Alterations at Dispersed Sites Cause Phosphorylation and Activation of SNF1 Protein Kinase during Growth on High Glucose

Marian Carlson,Milica Momcilovic

doi:10.1074/jbc.m111.244111

Abstract

The SNF1/AMP-activated protein kinases are central energy regulators in eukaryotes. SNF1 of Saccharomyces cerevisiae is inhibited during growth on high levels of glucose and is activated in response to glucose depletion and other stresses. Activation entails phosphorylation of Thr(210) on the activation loop of the catalytic subunit Snf1 by Snf1-activating kinases. We have used mutational analysis to identify Snf1 residues that are important for regulation. Alteration of Tyr(106) in the αC helix or Leu(198) adjacent to the Asp-Phe-Gly motif on the activation loop relieved glucose inhibition of phosphorylation, resulting in phosphorylation of Thr(210) during growth on high levels of glucose. Substitution of Arg for Gly(53), at the N terminus of the kinase domain, increased activation on both high and low glucose. Alteration of the ubiquitin-associated domain revealed a modest autoinhibitory effect. Previous studies identified alterations of the Gal83 (β) and Snf4 (γ) subunits that relieve glucose inhibition, and we have here identified a distinct set of Gal83 residues that are required. Together, these results indicate that alterations at dispersed sites within each subunit of SNF1 cause phosphorylation of the kinase during growth on high levels of glucose. These findings suggest that the conformation of the SNF1 complex is crucial to maintenance of the inactive state during growth on high glucose and that the default state for SNF1 is one in which Thr(210) is phosphorylated and the kinase is active.

Highlights

Deletion of the Gal83 glycogen-binding domain (GBD) and substitutions of single residues within the GBD that interact with Snf4 resulted in Thr210 phosphorylation and SNF1 catalytic activity during growth on high glucose [23]; several lines of evidence indicate that the effects of the GBD on SNF1 do not involve the binding of glycogen [13, 23]
SNF1 protein kinase containing the Snf1⌬347–398 catalytic subunit showed modestly increased Thr210 phosphorylation and kinase activity, relative to wildtype SNF1, when cells were grown on high levels of glucose, and catalytic activity increased further when cultures were depleted for glucose (Fig. 1, C and D)
In this study, we showed that residues in different regions of the Snf1 catalytic subunit are required to maintain inhibition of SNF1 during growth of cells on high levels of glucose (Fig. 5)

Summary

Introduction

Deletion of the Gal83 GBD and substitutions of single residues within the GBD that interact with Snf4 resulted in Thr210 phosphorylation and SNF1 catalytic activity during growth on high glucose [23]; several lines of evidence indicate that the effects of the GBD on SNF1 do not involve the binding of glycogen [13, 23]. We expressed each mutant Snf1 subunit in snf1⌬ cells and assessed regulation of the resulting mutant SNF1 protein kinase by monitoring phosphorylation of Thr210 and catalytic activity.

Results

Conclusion