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

Abstract

The SNF1/AMP-activated protein kinases are αβγ-heterotrimers that sense and regulate energy status in eukaryotes. They are activated by phosphorylation of the catalytic Snf1/α subunit, and the Snf4/γ regulatory subunit regulates phosphorylation through adenine nucleotide binding. In Saccharomyces cerevisiae, the Snf1 subunit is phosphorylated on the activation-loop Thr-210 in response to glucose limitation. To assess the requirement of the heterotrimer for regulated Thr-210 phosphorylation, we examined Snf1 and a truncated Snf1 kinase domain (residues 1-309) that has partial Snf1 function. Snf1(1-309) does not interact with the β and Snf4/γ regulatory subunits, and its activity was independent of them in vivo. Phosphorylation of both Snf1 and Snf1(1-309) increased in response to glucose limitation in wild-type cells and in cells lacking β-and Snf4/γ-subunits. These results indicate that glucose regulation of activation-loop phosphorylation can occur by mechanism(s) that function independently of the regulatory subunits. We further show that the Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-like phosphatase are largely responsible for dephosphorylation of Thr-210 of Snf1(1-309). Together, these findings suggest that these two phosphatases mediate heterotrimer-independent regulation of Thr-210 phosphorylation.

Ptc1 Protein Phosphatase 2C Contributes to Glucose Regulation of SNF1/AMP-activated Protein Kinase (AMPK) in Saccharomyces cerevisiae

“…2, C and D. An elevation of Thr-210 phosphorylation during growth on glucose was easily evident in sit4⌬ ptc1⌬ snf1⌬ cells expressing Snf1(1-309)-myc (Fig. 2E); in this case, phosphorylation increased robustly in response to glucose limitation, perhaps reflecting the fact that Snf1(1-309) has little activity (15), and thus, cells on high glucose are not pre-adapted for growth on low glucose. These results support a modest role for Ptc1 in regulating the phosphorylation state of Snf1 Thr-210 in sit4⌬ cells.…”

“…Plasmids-Snf1-8xmyc and Snf1(1-309)-8xmyc were expressed from the SNF1 promoter on centromeric plasmids pYL199, pYL411, and pYL414 (15,24). pXX7 and pYL200 are derivatives of pYL411 and pYL199 in vectors pRS316 (29) and YCp50 (30), respectively.…”

“…3A). However, Thr-210 may be more readily accessible to Sit4 in the context of the truncated kinase domain, which does not associate with ␤ or Snf4/␥ subunits (15), than in the context of the intact SNF1 heterotrimer.…”

“…2B and 3A), previous studies showed that in reg1⌬ sit4⌬ cells, phosphorylation of Snf1(1-309) was greatly elevated during growth on high glucose and increased Ͻ2-fold in response to glucose limitation (15). In an effort to detect a role of Ptc1 in regulating Snf1(1-309), we overexpressed HA-Ptc1 from the ADH1 promoter on plasmid pAR12 in reg1⌬ sit4⌬ snf1⌬ cells.…”

“…The sit4⌬ mutation similarly caused both a defect in Thr-210 dephosphorylation and inappropriate glycogen synthesis during growth on glucose, and blocking the latter restored dephosphorylation (11). The reg1⌬ sit4⌬ double mutation was lethal in cells expressing SNF1, but a truncated Snf1 kinase domain with reduced activity (residues 1-309; lacking the C-terminal regulatory region) could be expressed in glycogen-deficient reg1⌬ sit4⌬ snf1⌬ cells, and phosphorylation of Thr-210 was highly elevated during growth on glucose (15). These findings indicated that both Reg1-Glc7 and Sit4 function in glucose-regulated dephosphorylation of Thr-210 and do so independently of the intact SNF1 heterotrimer.…”