DIRECT DEMONSTRATION OF NFAT(P) DEPHOSPHORYLATION AND NUCLEAR-LOCALIZATION IN ACTIVATED HT-2 CELLS USING A SPECIFIC NFAT(P) POLYCLONAL ANTIBODY

Nuclear factor of activated T cells (NFAT) regulates transcription of a number of cytokine genes, and NFAT DNA binding activity is stimulate d following T cell activation. Several lines of evidence have suggeste d that NFAT is a substrate for calcineurin, a serine/threonine phospha tase. Using a polyclonal antibody to murine NFAT(p), Western blot anal ysis of various mouse tissues demonstrated that the 110-130-kDa NFAT(p ) protein was highly expressed in thymus and spleen. Treatment of immu noprecipitated NFAT(p) from untreated HT-2 cells with calcineurin resu lted in the dephosphorylation of NFAT(p), demonstrating that NFAT(p) i s an in vitro substrate for calcineurin. NFAT(p) immunoprecipitated fr om P-32-labeled HT-2 cells migrated as an approximately 120-kDa protei n that was localized to the cytosol of the cells. Treatment of the cel ls with ionomycin resulted in a decrease in the molecular weight of NF AT(p) and a loss of P-32, consistent with NFAT(p) dephosphorylation. T he dephosphorylation of NFAT(p) was accompanied by localization of the protein to the nuclear fraction. Both of these events were blocked by preincubation of the cells with FK506, a calcineurin inhibitor, consi stent with the hypothesis that NFAT(p) is a calcineurin substrate in c ells.