Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90

• Published in: The Journal of biological chemistry 1994-08, Vol.269 (32), p.20691-20699
• Main Authors: KAO, P. N, LEI CHEN, BROCK, G, NG, J, KENNY, J, SMITH, A. J, CORTHESY, B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; cDNA; Cloning, Molecular; control; Cyclosporine - pharmacology; DNA, Complementary; DNA-Binding Proteins - genetics; Fundamental and applied biological sciences. Psychology; gene expression; Humans; Immune Sera; interleukin 2; Jurkat cells; Lymphocyte Activation; lymphocytes T; man; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; NF45 protein; NF90 protein; NFAT protein; NFATC Transcription Factors; Nuclear Factor 45 Protein; Nuclear Factor 90 Proteins; nuclear factor of activated T cells; Nuclear Proteins - genetics; nucleotide sequence; prediction; RNA - genetics; RNA - metabolism; role; T-Lymphocytes - drug effects; T-Lymphocytes - metabolism; Tacrolimus - pharmacology; Transcription Factors - genetics; Transcription. Transcription factor. Splicing. Rna processing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)32048-3

Nuclear Factor of Activated T-cells (NF-AT) is a crucial transcription factor required for T-cell expression of interleukin 2. Purified NF-AT contains 45-kDa and 90-kDa subunits (Corthésy, B., and Kao, P. N. (1994) J. Biol. Chem. 269, 20682-20690). Partial internal amino acid sequences derived from each subunit indicate that these proteins are novel. The amino acid sequences were used to clone the cDNAs encoding each subunit. The cDNAs predict proteins of novel structures: NF45 has limited similarity to prokaryotic transcription factor sigma-54 and to human DNA topoisomerase II; NF90 has limited similarity to Drosophila Staufen 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 tag were transiently expressed and processed in the native environment of Jurkat T-cells. Histidine-tagged NF45 and NF90 proteins, affinity-purified on nickel chelate columns, encode a NF-AT DNA-binding activity that is enhanced following T-cell stimulation, and this enhancement is blocked when T-cells are stimulated in the presence of cyclosporin A or FK506.