title: AMP-activated protein kinase phosphorylates glutamine : fructose-6-phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity.
abstract: Glutamine : fructose-6-phosphate amidotransferase 1 (GFAT1) was identified as a protein phosphorylated in glucose-deprived cells by immunoprecipitation using the anti-phospho Akt substrates (PAS) antibody, which recognizes the phosphorylation motif site by AMP-activated protein kinase (AMPK), followed by mass fingerprinting analysis. Glucose depletion-induced phosphorylation of endogenous GFAT was potentiated by 2-deoxyglucose (2-DG), an AMPK activator, and the 2-DG-stimulated phosphorylation of FLAG-tagged GFAT1 in transfected cells was suppressed by Compound C, an AMPK inhibitor. The 2-DG induced phosphorylation of GFAT1 was attenuated by the introduction of the kinase-negative mutant of AMPK, and the phosphorylation was observed in the cells expressing the constitutively active mutant of AMPK even in the absence of 2-DG. Subsequent analysis revealed that the PAS antibody recognized GFAT1 phosphorylated at Ser243, which is conserved among different species. The assay of the GFAT enzymatic activity in the cell lysates indicated that the 2-DG-treatment inhibited the enzymatic activity, and Compound C-preincubation partially prevented the 2-DG-induced decrease of the activity. Furthermore, the mutant replacing Ser243 by alanine partially prevented the decrease of GFAT activity by 2-DG treatment. These results indicate that the phosphorylation of GFAT1 at Ser243 by AMPK has an important role in the regulation of the GFAT1 enzymatic activity.
authors.affiliation: Biosignal Research Center, Kobe University, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Eguchi
authors.forename: Noriko
authors.initials: N
authors.lastname: Oshiro
authors.forename: Takafumi
authors.initials: T
authors.lastname: Miyamoto
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Yoshino
authors.forename: Sumiko
authors.initials: S
authors.lastname: Okamoto
authors.forename: Takamasa
authors.initials: T
authors.lastname: Ono
authors.forename: Ushio
authors.initials: U
authors.lastname: Kikkawa
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Yonezawa
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism* / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D012694:Serine; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D055372:AMP-Activated Protein Kinases