Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1

Abstract

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

Fig. 1. Characterization of gfat-1(dh783)C. elegans mutants.

a Schematic representation of the hexosamine pathway (green box). The enzymes in the pathway are glutamine fructose-6-phosphate amidotransferase (GFAT-1/-2), glucosamine-6-phosphate N-acetyltransferase (GNA-1), phosphoglucomutase (PGM-3), UDP-N-acetylglucosamine pyrophosphorylase (UAP-1), and glucosamine-6-phosphate deaminase (GNPDA-1/-2). UDP-GlcNAc inhibits eukaryotic GFAT (red line). b Catalytic scheme of one GFAT monomer. (1) Before catalysis: The glutaminase domain does not adopt a fixed position. (2) Substrate binding: I. Frc6P binds, II. the glutaminase domain adopts a specific position, III. l-Gln binds. (3) Catalysis and UDP-GlcNAc inhibition. Catalysis: l-Gln is hydrolyzed to l-Glu and the released ammonia is shuttled through an ammonia channel from the glutaminase to the isomerase domain. There, Frc6P is isomerized to Glc6P and the ammonia is transferred to build GlcN6P. UDP-GlcNAc inhibition: UDP-GlcNAc binds to the isomerase domain, interacts with the interdomain linker, and inhibits the glutaminase function and thereby the GlcN6P production. c C. elegans (N2 wild type (WT) and gfat-1(dh783)) developmental resistance assay on NGM plates containing 10 µg/ml tunicamycin (TM) (mean + SEM, n = 5, *** p < 0.0001, unpaired two-sided t test). d Frequency plot of normalized parental alleles on chromosome II of dh783. The CloudMap Hawaiian Variant Mapping with WGS tool displays regions of linked loci where pure parental allele SNP positions instead of allele positions containing Hawaiian SNPs are over-represented. Gray bars represent 1 Mb and red bars represent 0.5 Mb sized bins. Table: Candidate non-synonymous SNPs between 10 and 12 Mb on chromosome II of gfat-1(dh783) animals. e C. elegans (N2 wild type (WT) and gfat-1(dh783)) developmental resistance assay on NGM plates containing 10 µg/ml tunicamycin (TM) upon RNAi treatment targeting gna-2 and control luciferase (luci) (mean + SEM, n = 4, * p = 0.0437, unpaired one-sided t test). f UDP-GlcNAc levels normalized to protein content in N2 wild type (WT) and gfat-1(dh783) animals (mean + SD, n = 5, *** p < 0.0001, unpaired two-sided t test). Source data are provided as a Source Data file.

Fig. 2. The GFAT-1 R203H gain-of-function substitution perturbs UDP-GlcNAc feedback inhibition.

a l-Gln kinetic of wild type (WT, black circle) and R203H (cyan square) GFAT-1 (mean ± SEM, WT n = 5, R203H n = 4). b Frc6P kinetic of wild type (WT, black circle) and R203H (cyan square) GFAT-1 (mean ± SEM, WT n = 5, R203H n = 4). c Representative UDP-GlcNAc dose response assay of wild type (WT, black circle) and R203H (cyan square) GFAT-1 (mean ± SD, n = 3). Table: IC₅₀ UDP-GlcNAc values (mean ± SEM, n = 4, ***p < 0.0001, unpaired two-sided t test). d, e Position of the R203H mutation in the structure of GFAT-1. Proteins are presented as cartoons. Superposition of wild type GFAT-1 (light gray/dark gray, PDB ID 6R4E) and R203H GFAT-1 (green–cyan, teal) with an RMSD of 0.38 Å over 1280 main chain residues. Glc6P (yellow sticks), l-Glu (violet sticks), and UDP-GlcNAc (white sticks) are highlighted, as well as the position of R203H (black box). d Overview. R203H (sticks) is located at the glutaminase domain of GFAT-1 (dashed box). e Close-up view of the position of R203H focusing on residues in close proximity. The R203H mutation and residues in close proximity to the mutation are highlighted with sticks. Arg203 interacts with the neighboring loops (dashed lines). Source data are provided as a Source Data file.

Fig. 3. The GFAT-1 gain-of-function substitution R203H disturbs PKA phosphorylation at Ser205.

a Position of gain-of-function mutation R203H in a protein sequence alignment of GFAT-1. R203H (cyan) disrupts the PKA consensus sequence (green box) at Ser205. b Workflow for the in vitro phosphorylation analysis. c Representative quantification of the light and heavy peptides by mass spectrometry. d Quantification of the Ser205 phosphorylation of wild type (WT, gray) and R203H (cyan) GFAT-1 before and after treatment with PKA (mean + SEM, n = 4, ** p = 0.0021, one-way ANOVA). Source data are provided as a Source Data file.

Fig. 4. PKA phosphorylation at Ser205 modulates UDP-GlcNAc inhibition of GFAT-1.

a l-Gln kinetic of wild type (WT, black circles) and S205D (red diamonds) GFAT-1 (mean ± SEM, WT n = 5, S205D n = 6). b Frc6P kinetic of wild type (WT, black circles) and S205D (red diamonds) GFAT-1 (mean ± SEM, WT n = 5, S205D n = 4). c Representative UDP-GlcNAc inhibition of wild type (WT, black circles) and S205D (red diamonds) GFAT-1 (mean ± SD, n = 3). Table: IC₅₀ UDP-GlcNAc values (mean ± SEM, n = 3). d Representative Western blot analysis of GFAT-1 protein levels in control HEK293 cells and HEK293 cells stably overexpressing the indicated GFAT-1 variants. The experiment was repeated three times with similar results. e LC/MS measurement of UDP-GlcNAc normalized to protein content presented as means + SEM with n = 4, *** p < 0.0001 versus wild type (WT), one-way ANOVA. f C. elegans (N2 wild type (WT) and heterozygous (het) gfat-1 S202D (syb3246)) developmental resistance assay on NGM plates containing 10 µg/ml tunicamycin (TM) (mean + SEM, n = 4, *** p < 0.0001, unpaired two-sided t test). Source data are provided as a Source Data file.

Fig. 5. Altered domain interactions of GFAT-1 after PKA phosphorylation at Ser205.

a Position of the phosphorylation site Ser205 in the structure of GFAT-1. Proteins are presented as cartoons. Superposition of wild type GFAT-1 (gray, PDB ID 6R4E) and S205D GFAT-1 (wheat) with an RMSD of 0.77 Å over 1312 main chain residues. Glc6P (yellow sticks), l-Glu (violet sticks), and UDP-GlcNAc (white sticks) are highlighted, as well as the position of S205D (sticks, dashed box). b Representative derivative melting curves of full-length GFAT-1 (black) and the isolated GFAT-1 isomerase domain (green) in standard SEC buffer without NaCl. c Melting temperatures of full-length GFAT-1 (black) and the isolated GFAT-1 isomerase domain (green) GFAT-1 in SEC buffer with rising NaCl concentrations (mean + SD, n = 3). d Melting temperatures of wild type GFAT-1 (black) and GFAT-1 S205D (red) (mean + SD, n = 3). e Positions of tryptophan residues and Ser205 in the structure of wild type GFAT-1. The protein is presented as a cartoon (light gray/dark gray & light blue/marine, PDB ID 6R4E). Glc6P (yellow sticks) and l-Glu (violet sticks) are highlighted, as well as the positions of Trp50, Trp96, and Ser205 (sticks). f Representative fluorescence emission spectra of W50F (blue) and W50F/S205D (orange) GFAT-1. g Maximal fluorescence emission of W50F (blue) and W50F/S205D (orange) GFAT-1. (mean + SD, n = 5). Source data are provided as a Source Data file.

Fig. 6. Altered domain interactions affect GFAT-1 activity.

a Superposition of the structures of wild type (gray) and R203H (green–cyan) GFAT-1 in cartoon representation. l-Glu (violet sticks) and UDP-GlcNAc (white sticks) are highlighted. Ser205, the R203H substitution, and residues that might form salt bridges in close proximity to the mutation are highlighted with sticks. b Schematic models of one GFAT monomer. (1) Catalysis: l-Gln is hydrolyzed to l-Glu and the released ammonia is shuttled through an ammonia channel from the glutaminase to the isomerase domain. (2) UDP-GlcNAc inhibition: UDP-GlcNAc binds to the isomerase domain, interacts with the interdomain linker, and inhibits the glutaminase function and thereby the GlcN6P production. (3) Ser205 phosphorylation: Upon phosphorylation, the catalytic activity is reduced and the UDP-GlcNAc inhibition is abolished.