NF-ATp, a T lymphocyte DNA-binding protein that is a target for calcineurin and immunosuppressive drugs

Abstract

The nuclear factor of activated T cells (NF-AT) is essential for transcription of the interleukin-2 gene upon T cell activation. Here we use a technique involving elution and renaturation of proteins from SDS-acrylamide gels to identify a DNA-binding component of NF-AT (NF-ATp) that is present in hypotonic extracts of T cells prior to activation and appears in nuclear extracts when T cells are activated. NF-ATp is present in resting T cells predominantly in a form migrating with an apparent molecular weight of 110,000-140,000, while NF-ATp from nuclear extracts of activated T cells migrates with a lower apparent molecular weight (90,000-125,000). This difference is likely to reflect dephosphorylation of NF-ATp, since treatment of NF-ATp with calf intestinal phosphatase or the calcium- and calmodulin-dependent phosphatase calcineurin in vitro results in a similar decrease in its apparent molecular weight. We show that NF-ATp is dephosphorylated in cell lysates by a calcium-dependent process that is blocked by inclusion of EGTA or a specific peptide inhibitor of calcineurin in the cell lysis buffer. Moreover, dephosphorylation of NF-ATp in cell extracts is inhibited by prior treatment of T cells with the immunosuppressive drugs cyclosporin A or FK506, which inhibit the phosphatase activity of calcineurin when complexed with their specific binding proteins, cyclophilin and FK506-binding protein. This work identifies NF-ATp as a DNA-binding phosphoprotein and a target for the drug/immunophilin/calcineurin complexes thought to mediate the inhibition of interleukin-2 gene induction by cyclosporin A and FK506.