The Snf1 protein kinase plays a central role in nutrient stress signaling in the budding yeast, Saccharomyces cerevisiae. The mammalian homologue of Snf1, the AMP-activated protein kinase (AMPK) plays an equally important role in mammalian cells. Recently AMPK was shown to be the target of metformin, the most widely prescribed drug used to treat type 2 diabetes. We propose to study the regulation of this medically important enzyme in yeast. In the previous grant period we developed an assay for the phosphorylation state of the Snf1 activation loop. We showed that phosphorylation of Snf1 was a critical step for the activation of the Snf1 kinase and that the phosphorylation of Snf1 was catalyzed by a distinct upstream kinase. Recently, we were able to demonstrate that there are three upstream kinases (Pakl, Tos3 and Elm1) that are capable of activating Snf1 in vivo. The identification of the Snf1 activating kinases was highly significant because it immediately led to the identification of the mammalian kinase that activates AMPK. In the next grant period we propose to continue our studies of the regulation of the Snf1 kinase signaling pathway. We will focus on upstream events in this pathway and determine the mechanism that regulate the activity of the three Snf1-activating kinases. We will determine whether there are distinct forms of the Snf1 kinase complex that are specialized for response to different stresses. We will assess the regulatory significance of phosphorylation sites in the Snf1 kinase complex that we have mapped by mass spectrometry. Finally, biochemical and genetic analyses have identified a number of candidates for regulators of the Snf1 kinase signaling pathway. These candidates proteins will be studied to determine if they associate with and regulate the activity of the Snf1 kinase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046443-16
Application #
7254131
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Anderson, James J
Project Start
1991-07-01
Project End
2008-08-31
Budget Start
2007-07-01
Budget End
2008-08-31
Support Year
16
Fiscal Year
2007
Total Cost
$301,989
Indirect Cost
Name
University of Pittsburgh
Department
Genetics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Offley, Sarah R; Schmidt, Martin C (2018) Protein phosphatases of Saccharomyces cerevisiae. Curr Genet :
McCartney, Rhonda R; Garnar-Wortzel, Leopold; Chandrashekarappa, Dakshayini G et al. (2016) Activation and inhibition of Snf1 kinase activity by phosphorylation within the activation loop. Biochim Biophys Acta 1864:1518-28
Chandrashekarappa, Dakshayini G; McCartney, Rhonda R; O'Donnell, Allyson F et al. (2016) The ? subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress. Cell Signal 28:1881-1893
O'Donnell, Allyson F; McCartney, Rhonda R; Chandrashekarappa, Dakshayini G et al. (2015) 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and ?-arrestin-mediated trafficking of hexose transporters 1 and 3. Mol Cell Biol 35:939-55
McCartney, Rhonda R; Chandrashekarappa, Dakshayini G; Zhang, Bob B et al. (2014) Genetic analysis of resistance and sensitivity to 2-deoxyglucose in Saccharomyces cerevisiae. Genetics 198:635-46
Schmidt, Martin C (2013) Signaling crosstalk: integrating nutrient availability and sex. Sci Signal 6:pe28
Chandrashekarappa, Dakshayini G; McCartney, Rhonda R; Schmidt, Martin C (2013) Ligand binding to the AMP-activated protein kinase active site mediates protection of the activation loop from dephosphorylation. J Biol Chem 288:89-98
Mayer, Faith V; Heath, Richard; Underwood, Elizabeth et al. (2011) ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase. Cell Metab 14:707-14
Chandrashekarappa, Dakshayini G; McCartney, Rhonda R; Schmidt, Martin C (2011) Subunit and domain requirements for adenylate-mediated protection of Snf1 kinase activation loop from dephosphorylation. J Biol Chem 286:44532-41
Zhang, Yuxun; McCartney, Rhonda R; Chandrashekarappa, Dakshayini G et al. (2011) Reg1 protein regulates phosphorylation of all three Snf1 isoforms but preferentially associates with the Gal83 isoform. Eukaryot Cell 10:1628-36

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