Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.
In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.
Thorens
B
B
Institute of Pharmacology and Toxicology, University of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland.
Dériaz
N
N
Bosco
D
D
DeVos
A
A
Pipeleers
D
D
Schuit
F
F
Meda
P
P
Porret
A
A
D019325:3-O-Methylglucose; D000595:Amino Acid Sequence; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D052216:Glucagon-Like Peptide 1; D051273:Glucose Transporter Type 2; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D013449:Sulfonamides; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D051273:Glucose Transporter Type 2; D007546:Isoquinolines; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D013449:Sulfonamides; D019325:3-O-Methylglucose; D005576:Colforsin; D052216:Glucagon-Like Peptide 1; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide