Calcineurin (CN) is a highly conserved ser/thr protein phosphatase that is regulated by Ca2+ and calmodulin, and thus mediates Ca2+-dependent changes in phosphorylation in vivo. In humans, CN regulates immune cell function, promotes development of muscle, vascular and pancreatic tissues, and modulates learning and memory in the brain. CN inhibitors, FK506 and cyclosporin A, are in wide clinical use as immunosupressants, and perturbation of CN-dependent signaling is associated with many pathophyisological conditions including Down's syndrome, heart disease, schizophrenia, cancer, and diabetes. Identification of CN substrates is key to understanding and modulating this wide range of physiological activities. This proposal seeks to identify the functions and substrates of CN in yeast. Previous studies demonstrated that CN promotes yeast cell survival during environmental stress, and identified several substrates required for this response. However, additional functions of CN, for which substrates have not yet been identified, are also indicated. Now approaches for global identification of CN substrates in yeast are proposed. This simple eukaryote offers many experimental advantages;these studies will provide insights into CN-dependent signaling that are relevant to all cells and will pave the way for global identification of phosphatase substrates in other organisms.
The specific aims of the proposed work are to: 1) Identify CN substrates comprehensively, using two complementary, proteomic approaches. First, proteins dephosphorylated by CN will be identified with a novel in vitro assay that uses phosphorylated protein microarrays. Second, phosphopeptides that are enriched in extracts of CN-deficient cells will be identified by label-free, quantitative mass spectrometry. Functional characterization of the resulting collection of substrates will expand our understanding of CN regulatory activities. Biochemical and bioinformatic analyses of these substrates will further define sequences involved in CN substrate recognition. 2) Identify critical regions of CN by identifying mutations that alter its activity and interactions with substrates in vivo. These mutants will provide new tools for investigating CN function and substrate specificity in many organisms. 3) Identify the role of CN-interacting proteins in CN signaling pathways. Characterization of CN substrates is essential for defining its physiological functions. The role of CN substrates, Slm1 and Slm2, in TOR-dependent activation of Ypk1/2 kinases during heat stress, will be investigated, and the interaction of CN with Whi3, an RNA binding protein that regulates entry into the cell cycle will be examined. Preliminary observations suggest roles for CN and Whi3 in yeast stress granules, which form in response to glucose starvation and contain non-translating mRNAs complexed with initiation factors.

Public Health Relevance

This project examines the functions of a highly conserved protein, calcineurin. Calcineurin regulates many processes in humans in response to changes in Ca2+, and alterations in calcineurin activity occur in immune disorders, diabetes, cancer, heart disease and schizophrenia. This research studies, at the biochemical level, the functions of calcineurin in a simple, experimentally tractable organism, i.e. yeast, and will serve as a model for understanding the actions of calcineurin in human cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048729-20
Application #
8299653
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Maas, Stefan
Project Start
1993-01-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
20
Fiscal Year
2012
Total Cost
$428,950
Indirect Cost
$157,032
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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
Grigoriu, Simina; Bond, Rachel; Cossio, Pilar et al. (2013) The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin. PLoS Biol 11:e1001492
Cyert, Martha S; Philpott, Caroline C (2013) Regulation of cation balance in Saccharomyces cerevisiae. Genetics 193:677-713
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
Rodriguez, Antonio; Roy, Jagoree; Martinez-Martinez, Sara et al. (2009) A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants. Mol Cell 33:616-26
Roy, Jagoree; Li, Huiming; Hogan, Patrick G et al. (2007) A conserved docking site modulates substrate affinity for calcineurin, signaling output, and in vivo function. Mol Cell 25:889-901
Bultynck, Geert; Heath, Victoria L; Majeed, Alia P et al. (2006) Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease. Mol Cell Biol 26:4729-45
Withee, J L; Sen, R; Cyert, M S (1998) Ion tolerance of Saccharomyces cerevisiae lacking the Ca2+/CaM-dependent phosphatase (calcineurin) is improved by mutations in URE2 or PMA1. Genetics 149:865-78