Snf1/AMPK family members mediate the nutrient stress response in eukaryotes. In yeast, Snf1 is required for survival during glucose deprivation. There are three different Snf1 isoforms; each contains the catalytic alpha subunit, one of three regulatory beta subunits, and the regulatory gamma subunit. In glucose limitation, Snf1 is stimulated by activation loop phosphorylation by three partially redundant Snf1-activating kinases (SAKs), Sak1, Tos3, and Elm1. In glucose abundance, the PP1 phosphatase Glc7 dephosphorylates Snf1. We investigated the relationships between the three SAKs and three Snf1 isoforms, the roles of the SAK nonkinase domains, and the mechanism(s) controlling glucose-regulated Snf1 phosphorylation.
Since Snf1 assembles into three complexes, the possibility existed that each SAK is dedicated to phosphorylating a single Snf1 isoform. To test this, we generated strains lacking different combinations of SAKs and beta subunits. While our results indicate that the SAKs are not dedicated to specific Snf1 isoforms, each SAK exhibited distinct abilities to activate Snf1, depending on the beta subunit and stress imposed on cells.
Domain deletion analysis was employed to characterize the SAKs nonkinase domains. Deletion of Sak1 and Tos3 (but not Elm1) C-termini diminished the ability to activate Snf1. Deletion of the Elm1 C-terminal domain abrogated Elm1s participation in morphogenetic signaling. Thus, the SAK C-terminal domains contribute to pathway specificity. Additional deletion mutants revealed an N-terminal Sak1 motif essential for Snf1 signaling. These data demonstrate that nonkinase N- and C-terminal domains are critical for SAK pathway specification.
Snf1 phosphorylation status is regulated by integration of the reaction rates of phosphorylation by the SAKs and dephosphorylation by Glc7. We sought to determine which reaction(s) is glucose-regulated. SAKs immunoprecipitated from extracts of cells grown in high- and low-glucose conditions exhibited similar activity toward Snf1, suggesting the SAKs exhibit constitutive activity. Snf1 dephosphorylation, however, is glucose-regulated. When de novo phosphorylation was inhibited, Snf1 phosphorylation was stable in low glucose but rapidly lost upon glucose addition. Glc7 catalytic activity is not glucose-regulated since another Glc7 substrate was dephosphorylated in both glucose conditions. Therefore, Snf1 phosphorylation status is controlled by Snf1s availability to serve as a Glc7 substrate.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-06272008-154806 |
| Date | 30 June 2008 |
| Creators | Rubenstein, Eric Meyer |
| Contributors | Martin Schmidt, PhD, Saleem Khan, PhD, Tom Smithgall, PhD, Karen Arndt, PhD, Ora Weisz, PhD |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-06272008-154806/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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