ANALYSIS OF THE PREEXISTING AND NUCLEAR FORMS OF NUCLEAR FACTOR OF ACTIVATED T-CELLS

The nuclear factor of activated T cells (NF-AT)3 is an inducible DNA-b inding 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 id entified as Fos and Jun proteins, and a preexisting, T cell-specific c omponent (NF-AT(p)) which is the target for the immunosuppressive agen ts cyclosporin A (CsA) and FK506. We have previously shown that nuclea r extracts from activated T cells form two inducible NF-AT complexes w ith an oligonucleotide corresponding to the distal NF-AT site of the m urine IL-2 promoter, although hypotonic extracts of unstimulated T cel ls form a single complex containing NF-AT(p). We show that the ability to detect NF-AT(p) in a gel shift assay, which is essential for purif ication and biochemical studies of this protein, is strikingly depende nt on the precise sequence of the NF-AT oligonucleotide used as the la beled probe. Moreover we present evidence that the component that form s the faster-migrating (''lower'') nuclear NF-AT complex is derived by a calcium-dependent, cyclosporin-sensitive, posttranslational modific ation of NF-AT(p), and that Fos and Jun proteins stabilize its interac tion with DNA. The results are discussed in the context of a model rel ating the two nuclear NF-AT complexes to NF-AT(p).