SIGNAL TRANSDUCTION AND GENE INDUCTION IN T LYMPHOCYTES

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DESCRIPTION (provided by applicant): Transcription factors of the NFAT family are highly-phosphorylated proteins whose functions are controlled by a balance between the calcium/ calmodulin-regulated serine/ threonine phosphatase calcineurin and several constitutive and inducible kinases. NFAT proteins play a major role in the regulating the transcription of inducible genes by cells of the immune system during an immune response, and have also been implicated in regulating various physiological and pathophysiological processes gene transcription in diverse non-immune cell types including heart, skeletal muscle, adipocytes, and certain neuronal cell types. The long-term goal of this project is to arrive at an understanding of the mechanisms by which the activation and function of NFAT transcription factors are regulated. Its specific aims are as follows. In Aim 1, we will develop reagents targeting the NFAT-calcineurin interaction, with which to explore NFAT functions in primary T cells. We will analyze transgenic mice in which a selective peptide inhibitor of the NFAT-calcineurin interaction, GFP-VIVIT, is expressed under tetracycline control; develop cell-permeant reagents that target the NFAT-calcineurin interaction, which can later be used to examine the role of NFAT in various biological processes; and collaborate with an established structural laboratory to obtain the X-ray crystal structure of the NFAT1-calcineurin complex. In Aim 2, we will identify the constitutive kinases that regulate the NFAT family member NFAT1, and determine whether they also phosphorylate one or more of the other calcium-regulated NFATs. Our objective is to ask whether the individual members of the NFAT family are regulated by common or distinct mechanisms. In Aim 3, we will define the mechanisms regulating transcription b y NFAT1, by defining the roles of inducible phosphorylation of the N-terminal transactivation domain, the LDFS motif in the N-terminal transactivation domain, and the modification that potentiates NFAT1 association with the histone acetyltransferases p300 and CBP. These experiments will increase our understanding of the molecular mechanisms of NFAT regulation. Therapeutic strategies that target NFAT proteins may have applicability in many types of pathological situations, including cardiac hypertrophy, transplant rejection, asthma and allergy, and autoimmune disease.