Nuclear factor of activated T cells 2 transactivation in mast cells: A novel isoform-specific transactivation domain confers unique FcεRI responsiveness

Murine nuclear factor of activated T cells (NFAT)2.α/β differ by 42 and 28 unique amino-terminal amino acids and are differentially expressed. Both isoforms share conserved domains that regulate DNA-binding and subcellular localization. A genetic "one-hybrid" assay was used to define two distinct transactivation (TA) domains: in addition to a conserved TAD present in both isoforms, a second, novel TAD exists within the β-specific amino terminus. Pharmacologic inhibitors Gö6976 and rottlerin demonstrate that both conventional and novel protein kinase C (PKC) family members regulate endogenous mast cell NFAT activity, and NFAT2 TA. Overexpression of dominant active PKCθ (which has been implicated in immune receptor signaling) induces NFAT2.α/β TA. Mutations within the smallest PKCθ-responsive transactivation domain demonstrate that the PKCθ effect is at least partially indirect. Significantly, the β-specific domain confers greater ability to TA in response to treatment with phorbol 12-myristate 13-acetate/ionomycin or lipopolysaccharide, and unique sensitivity to FcεRI signaling. Accordingly, overexpression of NFAT2.β results in significantly greater NFAT-and interleukin-4 reporter activity than NFAT2.α. These results suggest that whereas NFAT2 isoforms may share redundant DNA-binding preferences, there are specialized functional consequences of their isoform-specific domains.