Current Research and Scholarly Interests
The Cyert lab studies Ca2+-dependent signal transduction mediated by calcineurin, the conserved Ca2+/calmodulin-regulated protein phosphatase. Calcineurin, the target of immunosuppressants FK506 and cyclosporin A, regulates many processes in mammals, including T-cell activation, heart valve development, cardiac hypertrophy and some aspects of learning and memory. In several pathogenic fungi, calcineurin is required for virulence. In Saccharomyces cerevisiae, calcineurin promotes cell survival during environmental stress. Research in the lab has three major goals:
1) Elaborate calcineurin signaling networks. 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.
2) Elucidate calcineurin function. Calcineurin regulates a range of processes: Gene expression, through activation of the Crz1 transcription factor; membrane trafficking, through regulation of alpha-arrestins, Aly1/Art6 and Rod1/Art4, and Hph1, an ER protein that regulates post-translational import into the ER, membrane structure and function through regulation of the synaptojanin Inp53/Sjl3 and eisosome proteins Slm1 and Slm2; polarized growth via dephosphorylation of Elm1, Rga2, and Boi2. Calcineurin also negatively regulates the yeast mating response by stimulating endocytosis of the pheromone receptor, and by activating Dig2, the transcriptional repressor. We are currently studying additional roles for calcineurin in membrane trafficking and polarized growth.
3) Define how calcineurin recognizes and dephosphorylates substrates. Calcineurin interacts with at least two distinct structural motifs in its substrates: The PxIxIT motif determines substrate affinity for calcineurin. Changing the affinity of the PxIxIT motif in Crz1 for calcineurin changes the Ca2+ concentration dependence of Crz1-dependent gene expression in vivo.Furthermore, calcineurin and MAPK directly compete for access to some substrates, i.e. Dig2 in yeast and JunB in mammals, via a composite docking sequence that encodes both a PxIxIT motif and a MAPK docking site (D-site). We are currently studying the regulatory properties conferred by this competitive binding between a kinase and phosphatase. Calcineurin also recognizes a second motif in substrates, φLxVP. 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. This first structure of φLxVP bound to calcineurin established its binding surface on calcineurin, and showed that three unrelated inhibitors: FK506, cyclosporin A, and A238L all inhibit calcineurin by preventing this interaction. This suggests that substrates must engage calcineurin via an φLxVP-type interaction during dephosphorylation. We are now studying how the spacing and orientation of φLxVP and PxIxIT sites in calcineurin substrates determine its selection of specific dephosphorylation sites. Using our large collection of calcineurin-dependent phosphoarylation sites, we aim to develop bioinformatic tools for de novo identification of calcineurin substrates.