REGULATION OF GLUTAMINE-FRUCTOSE-6-PHOSPHATE AMIDOTRANSFERASE BY CAMP-DEPENDENT PROTEIN-KINASE

Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limi ting enzyme in hexosamine biosynthesis, an important pathway for cellu lar glucose sensing. Human GFA has two potential sites for phosphoryla tion by cAMP-dependent protein kinase A (PKA). To test whether GFA act ivity is regulated by cAMP-dependent phosphorylation, rat aortic smoot h muscle cells were treated in vivo with cAMP-elevating agents, 10 mu mol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. A U treatments resulted in rapid and significant increases (2- to 2.4-fo ld) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 mu mol/l and 60 min. Preincubation of ce lls with cycloheximide did not abolish the effect of forskolin. Incuba tion of cytosolic extracts at 37 degrees C for 10 min in a buffer with out phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibit ors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mm ol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA under goes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold in crease in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We, conclude that GFA activity may be modulated by cA MP-dependent phosphorylation.