Regulation of glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase.
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.
Zhou
J
J
Department of Medicine, University of Mississippi Medical Center, Jackson 39216, USA.
Huynh
Q K
QK
Hoffman
R T
RT
Crook
E D
ED
Daniels
M C
MC
Gulve
E A
EA
McClain
D A
DA
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017386:Sequence Homology, Amino Acid
D004791:Enzyme Inhibitors; D005576:Colforsin; D000242:Cyclic AMP; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D017868:Cyclic AMP-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases; D000262:Adenylyl Cyclases