Regulation of glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase.

Abstract

Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme in hexosamine biosynthesis, an important pathway for cellular glucose sensing. Human GFA has two potential sites for phosphorylation by cAMP-dependent protein kinase A (PKA). To test whether GFA activity is regulated by cAMP-dependent phosphorylation, rat aortic smooth muscle cells were treated in vivo with cAMP-elevating agents, 10 micromol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. All treatments resulted in rapid and significant increases (2- to 2.4-fold) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 micromol/l and 60 min. Preincubation of cells with cycloheximide did not abolish the effect of forskolin. Incubation of cytosolic extracts at 37 degrees C for 10 min in a buffer without phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibitors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mmol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA undergoes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold increase in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We conclude that GFA activity may be modulated by cAMP-dependent phosphorylation.

Palmitate-Induced Activation of the Hexosamine Pathway in Human Myotubes

“…We also observed no high glucose effect on the amount of UDP-GlcNAc, while in other reports a hyperglycemia-induced increase in UDP-GlcNAc concentrations in skeletal muscle has been described (41,42). However, the flux through the HBP is tightly regulated-GFAT activity is inhibited in an autocrine feedback loop by UDP-GlcNAc (13) and GlcN-6-P (43) and is posttranslationally modified by protein kinase A phosphorylation (44). A recently described striated muscle-specific splice variant of GFAT exhibits a markedly increased susceptibility to inhibition by UDP-GlcNAc (45).…”

Functional regulation of glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1) of Drosophila melanogaster in a UDP-N-acetylglucosamine and cAMP-dependent manner

“…However, the effect was not normalized to their standard conditions except the depletion or addition of the PKA. The stimulation of GFAT from rat aortic smooth-muscle cells via in i o induction of endogenous PKA has also been shown by Zhou et al [10]. This activation was approx.…”

“…In B. emersonii protein phosphatases 2A and 2C are involved in this process [9]. The stimulation of rat GFAT by protein kinase A (PKA) has been demonstrated in i o and in itro [10]. However, the K i value for the feedback inhibitor UDPNG was not altered by PKA treatment of GFAT, suggesting that PKA is not involved in the feedback regulation of GFAT mentioned above.…”

Identification of GFAT1-L, a novel splice variant of human glutamine: fructose-6-phosphate amidotransferase (GFAT1) that is expressed abundantly in skeletal muscle

“…Also, the unique tissue distribution makes GFAT1-L a very interesting target for further research on the tissue-specific regulation of hexosamine generation and the potential link to insulin resistance. Zhou et al (1998) have shown that GFAT1 activity is regulated through phosphorylation by cAMP-dependent protein kinase A (PKA). Human GFAT1 contains two potential PKA phosphorylation sites.…”

“…The GFAT1-L open reading frame consists of 2097 nucleotides, encoding a 699-aminoacid protein with a predicted molecular mass of 78790.38 Da (GFAT1: 2043 bp, 681 amino acids, and 76743.22 Da). The GFAT1-L insertion occurs within a sequence corresponding to a hinge region in the GFAT1 protein (Zhou et al 1998). …”