2) What processes does calcineurin regulate in vivo?.
In yeast, calcineurin regulates a range of processes: Gene expression, through activation of the Crz1 transcription factor; membrane trafficking, through regulation of Hph1, an ER protein that regulates post-translational  import into the ER (Piña et al 2011) and alpha-arrestins, Aly1/Art6 and Rod1/Art4 (O’Donnell et al. 2013, Alvaro et al. 2014, Goldman et al. 2014). Calcineurin negatively regulates the yeast response to mating factor both by stimulating endocytosis of the pheromone receptor via Rod1, and by activating Dig2, the transcriptional repressor (Alvaro et al. 2014, Goldman et al. 2014).  Calcineurin regulates membrane structure and function by dephosphorylating eisosome proteins Slm1 and Slm2 and synaptojanin, Inp53/Sjl3 (Guiney et al 2014, Bultynck et al. 2006). Regulation of synaptojanin is particularly interesting, as it is one of only two proteins dephosphorylated by calcineurin in both yeast and mammals. During osmotic stress, when cell shrinkage causes plasma membrane excess (Dupont et al 2010), calcineurin induces Inp53 association with new protein partners, including intersectin and syndapin (Guiney et al, 2014)--a response that has striking parallels to activity dependent bulk endocytosis, a calcineurin-activated process in neurons that rapidly retrieves excess plasma membrane caused by multiple rounds of synaptic vesicle exocytosis (Clayton and Cousin 2009).  Current work in the lab focuses on additional roles for calcineurin in membrane trafficking and in polarized growth, where it regulates Elm1, Rga2 and Boi2 (Goldman et al, 2014).
We  study Ca2+-dependent signaling networks
The Cyert lab studies Ca2+-dependent signal transduction mediated by calcineurin, the highly conserved Ca2+/calmodulin-regulated protein phosphatase. Calcineurin, is the target of immunosuppressant drugs, FK506 and cyclosporin A, and regulates many Ca2+-dependent processes in mammalian cells, including T-cell activation, heart valve development, cardiac hypertrophy and some aspects of learning and memory (Roy and Cyert 2013). In several pathogenic fungi, calcineurin is required for virulence. In baker’s yeast, Saccharomyces cerevisiae, calcineurin promotes cell survival during environmental stress (Cyert and Philpott, 2013).
Research in the lab has three major goals:
We identified 39 proteins in S. cerevisiae that are either calcineurin substrates or calcineurin-interacting proteins, using a combination of phosphoproteomic, bioinformatics and experimental analyses.  This calcineurin signaling network, the most complete in any organism, provides new insights into calcineurin function, and into the evolution of phosphorylation networks. Analyses of closely related yeasts show that many proteins recently became calcineurin substrates by acquiring a calcineurin-recognition motif (i.e. a PxIxIT site). Unexpectedly, however, a very similar set of kinases phosphorylate calcineurin substrates in yeast and mammals, despite little conservation in substrate identity. Thus, we propose that signaling networks evolve via conserved kinase-phosphatase pairs that are maintained, despite rapid change in the proteins they regulate (Goldman et al., 2014). These kinase-phosphatase modules may be maintained through common recognition of substrate features, including docking sits, on example being joint docking sits for MAPK and calcineurin (see below). Future work will identify mammalian calcineurin substrates using similar approaches to those we used for yeast.
Calcineurin interacts with at least two distinct peptide motifs in its substrates, which are examples of Short Linear Motifs, or SLiMs, that occur in disordered protein  domains and mediate millions of specific protein-protein interactions (Roy and Cyert 2009) (Tompa et al, 2014). Each yeast substrate contains a conserved sequence, termed the PxIxIT motif, which determines its affinity for calcineurin. For Crz1, changing the affinity of the PxIxIT motif for calcineurin changes the Ca2+ concentration dependence of calcineurin/Crz1-dependent gene expression in vivo (Roy et al. 2007). Furthermore, calcineurin and MAPK directly compete for access to some substrates, including Dig2 from yeast and JunB from mammals, via a composite docking sequence that encodes both a PxIxIT motif and a MAPK docking site (D-site) (Goldman et al. 2014). We are currently studying the regulatory properties that are conferred by this competitive binding between a kinase and phosphatase.
Calcineurin also recognizes a second motif in substrates, φLxVP (Rodriguez et al 2009). Our recent biochemical and structural analyses of A238L, a viral protein inhibitor of calcineurin, show that A238L inhibits calcineurin by blocking the φLxVP and PxIxIT binding surfaces on the enzyme, while leaving the catalytic center of the enzyme unoccluded (Grigoriu et al. 2013). This first structure of φLxVP bound to calcineurin establishes its binding surface on calcineurin, and shows that three unrelated inhibitors, the immunosuppressants, FK506 and cyclosporin A and A238L, all inhibit calcineurin by preventing this interaction. Thus, we propose that substrates must engage calcineurin via an φLxVP-type interaction during dephosphorylation. Currently, we are using both natural and model substrates to determine how the spacing and orientation of φLxVP and PxIxIT sites in calcineurin substrates determine its selection of specific dephosphorylation sites. We are also employing better recognition of this critical SLiM to identify calcineurin substrates in the mammalian cardiovascular system, and studying the regulation of a human calcineurin isoform that is regulated by an autoinhibitory φLxVP sequence.
1) Calcineurin signaling networks: identification & evolution
3) How does calcineurin select its substrates?