Identification of a new calcineurin‐binding region in nuclear factor of activated T cells

Calcineurin (CN), a Ca 2+ /calmodulin‐dependent protein phosphatase, plays an important role in immunological and other biological functions. CN dephosphorylates nuclear factor of activated T cells (NFAT), which regulates transcription of numerous genes in T cells and other cell types/tissues. Two CN‐binding regions have been identified in N‐ (CNBR1) and C‐ (CNBR2) terminuses of Ca 2+ ‐regulatory domain (CRD) of NFAT. This study was performed to elucidate the relative contribution of known and unknown CNBRs to CN/NFAT‐binding. CN‐binding activity with recombinant GST‐fusion proteins of whole and partial domains of NFAT‐CRD was measured by semi‐quantitative immunoprecipitation assay. Besides CNBR1 and CNBR2, the region between CNBR1 and CNBR2 (CNBR3) was newly identified as a CN‐binding domain of NFAT. CN‐binding activity of CNBR3 was equivalent to that of CNBR1 and CNBR2. In comparison among NFAT subtypes, the potency of CN‐binding activity of CNBR3 was NFAT2=NFAT3>NFAT4=NFAT1. Further mapping studies demonstrated that the critical CN‐binding region of NFAT‐CNBR3 potentially exists in and around its conserved Ser‐Pro repeat‐associated motif. Our present findings are useful to develop a novel means to regulate CN/NFAT‐dependent biological reactions.