FIG. 6.

Differential regulation of NFATc1 isoforms by JNK. (A) The NH₂-terminal regions of NFATc1 (18), NFATc1-α (25), and NFATc1-β (26, 33) are illustrated schematically. The PxIxIT motif (hatched box), the NFAT homology domain (NHD), and the alternative NFATc1 NH₂ termini are indicated. Immobilized GST, GST-NFATc1 (residues 1 to 126), GST–NFATc1-α (residues 1 to 202), and GST–NFATc1-β (residues 1 to 189) was incubated with extracts prepared from COS cells transfected with HA-JNK1. The binding of HA-JNK1 was examined by immunoblot analysis. (B) Comparison of the phosphorylation of NFATc1 isoforms by JNK1 in vitro. Immunocomplex kinase assays were performed using Flag epitope-tagged JNK1 activated without (−) and with (+) MLK3, using recombinant NFATc1 isoforms as the substrate. (C and D) Subcellular distribution of NFATc1 isoforms, examined by immunofluorescence analysis in transfected BHK cells. The NFAT proteins (red; right) and nucleus (blue; left) were visualized (C). The effect of constitutively activated calcineurin (ΔCN) or MKK7 plus JNK1 (JNK) was examined. Arrowheads indicate the nuclei of cells expressing transfected proteins. The data were quantitated (D) following examination of 300 transfected cells and are presented as the percentage of cells with nuclear NFATc1 (mean ± standard deviation; n = 3).