Phosphorylation of Glutamine Synthetase on Threonine 301 Contributes to Its Inactivation During Epilepsy.

Deborah Huyghe,Stephen J Moss,Nicholas Brandon,Helle S Waagepetersen,Menelas Pangalos,Andrew D Ferguson,Ning Gao,Caroline M Voss,Peter Doig,Pernille Frank,Andrew R Denninger

doi:10.3389/fnmol.2019.00120

Abstract

The astrocyte-specific enzyme glutamine synthetase (GS), which catalyzes the amidation of glutamate to glutamine, plays an essential role in supporting neurotransmission and in limiting NH4+ toxicity. Accordingly, deficits in GS activity contribute to epilepsy and neurodegeneration. Despite its central role in brain physiology, the mechanisms that regulate GS activity are poorly defined. Here, we demonstrate that GS is directly phosphorylated on threonine residue 301 (T301) within the enzyme’s active site by cAMP-dependent protein kinase (PKA). Phosphorylation of T301 leads to a dramatic decrease in glutamine synthesis. Enhanced T301 phosphorylation was evident in a mouse model of epilepsy, which may contribute to the decreased GS activity seen during this trauma. Thus, our results highlight a novel molecular mechanism that determines GS activity under both normal and pathological conditions.

Highlights

The glutamate/GABA-glutamine cycle plays a fundamental role in the central nervous system (CNS) by detoxifying the brain of excess neurotransmitters and ammonia as well as supplying neurons with glutamine, which is a precursor of both GABA and glutamate
Both species exhibit two consensus sites for protein kinase (PKA)-dependent phosphorylation near the active site of the protein localized on threonine 301 and serine 343 (T301 and S343, Figure 1A)
In order to assess the possible role that phosphorylation plays in determining glutamine synthetase (GS) activity, we initially expressed WT and mutant versions of mouse GS modified with an N-terminal 6× His-Avi-TEV tag in E. coli

Summary

Introduction

The glutamate/GABA-glutamine cycle plays a fundamental role in the central nervous system (CNS) by detoxifying the brain of excess neurotransmitters and ammonia as well as supplying neurons with glutamine, which is a precursor of both GABA and glutamate. Glutamine synthetase (GS) catalyzes an essential step in this pathway, the ATP-dependent condensation of glutamate and ammonia into glutamine (Schousboe et al, 2013). Previous studies have shown that GS is essential to survival, both in rodents and in humans (Häberle et al, 2005; He et al, 2010). Consistent with its central role in neurotransmitter recycling and neurotransmission, GS expression and/or activity are dramatically decreased in several neurological disorders, including mesial temporal lobe epilepsy

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