT complex formation
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