The highly conserved SNF1/AMP-activated protein kinase (AMPK) is a central energy regulator in eukaryotes. In humans, AMPK is implicated in the pathogenesis of disease, including type 2 diabetes, obesity and cancer, and AMPK is a target of anti-diabetic drugs. The yeast SNF1 pathway has provided a paradigm for understanding the human pathway. We propose to continue our functional analysis of the SNF1/AMPK pathway in the yeast system, which offers the advantages of powerful genetics. SNF1/AMPK, a heterotrimer, is activated by phosphorylation of the activation loop on the catalytic subunit in response to metabolic and other stress. Accumulating evidence points to the importance of protein phosphatases in regulating SNF1/AMPK, but the phosphatases that are involved and the mechanisms regulating dephosphorylation are not well understood. We propose studies to elucidate the roles of phosphatases in the SNF1 pathway. We recently found that, in addition to Reg1-Glc7 protein phosphatase 1 (PP1), type 2A-like phosphatase Sit4 has a role in the SNF1 pathway. We propose genetic and biochemical approaches to determine whether these two phosphatases together are responsible for dephosphorylation of SNF1 during growth on high levels of glucose and to address their roles in different stress responses. The interactions of these phosphatases with SNF1 will be investigated under different conditions. The roles of the three SNF1 subunits in determining binding specificity for each phosphatase will be assessed, and various alterations of SNF1 that prevent dephosphorylation of the activation loop will be tested for effects on interactions with phosphatases. The proposed studies should provide insight into mechanisms that control phosphatase function under different conditions. Finally, it will be important to identify all the major phosphatases that regulate the SNF1 pathway. Evidence suggests Ptc1 as a promising candidate, and we plan to assess its role in regulation of SNF1. We will also assess the roles of several other candidates, and, if appropriate, undertake a search for other phosphatases. The proposed studies will provide new insights into regulatory mechanisms of general relevance for SNF1/AMPK pathways and will inform efforts to identify human phosphatases for AMPK. We further anticipate that this work will point to new strategies for preventive interventions for metabolic disorders and cancer, by targeting phosphatases and their interactions with AMPK to activate the kinase.

Public Health Relevance

The highly conserved SNF1/AMPK pathway is essential for control of metabolism and energy homeostasis, and in humans has roles in type 2 diabetes, obesity, cancer, and hereditary heart disease. The proposed studies on regulation of this pathway will provide insight into human disease and identify new targets for development of drugs for treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM034095-27A1
Application #
8371829
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Maas, Stefan
Project Start
1984-07-01
Project End
2015-06-30
Budget Start
2012-07-05
Budget End
2013-06-30
Support Year
27
Fiscal Year
2012
Total Cost
$352,800
Indirect Cost
$132,300
Name
Columbia University (N.Y.)
Department
Genetics
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Ruiz, Amparo; Xu, Xinjing; Carlson, Marian (2013) Ptc1 protein phosphatase 2C contributes to glucose regulation of SNF1/AMP-activated protein kinase (AMPK) in Saccharomyces cerevisiae. J Biol Chem 288:31052-8
Ruiz, Amparo; Xu, Xinjing; Carlson, Marian (2011) Roles of two protein phosphatases, Reg1-Glc7 and Sit4, and glycogen synthesis in regulation of SNF1 protein kinase. Proc Natl Acad Sci U S A 108:6349-54
Momcilovic, Milica; Carlson, Marian (2011) Alterations at dispersed sites cause phosphorylation and activation of SNF1 protein kinase during growth on high glucose. J Biol Chem 286:23544-51
Liu, Yang; Xu, Xinjing; Carlson, Marian (2011) Interaction of SNF1 protein kinase with its activating kinase Sak1. Eukaryot Cell 10:313-9
Momcilovic, Milica; Iram, Surtaj H; Liu, Yang et al. (2008) Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase. J Biol Chem 283:19521-9
Hedbacker, Kristina; Carlson, Marian (2008) SNF1/AMPK pathways in yeast. Front Biosci 13:2408-20
Rudolph, Michael J; Amodeo, Gabriele A; Iram, Surtaj H et al. (2007) Structure of the Bateman2 domain of yeast Snf4: dimeric association and relevance for AMP binding. Structure 15:65-74
Hong, Seung-Pyo; Carlson, Marian (2007) Regulation of snf1 protein kinase in response to environmental stress. J Biol Chem 282:16838-45
Orlova, Marianna; Kanter, Ellen; Krakovich, David et al. (2006) Nitrogen availability and TOR regulate the Snf1 protein kinase in Saccharomyces cerevisiae. Eukaryot Cell 5:1831-7
Berkey, Cristin D; Carlson, Marian (2006) A specific catalytic subunit isoform of protein kinase CK2 is required for phosphorylation of the repressor Nrg1 in Saccharomyces cerevisiae. Curr Genet 50:1-10

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