Abstract
The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.
Highlights
SNF1/AMP-activated protein kinases (AMPKs) are central energy regulators that are inactivated by dephosphorylation of the activation loop
Growth of Cultures—Cells were grown to mid-log phase in selective synthetic complete medium (SC) containing 2% glucose, and an aliquot of the culture was harvested by rapid filtration to preserve the phosphorylation state of Thr-210 and was frozen in liquid nitrogen
We present genetic evidence that Ptc1, a member of the phosphatase 2C (PP2C) family, has a role in regulating the dephosphorylation of Thr-210 on the activation loop of Snf1 and the inactivation of SNF1
Summary
SNF1/AMP-activated protein kinases (AMPKs) are central energy regulators that are inactivated by dephosphorylation of the activation loop. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc protein phosphatase 1 and Sit type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf catalytic subunit during growth on high concentrations of glucose; reg1⌬ and sit4⌬ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, caused by these mutations, is blocked. The reg1⌬ ptc1⌬ mutant was viable only when expressing mutant Snf proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose This evidence, together with findings on the reg1⌬ sit4⌬ mutant, indicates that Reg1-Glc plays the major role, all three phosphatases contribute to maintenance of the Snf activation loop in the dephosphorylated state during growth on high glucose. We here examine the effects of the ptc1⌬ mutation and Ptc overexpression, in combination with reg1⌬ and/or sit4⌬ mutations, on Thr-210 phosphorylation of wild-type and mutant forms of SNF1
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