The NFAT family of Ca2+-regulated transcription factors has a critical role in vertebrate development and function. NFAT proteins regulate T cell activation;for T cell differentiation, T regulatory function and T cell anergy;activation of other immune-system cells (e.g. mast cells, NK cells) in response to stimulation;cardiac valve development, neuronal and vascular patterning, osteoclast, osteoblast, keratinocyte and chondrocyte differentiation;differentiation of slow-twitch skeletal muscle fibers;and other facets of skeletal, cardiac and smooth muscle function. They are also important modulators of cancer progression and metastasis, and are implicated in many pathological processes including transplant rejection, inflammation, osteoporosis, myocardial hypertrophy, allergy and autoimmune disease. Although the mechanisms of NFAT activation and deactivation have been extensively investigated, much remains to be understood. NFAT proteins are regulated by Ca2+ and the Ca2+ calmodulin-dependent serine phosphatase calcineurin. In resting cells, NFAT proteins are phosphorylated and reside in the cytoplasm;upon stimulation, they are dephosphorylated by calcineurin, translocate to the nucleus and become transcriptionally active. The overall goal of this project is to understand, at a detailed biochemical level, the mechanisms underlying NFAT activation, so that steps of the process can be manipulated for possible therapeutic benefit. Specifically, the objective of Aim 1 is to generate an integrated picture of how NFAT dephosphorylation leads to its activation, and how this process is countered by NFAT kinases, particularly DRYK and PKD, which emerged as novel NFAT regulators from a genome-wide RNAi screen recently performed in Drosophila. The goal of Aim 2 is to follow up on recent data indicating that NFAT nuclear import is regulated by a large cytoplasmic scaffold complex, which contains both proteins and noncoding RNA. The goal of Aim 3 is to elucidate the structural basis for calcineurin/ NFAT signalling, and investigate the mechanisms of information transfer from Ca2+ to calcineurin to NFAT within the cell. Techniques will be developed to monitor conformational changes occurring in calcineurin and NFAT during their activation, and to analyze at a structural level the interaction between calcineurin and NFAT.