Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells

Abstract

The nuclear factor of activated T cells (NF-AT)3 is an inducible DNA-binding protein that is essential for transcriptional induction of the IL-2 gene during T cell activation. NF-AT is thought to consist of two components: a ubiquitous, inducible nuclear component that we have identified as Fos and Jun proteins, and a preexisting, T cell-specific component (NF-ATp) which is the target for the immunosuppressive agents cyclosporin A (CsA) and FK506. We have previously shown that nuclear extracts from activated T cells form two inducible NF-AT complexes with an oligonucleotide corresponding to the distal NF-AT site of the murine IL-2 promoter, although hypotonic extracts of unstimulated T cells form a single complex containing NF-ATp. We show that the ability to detect NF-ATp in a gel shift assay, which is essential for purification and biochemical studies of this protein, is strikingly dependent on the precise sequence of the NF-AT oligonucleotide used as the labeled probe. Moreover we present evidence that the component that forms the faster-migrating ("lower") nuclear NF-AT complex is derived by a calcium-dependent, cyclosporin-sensitive, posttranslational modification of NF-ATp, and that Fos and Jun proteins stabilize its interaction with DNA. The results are discussed in the context of a model relating the two nuclear NF-AT complexes to NF-ATp.