Global identification and functional analysis of calcineurin substrates in yeast
Abstract/Contents
- Abstract
- The regulation of the phosphoproteome by kinases and phosphatases is a critical component of complex signaling networks. Many tools for identification of kinase substrates cannot be applied to phosphatases, creating a disparity in kinase versus phosphatase substrate identification. In this work, we describe two complementary approaches for identifying phosphatase substrates, both using the calcium-dependent phosphatase calcineurin in S. cerevisiae as a model. First, mass spectrometry-based analysis of calcineurin-deficient and calcineurin-activated yeast, in silico screening to identify calcineurin-docking motifs, and biochemical verification led to the identification of 15 novel calcineurin substrates from a set of 29 novel interactors for calcineurin in yeast. This represents the first successful comprehensive identification of phosphatase substrates in any organism and significantly advances our understanding of calcium/calcineurin signaling. Substrate identification has suggested new roles for calcium/calcineurin signaling in polarized growth, autophagy, and zinc homeostasis. Specific analysis suggests that calcineurin negatively regulates the kinase Elm1 in the alkaline pH stress response and early G1 of the cell cycle. Second, a protein microarray phosphatase assay was used to identify calcineurin substrates in vitro. This assay uses a mixture of purified yeast kinases to radiolabel phosphorylation sites that have been dephosphorylated by calcineurin. Our pilot screen identified approximately 90 potential calcineurin substrates with Hel2, Sgt1, and Skg1 being verified by secondary assays. The methodologies presented in this work may be generalizable to identify substrates for phosphatases in yeast and other organisms and help lead to a better understanding of complex signaling networks.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Goldman, Aaron Robert |
|---|---|
| Associated with | Stanford University, Department of Biology. |
| Primary advisor | Cyert, Martha S, 1958- |
| Thesis advisor | Cyert, Martha S, 1958- |
| Thesis advisor | Kopito, Ron Rieger |
| Thesis advisor | Snyder, Michael, Ph. D |
| Thesis advisor | Wang, Zhi-Yong, Dr |
| Advisor | Kopito, Ron Rieger |
| Advisor | Snyder, Michael, Ph. D |
| Advisor | Wang, Zhi-Yong, Dr |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Aaron Robert Goldman. |
|---|---|
| Note | Submitted to the Department of Biology. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2012. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Aaron Robert Goldman
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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