PHOSPHORYLATION OF THE TRANSCRIPTION FACTOR NFATP INHIBITS ITS DNA-BINDING ACTIVITY IN CYCLOSPORINE-A-TREATED HUMAN B-CELLS AND T-CELLS

Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preve nting transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phospha tase calcineurin, which in turn inhibits translocation of an NFAT comp onent 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 inhi bited in DNA-binding and in its ability to form an NFAT complex with F os and Jun. Immunoblot analyses and metabolic labeling with [P-32]orth ophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without af fecting subcellular distribution. Dephosphorylation by in vitro treatm ent with calcineurin or alkaline phosphatase restores NFATp DNA bindin g activity and its ability to reconstitute an NFAT complex with Fos an d Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA bin ding.