CLONING AND EXPRESSION OF CYCLOSPORINE-A-SENSITIVE AND FK506-SENSITIVE NUCLEAR FACTOR OF ACTIVATED T-CELLS - NF45 AND NF90

Nuclear Factor of Activated T-cells (NF-AT) is a crucial transcription factor required for T-cell expression of interleukin 2. Purified NF-A T contains 45-kDa and 90-kDa subunits (Corthesy, B., and Kao, P. N. (1 994) J. Biol. Chem. 269, 20682-20690). Partial internal amino acid seq uences derived from each subunit indicate that these proteins are nove l, The amino acid sequences were used to clone the cDNAs encoding each subunit. The cDNAs predict proteins of novel structures: NF45 has lim ited similarity to prokaryotic transcription factor sigma-54 and to hu man DNA topoisomerase II; NF90 has limited similarity to Drosophila St aufen in a domain predicted to bind double-stranded RNA. RNA encoding NF45 and NF90 exists in nonstimulated Jurkat T-cells and in all other cell types examined (HeLa, HepG2, K562). Immunofluorescence microscopy was used to demonstrate that both proteins are located in the nucleus of Jurkat T-cells. Clones NF45 and NF90 with a polyhistidine fusion t ag were transiently expressed and processed in the native environment of Jurkat T-cells. Histidine-tagged NF45 and NF90 proteins, affinity-p urified on nickel chelate columns, encode a NF-AT DNA-binding activity that is enhanced following T-cell stimulation, and this enhancement i s blocked when T-cells are stimulated in the presence of cyclosporin A or FK506.