Regulation of glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase
Autor: | E A Gulve, Jianxin Zhou, Q K Huynh, R T Hoffman, Donald A. McClain, Marc C. Daniels, Errol D. Crook |
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Rok vydání: | 1998 |
Předmět: |
Endocrinology
Diabetes and Metabolism Phosphatase Molecular Sequence Data Biology Dephosphorylation chemistry.chemical_compound Internal Medicine Cyclic AMP Animals Amino Acid Sequence Enzyme Inhibitors Phosphorylation Protein kinase A Sodium orthovanadate Protein kinase C Cells Cultured Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) Forskolin Cell-Free System Sequence Homology Amino Acid Colforsin Okadaic acid Molecular biology Cyclic AMP-Dependent Protein Kinases Phosphoric Monoester Hydrolases Rats Enzyme Activation chemistry Biochemistry Adenylyl Cyclases |
Zdroj: | Diabetes. 47(12) |
ISSN: | 0012-1797 |
Popis: | 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. |
Databáze: | OpenAIRE |
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