Roles of two protein phosphatases, Reg1-Glc7 and Sit4, and glycogen synthesis in regulation of SNF1 protein kinase

Abstract

The SNF1 protein kinase of Saccharomyces cerevisiae is a member of the SNF1/AMP-activated protein kinase family, which is essential for metabolic control, energy homeostasis, and stress responses in eukaryotes. SNF1 is activated in response to glucose limitation by phosphorylation of Thr210 on the activation loop of the catalytic subunit Snf1. The SNF1 β-subunit contains a glycogen-binding domain that has been implicated in glucose inhibition of Snf1 Thr210 phosphorylation. To assess the role of glycogen, we examined Snf1 phosphorylation in strains with altered glycogen metabolism. A reg1Δ mutant, lacking Reg1-Glc7 protein phosphatase 1, exhibits elevated glycogen accumulation and phosphorylation of Snf1 during growth on high levels of glucose. Unexpectedly, mutations that abolished glycogen synthesis also restored Thr210 dephosphorylation in glucose-grown reg1Δ cells, indicating that elevated glycogen synthesis contributes to activation of SNF1 and that another phosphatase acts on Snf1. We present evidence that Sit4, a type 2A-like protein phosphatase, contributes to dephosphorylation of Snf1 Thr210. Finally, evidence that the effects of glycogen are not mediated by binding to the β-subunit raises the possibility that elevated glycogen synthesis alters glucose metabolism and thereby reduces glucose signaling to the SNF1 pathway.glucose regulation | signal transduction | yeast T he SNF1/AMP-activated protein kinase (AMPK) family is conserved from yeast to mammals and is essential for metabolic control and energy homeostasis (1, 2). The Saccharomyces cerevisiae SNF1 protein kinase is required for adaptation to glucose limitation and other stresses and for growth on carbon sources that are less preferred than glucose; it is named for the sucrose-nonfermenting phenotype of the snf1 mutant (2). SNF1 regulates transcription of a large set of genes (3) and the activity of metabolic enzymes involved in carbohydrate storage and fatty acid metabolism (4-6).

Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases

“…dephosphorylation; a partial defect in dephosphorylation upon glucose replenishment of glucose-limited cells was still evident in the absence of glycogen synthesis (15). Glycogen binding to the β-subunit was not involved (15,37).…”

“…Reg1-Glc7 PP1 and the type 2A-like Sit4 phosphatase have roles in dephosphorylation of Thr-210 of SNF1 (9, 10, 12-15, 54), and both Reg1 and Sit4 associate with Snf1 (12,13,15). The reg1Δ and sit4Δ mutants exhibited elevated Thr-210 phosphorylation during growth on high glucose, but glycogen levels were also elevated, and abolishing glycogen synthesis restored Thr-210 Fig.…”

“…Reg1-Glc7 protein phosphatase 1 (PP1) has been thought to be solely responsible for dephosphorylating Thr-210 (9)(10)(11)(12)(13)(14), and it has been proposed that access of Reg1-Glc7 to Thr-210 is restricted during growth on limiting glucose (9). However, recent evidence has also implicated the type 2A-like protein phosphatase Sit4 in Thr-210 dephosphorylation (15). Both the reg1Δ and sit4Δ mutants have defects in dephosphorylation, but they also have elevated levels of glycogen, and abolishing glycogen synthesis restored Thr-210 dephosphorylation during growth on high levels of glucose (15).…”

“…However, recent evidence has also implicated the type 2A-like protein phosphatase Sit4 in Thr-210 dephosphorylation (15). Both the reg1Δ and sit4Δ mutants have defects in dephosphorylation, but they also have elevated levels of glycogen, and abolishing glycogen synthesis restored Thr-210 dephosphorylation during growth on high levels of glucose (15). The reg1Δ sit4Δ double mutant is not viable due to inappropriate activation of SNF1, which precluded studies to demonstrate that both Reg1-Glc7 and Sit4 function in dephosphorylation of Thr-210.…”

“…The β-subunits specify subcellular localization (31), affect access to substrates (32,33), and contain glycogen-binding domains (34,35). Glycogen binding is inhibitory to AMPK in vitro (36), whereas the glycogen-binding domain of the major SNF1 β-subunit is required for dephosphorylation of Thr-210 during growth of cells on high levels of glucose, independent of glycogen (15,37). Finally, substitutions of residues at dispersed sites in all three subunits of SNF1 result in Thr-210 phosphorylation during growth on high levels of glucose, suggesting that the conformation of the heterotrimer is important to maintaining the dephosphorylated state (37,38).…”