AMP‐activated protein kinase phosphorylates glutamine : fructose‐6‐phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity

Abstract

Glutamine : fructose-6-phosphate amidotransferase 1 (GFAT1) was identified as a protein phosphorylated in glucose-deprived cells by immunoprecipitation using the anti-phospho Akt substrates (PAS) antibody, which recognizes the phosphorylation motif site by AMP-activated protein kinase (AMPK), followed by mass fingerprinting analysis. Glucose depletion-induced phosphorylation of endogenous GFAT was potentiated by 2-deoxyglucose (2-DG), an AMPK activator, and the 2-DG-stimulated phosphorylation of FLAG-tagged GFAT1 in transfected cells was suppressed by Compound C, an AMPK inhibitor. The 2-DG induced phosphorylation of GFAT1 was attenuated by the introduction of the kinase-negative mutant of AMPK, and the phosphorylation was observed in the cells expressing the constitutively active mutant of AMPK even in the absence of 2-DG. Subsequent analysis revealed that the PAS antibody recognized GFAT1 phosphorylated at Ser243, which is conserved among different species. The assay of the GFAT enzymatic activity in the cell lysates indicated that the 2-DG-treatment inhibited the enzymatic activity, and Compound C-preincubation partially prevented the 2-DG-induced decrease of the activity. Furthermore, the mutant replacing Ser243 by alanine partially prevented the decrease of GFAT activity by 2-DG treatment. These results indicate that the phosphorylation of GFAT1 at Ser243 by AMPK has an important role in the regulation of the GFAT1 enzymatic activity.

New Aspects of Cellular Cholesterol Regulation on Blood Glucose Control—Review and Perspective on the Impact of Statin Medications on Metabolic Health

“…90 Mechanistically, adiponectin stimulates AMPK, a kinase that suppresses energy-consuming pathways such as hexosamine and cholesterol biosynthesis. [91][92][93] We have found that AMPK stimulation improves GLUT4-mediated glucose transport and ABCA1/ ApoA1-mediated cholesterol efflux from insulin-resistant 3T3-L1 adipocytes via lowering membrane cholesterol levels. 41 This factor was not found to differ before and after administration of pitavastatin in overall analysis of all the subjects.…”

Fission yeast TORC1 regulates phosphorylation of ribosomal S6 proteins in response to nutrients and its activity is inhibited by rapamycin

“…The anti-PAS antibody has been used to screen for growth-factor-regulated and nutrient-regulated phosphoproteins, which include not only the actual substrates of Akt but also substrates of S6K and AMP-activated protein kinase in mammalian cells (Eguchi et al, 2009;Kane et al, 2002;Manning et al, 2002;Miyamoto et al, 2008). To examine nitrogen-regulated phosphoproteins by using the anti-PAS antibody, fission yeast cells were cultured in medium in the presence or absence of ammonium as the sole nitrogen source.…”

Physiological regulation of the heat shock response by glutamine: implications for chronic low-grade inflammatory diseases in age-related conditions

“…In fact, chronic glutamine supplementation to trained rats has shown to increase muscle GSH contents and GS at the same time that it has enhanced HS response [49]. Corroborating this assumption is the fact that the activation of GFAT depletes intracellular glucose stores [239]. Oppositely, GSH depletion, per se, increases OGT gene expression leading to O-GlcNAcylation of different regulatory proteins [251], thus strongly suggesting that HBP has a short-loop positive feedback system devoted to assist in cytoprotection via HS response soon as some redox threatening situation is ongoing.…”

“…HBP is a nutrient-sensing pathway [226] that presents multiple connections with energy metabolism, not only with glycogen synthesis [219]. AMPK, which occupies a central position in metabolic regulation in order to avoid inflammatory dysregulation, phosphorylates and inhibits GFAT1, the flux-generating step of HBP, thus allowing for the downregulation of such a shunt from the glycolysis under low glucose situations [239]. Conversely, chronic hexosamine flux stimulates fatty acid oxidation by activating AMPK [240].…”

“…As depicted in Fig. 5, glutamine may divert muscle glycolysis and glycogenesis favoring the formation of UDP-Nacetylglucosamine, the end-product of HBP [55,239]. When performing a flux balance analysis, based on metabolite concentrations of the rat muscle [49], it is predicted that the flux through G6PDH should be around 700 μmol min −1 g tissue −1 (corresponding to 3.3 % of incoming glucose after hexokinase reaction), whereas the flux through GFAT, 80 μmol/min/g tissue, responds to 4.3 % of fructose-6-phsophate utilized by the muscle (or 0.9 % of total incoming glucose).…”