title: Identification of a novel serine phosphorylation site in human glutamine:fructose-6-phosphate amidotransferase isoform 1.
abstract: Glutamine:fructose-6-phosphate amidotransferase (Gfat) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway. The increasing amount of evidence that links excess hexosamine biosynthesis with pathogenic complications of type II diabetes highlights the need to understand the regulation of Gfat. Previous studies showed that eukaryotic Gfat is subjected to feedback inhibition by UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and to phosphorylation by cAMP-activated protein kinase A (PKA). In this study, overexpression of human Gfat isoform 1 (hGfat1) in insect cells revealed that hGfat1 is phosphorylated in vivo. Using matrix-assisted laser desorption/ionization and electrospray tandem mass spectrometry, we have identified Ser243 as a novel phosphorylation site. Biochemical properties of the wild type and the Ser243Glu mutant of hGfat1 overexpressed in Escherichia coli were compared. Our results provide evidence that phosphorylation at Ser243 stimulates glucosamine 6-phosphate-synthesizing activity, lowers amidohydrolyzing activity in the absence of fructose 6-phosphate (F6P) (glutaminase activity), and lowers Km(F6P) 2-fold, but has no effect on UDP-GlcNAc inhibition. On the basis of the sequence consensus, AMP-activated protein kinase and calcium/calmodulin-dependent kinase II were identified to phosphorylate specifically Ser243 in vitro. Phosphorylation by these two kinases results in an increase of enzymatic activity by 1.4-fold. These findings suggest for the first time that hGfat1 may be regulated by kinases other than PKA.
authors.affiliation: CNRS, Institut de Chimie des Substances Naturelles, 91198 Gif-Sur-Yvette, France.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Li
authors.forename: Céline
authors.initials: C
authors.lastname: Roux
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lazereg
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: LeCaer
authors.forename: Olivier
authors.initials: O
authors.lastname: Laprévote
authors.forename: Bernard
authors.initials: B
authors.lastname: Badet
authors.forename: Marie-Ange
authors.initials: MA
authors.lastname: Badet-Denisot
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D053719:Tandem Mass Spectrometry
chemical_list: D012694:Serine; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)