Identification of a Novel Serine Phosphorylation Site in Human Glutamine:Fructose-6-phosphate Amidotransferase Isoform 1

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• 作者：Yanyan Li ; C&eacute ; line Roux ; Sylvie Lazereg ; Jean-Pierre LeCaer ; Olivier Lapr&eacute ; vote ; Bernard Badet ; Marie-Ange Badet-Denisot
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：November 13, 2007
• 年：2007
• 卷：46
• 期：45
• 页码：13163 - 13169
• 全文大小：314K
• 年卷期：v.46,no.45(November 13, 2007)
• ISSN：1520-4995

文摘

Glutamine:fructose-6-phosphate amidotransferase (Gfat) catalyzes the first and rate-limitingstep in the hexosamine biosynthetic pathway. The increasing amount of evidence that links excesshexosamine biosynthesis with pathogenic complications of type II diabetes highlights the need to understandthe regulation of Gfat. Previous studies showed that eukaryotic Gfat is subjected to feedback inhibitionby UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) and to phosphorylation by cAMP-activated protein kinaseA (PKA). In this study, overexpression of human Gfat isoform 1 (hGfat1) in insect cells revealed thathGfat1 is phosphorylated in vivo. Using matrix-assisted laser desorption/ionization and electrospray tandemmass spectrometry, we have identified Ser243 as a novel phosphorylation site. Biochemical properties ofthe 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 K_m(F6P) 2-fold, but has no effect on UDP-GlcNAc inhibition. On thebasis of the sequence consensus, AMP-activated protein kinase and calcium/calmodulin-dependent kinaseII were identified to phosphorylate specifically Ser243 in vitro. Phosphorylation by these two kinasesresults in an increase of enzymatic activity by 1.4-fold. These findings suggest for the first time thathGfat1 may be regulated by kinases other than PKA.