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Satoshi Eguchi^a, Noriko Oshiro, Takafumi Miyamoto^b, Ken-ichi Yoshino, Sumiko Okamoto, Takamasa Ono, Ushio Kikkawa^* and Kazuyoshi Yonezawa

Biosignal Research Center, Kobe University, Kobe 657-8501, Japan

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.

^aPresent address: Department of Pathology and Immunology, Akita University School of Medicine, Akita 010-8543, Japan.

^bPresent address: Cancer Medicine and Biophysics Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan.

Deceased on 8 July 2005

^* Correspondence: ukikkawa{at}kobe-u.ac.jp