In yeast, calcineurin is activated in response to a number of different environmental conditions, including elevated temperature, exposure to ions (Na+/Li+, Mn2+, OH-), ER stress and cell wall damage, and is required for cells to survive these conditions (reviewed in Cyert, 2003). A major function of calcineurin is to activate gene expression, and studies using DNA microarrays identified more than one hundred genes whose transcription is induced by calcineurin (Yoshimoto et al. 2002). These genes encode products that function in cell wall biosynthesis, secretion, signal transduction and ion homeostasis, and allow cells to survive environmental stress.

Calcineurin alters gene expression by regulating the transcription factor, Crz1p (Stathopoulos and Cyert, 1997). In response to stress, calcineurin dephosphorylates Crz1p, causing its rapid relocalization from the cytosol to the nucleus (Stathopoulos and Cyert 1999). This role of calcineurin is conserved in higher eukaryotes, where calcineurin similarly dephosphorylates the NFAT transcription factor to effect its translocation to the nucleus. Surprisingly, Crz1p and NFAT share little sequence similarity, although notably both contain a conserved calcineurin-docking motif (see below).

Recently, biochemical and proteomic approaches were used to identify two kinases that phosphorylate and negatively regulate Crz1p (Kafadar et al. 2003, Kafadar and Cyert 2004). These kinases modulate the signaling threshold required to activate calcineurin/Crz1p –dependent gene expression. One of these kinases, PKA, responds to the nutritional status of the cell. Thus, these findings identify one mechanism by which nutrient and stress signaling pathways are coordinated in vivo. The lab is currently investigating additional mechanisms of “cross-talk” by which calcineurin signaling is integrated with the activity of other signal transduction pathways. Specifically, we are examining the relationship between calcineurin and the cell integrity pathway, which is regulated by protein kinase C.

Genetic studies indicate that calcineurin performs other roles in addition to regulating Crz1p, and current efforts in the lab are aimed at identifying additional substrates of calcineurin. Mammalian and yeast calcineurin interact directly with their substrates through a conserved docking motif: PxIxIT in NFAT, and PIISIQ in Crz1p (Boustany and Cyert, 2002). Disruption of this docking motif in the substrate prevents its dephosphorylation and regulation by calcineurin. We are characterizing proteins that interact with calcineurin, and have identified several new targets of the phosphatase. Currently, we are investigating the functions of these novel substrates, which include Hph1, an integral ER protein that may regulate protein trafficking during stress (Heath et al. 2004). We are also using biochemical approaches to study further the interaction of calcineurin with its substrates. Genomic approaches, employing parallel analysis of the genome-wide collection of yeast deletion mutants, and screens with small molecule inhibitors, are being used to identify and characterize additional downstream components of calcineurin-regulated signaling pathways.

Other projects in the lab address additional aspects of calcium-dependent signal transduction in yeast, including characterization of Yvc1p, an mechanosensitive ion channel that is responsible for intracellular Ca2+ release from the yeast vacuole and is a member of the TRP (transient receptor potential) family of ion channels (Denis and Cyert, 2002).