Structural Studies Of The Calcineurin/NFAT Interaction

Calcium signaling controls the proliferation,differentiation,apoptosis,and a variety of transcriptional programs in immune ceils including T cells,B cells,mast cells,etc.The calcineurin/NFAT interaction is a pivotal calcium switch controlling the transcription of cytokine genes as well as many other immune response-related genes.Immunosuppressive drugs like cyclosporin A and FK506 have been developed to inhibit calcineurin phosphatase activity so that NFAT remains inactive and NFAT-induced gene expression is impaired.But this "deadly" measure simultaneously blocks other signaling pathways mediated by calcineurin leading to severe complications.Hence,research on the interaction between calcineurin and NFAT shall unravel the mechanism of this calcium switch and provide insight to new therapeutic approaches that can specifically target on this complex without affecting other signaling pathways mediated by calcineurin.Expression and purification of the highly phosphorylated NFAT protein has been a major challenge to structural biologists.Therefore,we took a novel approach to co-express an NFAT truncated protein and a catalytically dead calcineurin in Sf9 insect cells using the baculovirus expression system.Using this strategy,we were able to stabilize the truncated NFAT protein in its phosphorylated state.Co-immunoprecipitation using anti-NFATc2 antibodies or pull-down with CaM-sepharose showed that the triple co-expressed proteins existed as a ternary complex. More amazingly,preliminary crystals of phosphorylated NFAT were obtained.These results provided the foundation for further studies of the calcineurin/NFAT interaction.