Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program

Ritu Gupta,Sreesa Sreedharan,Adhish S Walvekar,Rajalakshmi Srinivasan,Ganesh Kadamur,Sunil Laxman

doi:10.1074/jbc.ra120.014248

Abstract

Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, and amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (in which the roles of Gcn4 are not well-studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase 2A (PP2A); our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 toward a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs the conserved methyltransferase Ppm1 via its target phosphatase PP2A to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master regulator and drive anabolism.

Highlights

Cellular commitments to different states, such as growth, survival, or selfdestruction depend on multiple cues
Methionine transcriptionally induces ribosomal genes, suggesting that it upregulates overall protein synthesis. To test if this transcriptional induction of ribosomal genes was functionally observed as increased translation, we examined the impact of methionine addition on global translation using polysome profiling, with cells in rich medium as a control
Methionine supplementation during a shift to MM medium (MM+Met) significantly restored polysome levels, resulting in a polysome:monosome ratio of ~1.7:1 (Figure 1A). This increase in polysome levels is greater than that observed with supplementing all non-sulphur amino acids combined except tyrosine. These data confirm that methionine supplementation increases global translation in otherwise amino acid limited conditions, consistent with the expectations from the observed anabolic program

Summary

Introduction

Cellular commitments to different states, such as growth, survival, or selfdestruction depend on multiple cues. Using simple eukaryotic model organisms like yeast, several comprehensive, systems-level studies have investigated responses of cells to distinct nutrient availabilities, to define transcriptional programs that indicate growth or starvation states [1,2,3,4,5,6]. When cells commit to mitotic division, multiple signalling cascades and transcriptional responses control overall ‘growth programs’ [1, 3, 6,7,8,9,10]. While global responses to nutrient changes are well documented, specific signalling and regulatory mechanisms that directly couple the sensing of specific metabolites to metabolic programs remain more poorly studied. There is a need to identify these regulatory mechanisms, in order to mechanistically understand growth programs

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