The Ca2+/calmodulin dependent protein phosphatase, calcineurin, is a critical component of Ca2+-dependent signal transduction. In mammals, calcineurin regulates immune cell function, promotes heart and blood vessel development, allows heart cells to respond to stress, and modulates learning and memory. In vitro, calcineurin catalytic activity requires only the addition of Ca2+ and calmodulin. However, in vivo the interaction of calcineurin with its substrates and with regulatory proteins has a profound impact on its activity. In budding yeast, calcineurin allows cells to adapt to several environmental stresses, including exposure to ions (Na+, Mn2+, OH-), cell wall damage, and prolonged incubation with mating factor. Under these conditions, calcineurin dephosphorylates the Crz1p transcription factor, and causes its rapid translocation from the cytosol to the nucleus where it activates gene expression. How are calcineurin-dependent responses modulated in vivo, and how are they coordinated with other signaling mechanisms to achieve the proper physiological response to a particular environmental condition? To address these questions, we will examine mechanisms that regulate calcineurin and/or Crz1 function, study the interactions of other signaling pathways with Crz1/calcineurin, and characterize the association of calcineurin and Crz1 p with other proteins during signaling. Specifically, we will 1) Identify protein kinases that phosphorylate Crz1 p and determine the role of these kinases in Crz1p regulation, 2) Identify and mutagenize Crz1p phosphorylation sites and determine their roles in regulating Crz1p localization and activity, 3) Examine the mechanism by which Skn7p, a key protein in the yeast response to oxidative stress, influences catcineurin/Crz1p function to mediate cross-talk between these signaling pathways, and 4) Characterize calcineurin- and Crz1 p-containing protein complexes to define the role of specific protein-protein interactions in modulating calcineurin and Crz1 p-dependent signaling in vivo. The broad goal of this research is to elucidate the mechanisms and functions of Ca2+/calcineurin dependent signal transduction. We address these questions using S. cerevisiae, because of the many experimental advantages offered by this organism. However, we believe our findings will establish general principles that will apply to calcineurin-dependent signaling in all cells.
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