In mammals, calcineurin, the Ca2+/calmodulin dependent protein phosphatase, regulates immune cell activity, promotes heart and blood vessel development, mediates cardiac muscle response to stress, and modulates learning and memory in the brain. Calcineurin inhibitors, FK506 and cyclosporin A, are used clinically as immunosupressants, and, in animal models, reduce cardiac hypertrophy. As a key effector of Ca2+/calmodulin dependent signaling, detailed analysis of calcineurin function has the potential to impact many aspects of human health and development. In S. cerevisiae, calcineurin promotes survival during environmental stress, and in response to cell wall damage. A major role of calcineurin is to dephosphorylate and activate the Crz1p transcription factor, using mechanism analogous to calcineurin regulation of the mammalian transcription factor, NFAT. Additional calcineurin-mediated events that promote yeast survival during environmental stress are less well characterized, and are the focus of this application.
This research aims to identify comprehensively the functions and components of calcineurin-dependent signaling pathways. We address these questions using S. cerevisiae, because of many experimental advantages offered by this simple eukaryotic organism, but strive to establish general principles that apply to calcineurin-dependent signaling in all cells. Previously, we exploited a particular feature of calcineurin signaling, i.e. the requirement for calcineurin to interact directly with its substrates via a docking site that is distinct from residues that are dephosphorylated, to identify several new components of calcineurin-mediated signaling pathways including 3 novel substrates: Slm1p, Slm2p and Hph1p. Here we propose to characterize further the mechanism by which calcineurin interacts with its substrates, which is evolutionary conserved. We will also apply genetic, genomic, and proteomic approaches to the identification of additional substrates and regulators of calcineurin. Specifically we will 1) Characterize calcineurin-docking sites in substrates and examine the impact of calcineurin-substrate affinity on signaling. 2) Examine the effect of specific point mutations in calcineurin on substrate interaction. 3) Identify the role of calcineurin interacting proteins in calcineurin signaling pathways. 4) Identify additional calcineurin substrates using novel proteomic and genomic screening methods. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048729-16
Application #
7452288
Study Section
Cellular Signaling and Dynamics Study Section (CSD)
Program Officer
Anderson, Richard A
Project Start
1993-01-01
Project End
2010-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
16
Fiscal Year
2008
Total Cost
$378,690
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Ly, Nina; Cyert, Martha S (2017) Calcineurin, the Ca2+-dependent phosphatase, regulates Rga2, a Cdc42 GTPase-activating protein, to modulate pheromone signaling. Mol Biol Cell 28:576-586
Guiney, Evan L; Goldman, Aaron R; Elias, Joshua E et al. (2015) Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress. Mol Biol Cell 26:769-85
Arsenault, Heather E; Roy, Jagoree; Mapa, Claudine E et al. (2015) Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation. Mol Biol Cell 26:3570-7
Goldman, Aaron; Roy, Jagoree; Bodenmiller, Bernd et al. (2014) The calcineurin signaling network evolves via conserved kinase-phosphatase modules that transcend substrate identity. Mol Cell 55:422-435
Alvaro, Christopher G; O'Donnell, Allyson F; Prosser, Derek C et al. (2014) Specific ?-arrestins negatively regulate Saccharomyces cerevisiae pheromone response by down-modulating the G-protein-coupled receptor Ste2. Mol Cell Biol 34:2660-81
Cyert, Martha S; Philpott, Caroline C (2013) Regulation of cation balance in Saccharomyces cerevisiae. Genetics 193:677-713
Grigoriu, Simina; Bond, Rachel; Cossio, Pilar et al. (2013) The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin. PLoS Biol 11:e1001492
Holmes, Kristen J; Klass, Daniel M; Guiney, Evan L et al. (2013) Whi3, an S. cerevisiae RNA-binding protein, is a component of stress granules that regulates levels of its target mRNAs. PLoS One 8:e84060
Pina, Francisco J; O'Donnell, Allyson F; Pagant, Silvere et al. (2011) Hph1 and Hph2 are novel components of the Sec63/Sec62 posttranslational translocation complex that aid in vacuolar proton ATPase biogenesis. Eukaryot Cell 10:63-71
Rodríguez, Antonio; Roy, Jagoree; Martínez-Martínez, Sara et al. (2009) A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants. Mol Cell 33:616-26

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