This is a competitive renewal from Dr. Marian Carlson to study the glucose regulation of the invertase gene, SUC2, in baker's yeast. This application focuses on the Snf1 protein kinase complex that plays a central role in the signal transduction pathway that senses glucose and alters transcription of a large number of glucose repressed and activated genes. The PI's current model for glucose regulation of SUC2 involves three tiers of complexes. 1). The presence of glucose is sensed through the Reg1-PP1 (protein phosphatase I) complex which dephosphorylates 2) a member of the Snf1 kinase complex. The Snf1 kinase complex includes at least the Snf4, and the Sip1, 2, and Gal83 proteins. When dephosphorylated by the glucose signal it isnon-functional, but when glucose is absent, it phosphorylates 3) the Mig1 repressor, inactivating it. When active, the Mig1 repressor complexes with the up1-Ssn6 general repressor to repress transcription of SUC2. The link between each of these tiers is tenuous, although the evidence suggests that each interacts as described. However, there are a number of missing factors including: the proposed Snf1 kinase kinase, the glucose sensing mechanism, and the transcriptional activators of SUC2 which may well be glucose regulated through Snf1, too. This application proposes to detail the known interactions and identify and characterize the missing factors. A combination of biochemical and genetic approaches will be used. The Snf1 kinase will be purified for both in vitro phosphorylation studies, especially focused on Mig1, and protein- protein interaction studies. The latter will be carried out using fusion proteins which will allow tethering one member of the complex to a column. The genetic studies place heavy reliance on the two-hybrid system to isolate factors that interact with Snf1 and members of the SNf1 complex as well as to detail the interacting regions. This approach will be complemented with mutant and high copy suppression hunts designed to identify factors that function upstream of the Snf1 kinase. For analyses of the downstream targets, phosphorylation of both wildtype Mig1 and mutant versions altered in the putative Snf1 target sites, will be studied in vitro and in vivo. Also a strategy is described using a modified two-hybrid approach to identify the SUC2 activator(s) based upon the assumption that these proteins are either essential or redundant since they were never identified in the original SUC2 uninducible mutant hunts.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (ZRG2-GEN (05))
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Anderson, James J
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Columbia University (N.Y.)
Internal Medicine/Medicine
Schools of Medicine
New York
United States
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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; Liu, Yang; Xu, Xinjing et al. (2012) Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases. Proc Natl Acad Sci U S A 109:8652-7
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
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
Liu, Yang; Xu, Xinjing; Carlson, Marian (2011) Interaction of SNF1 protein kinase with its activating kinase Sak1. Eukaryot Cell 10:313-9
Amodeo, Gabriele A; Momcilovic, Milica; Carlson, Marian et al. (2010) Biochemical and functional studies on the regulation of the Saccharomyces cerevisiae AMPK homolog SNF1. Biochem Biophys Res Commun 397:197-201
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
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
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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