GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis

Abstract

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by -linked β--acetylglucosamine (-GlcNAc). In the present study, we tested the hypothesis that AMPK controls -GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced -GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

AMPK: guardian of metabolism and mitochondrial homeostasis

“…AMPK also prevents the storage of glycogen by inhibitory phosphorylation of the glycogen synthases GYS1 and GYS2 (REF. 87) and inhibits hexosamine synthesis and O-GlcNAc modification of cellular proteins via phosphorylation of GFAT1 (REFS 88,89) (FIG. 2).…”

Infection‐driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells

“…This resulted in an increase in O ‐GlcNAcylation, since this post‐translational modification of proteins is directly dependent on the concentration of UDP‐GlcNAc (Kreppel & Hart, ). GFPT acts as a tetramer and is negatively regulated by several post‐translational modifications (Chang et al , ; Zibrova et al , ) and by UDP‐GlcNAc (Assrir et al , ). Transamidation on Gln328 could interfere with these down‐regulation mechanisms and thereby unleash the HBP.…”

The hexosamine biosynthesis pathway is a targetable liability in lung cancers with concurrent KRAS and LKB1 mutations

“…To fully understand the mechanism by which LKB1 inhibits GFPT2, further work will be required. AMPK is a major metabolic effector and it has been reported to inhibit GFPT1 enzyme activity by phosphorylation 34 . This raises the possibility that AMPK may also be involved in GFPT2 regulation.…”