DYNAMIC EQUILIBRIUM BETWEEN CALCINEURIN AND KINASE-ACTIVITIES REGULATES THE PHOSPHORYLATION STATE AND LOCALIZATION OF THE NUCLEAR FACTOR OFACTIVATED T-CELLS

The nuclear factor of activated T-cells (NFAT(p)) is a phosphorylated transcription factor that resides in the cytoplasm of unactivated T-ce lls. T-cell activation results in the activation of the phosphatase ca lcineurin (CaN), which leads to the dephosphorylation and subsequent n uclear localization of NFAT(p). We have investigated the role of kinas es in the phosphorylation state and subcellular localization of NFAT,. The phosphorylation state and nuclear/cytoplasmic location of NFAT(p) were determined in unstimulated murine HT-2 cells treated with a pane l of kinase inhibitors. Two of the seven kinase inhibitors, staurospor ine (St) and bisindolylnaleimide I (BI), resulted in the dephosphoryla tion and nuclear localization of NFAT(p). These St-induced effects wer e inhibited by pretreatment with FK506, indicating that CaN activity w as required for the observed effects on NFAT(p). Treatment of cells wi th ionomycin resulted in NFAT(p) dephosphorylation and nuclear localiz ation. Removal of ionomycin from the cells resulted in the reappearanc e of phosphorylated NFAT(p) in the cytosol. St and BI also inhibited t he re-accumulation of NFAT(p) in the cytoplasm and its re-phosphorylat ion after ionomycin removal. The re-accumulation of NFAT(p) in the cyt osol after ionomycin withdrawal was shown to be energy- and temperatur e-dependent. Taken together, these results suggest that in unstimulate d cells NFAT(p) is actively maintained in the cytoplasm by kinases act ing in opposition to basal CaN activity.