Calcineurin (CN) is a highly conserved ser/thr protein phosphatase that is regulated by Ca2+ and calmodulin, and thus mediates Ca2+-dependent changes in phosphorylation in vivo. In humans, CN regulates immune cell function, promotes development of muscle, vascular and pancreatic tissues, and modulates learning and memory in the brain. CN inhibitors, FK506 and cyclosporin A, are in wide clinical use as immunosupressants, and perturbation of CN-dependent signaling is associated with many pathophyisological conditions including Down's syndrome, heart disease, schizophrenia, cancer, and diabetes. Identification of CN substrates is key to understanding and modulating this wide range of physiological activities. This proposal seeks to identify the functions and substrates of CN in yeast. Previous studies demonstrated that CN promotes yeast cell survival during environmental stress, and identified several substrates required for this response. However, additional functions of CN, for which substrates have not yet been identified, are also indicated. Now approaches for global identification of CN substrates in yeast are proposed. This simple eukaryote offers many experimental advantages; these studies will provide insights into CN-dependent signaling that are relevant to all cells and will pave the way for global identification of phosphatase substrates in other organisms. The specific aims of the proposed work are to: 1) Identify CN substrates comprehensively, using two complementary, proteomic approaches. First, proteins dephosphorylated by CN will be identified with a novel in vitro assay that uses phosphorylated protein microarrays. Second, phosphopeptides that are enriched in extracts of CN-deficient cells will be identified by label-free, quantitative mass spectrometry. Functional characterization of the resulting collection of substrates will expand our understanding of CN regulatory activities. Biochemical and bioinformatic analyses of these substrates will further define sequences involved in CN substrate recognition. 2) Identify critical regions of CN by identifying mutations that alter its activity and interactions with substrates in vivo. These mutants will provide new tools for investigating CN function and substrate specificity in many organisms. 3) Identify the role of CN-interacting proteins in CN signaling pathways. Characterization of CN substrates is essential for defining its physiological functions. The role of CN substrates, Slm1 and Slm2, in TOR-dependent activation of Ypk1/2 kinases during heat stress, will be investigated, and the interaction of CN with Whi3, an RNA binding protein that regulates entry into the cell cycle will be examined. Preliminary observations suggest roles for CN and Whi3 in yeast stress granules, which form in response to glucose starvation and contain non-translating mRNAs complexed with initiation factors.