The incidence of obesity has increased dramatically over the last fifty years. Over half of all Americans are over- weight and almost one quarter are classified as clinically obese. The health ramifications of this epidemic of obesity are seen in the concomitant increase in the incidence of type 2 diabetes. An estimated 20 million Americans have diabetes with 80% being type 2. The most commonly prescribed therapeutic drugs used for type 2 diabetes target the AMP-activated protein kinase (AMPK). Therefore, understanding how AMPK is normally regulated is of great significance to human health. This proposal uses baker's yeast as its model system to study the regulation of the yeast AMPK. In both yeast and human cells, AMPK is activated under conditions of energy stress. Past studies from our lab have shown that the activation of Snf1, the yeast AMPK, involves at least two steps: phosphorylation of the Snf1 activation loop and activation mediated by the gamma subunit of the Snf1 complex. More recently, we have shown that the first step, control of activation loop phosphorylation, is not regulated at the level of phosphate addition. Surprisingly, it is the dephosphorylation step that responds to cellular energy status.
Specific aim 1 of this proposal will determine the molecular mechanism by which glucose regulates the dephosphorylation of Snf1. The protein phosphatase which acts on Snf1 will be purified and characterized. We will determine whether small molecule ligands determine the rate of Snf1 dephosphorylation.
Specific aim 2 will investigate the role of the gamma subunit plays in activation of Snf1 kinase complex. The mammalian gamma subunit is thought to be the site of AMP binding. Whether the yeast gamma subunit binds AMP or some other ligand is not known. Site directed and random mutagenesis will be used to identify the residues and regions of the gamma subunit that are important for regulation of Snf1. Mutations in the gamma subunit will be characterized genetically and biochemically. Finally, we have uncovered evidence that Snf1 is a potential regulator of the TOR kinase. In humans, mTOR (target of rapamycin) is a conserved protein kinase that is an important new therapeutic target for many cancers.
Specific aim 3 will determine the role of the Snf1 in controlling TOR activity.

Public Health Relevance

The incidence of obesity and type 2 diabetes have dramatically increased in the last fifty years. A human protein called AMPK is the site of action for the most widely prescribed drug used to treat type 2 diabetes. This project will elucidate the normal regulation of AMPK in baker's yeast and may reveal additional targets for novel therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046443-20
Application #
8094292
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Maas, Stefan
Project Start
1991-07-01
Project End
2013-03-31
Budget Start
2011-07-01
Budget End
2013-03-31
Support Year
20
Fiscal Year
2011
Total Cost
$349,819
Indirect Cost
Name
University of Pittsburgh
Department
Genetics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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
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
Schmidt, Martin C (2013) Signaling crosstalk: integrating nutrient availability and sex. Sci Signal 6:pe28
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
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
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
Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R et al. (2010) Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex. Eukaryot Cell 9:173-83

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