AMP-activated protein kinase phosphorylates glutamine : fructose-6-phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity
AMP-activated protein kinase phosphorylates glutamine : fructose-6-phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity
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DOI：
10.1111/j.1365-2443.2008.01260.x
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发表时间：
2009-02-01
期刊：
影响因子：
2.1
通讯作者：
Yonezawa, Kazuyoshi
中科院分区：
文献类型：
--
作者：
Eguchi, Satoshi;Oshiro, Noriko;Yonezawa, Kazuyoshi
谷氨酰胺：通过使用抗磷酸Akt底物（PAS）抗体的免疫沉淀，随后通过质量指纹分析，将果糖-6-磷酸酰胺转移酶1（GFAT 1）鉴定为葡萄糖剥夺细胞中磷酸化的蛋白质，所述PAS抗体识别AMP活化蛋白激酶（AMPK）的磷酸化基序位点。葡萄糖消耗诱导的内源性GFAT磷酸化被2-脱氧葡萄糖（2-DG）（一种AMPK激活剂）增强，并且转染细胞中FLAG标记的GFAT 1的2-DG刺激的磷酸化被化合物C（一种AMPK抑制剂）抑制。2-DG诱导的GFAT 1磷酸化被减弱的激酶阴性突变体的AMPK的引入，和磷酸化中观察到的细胞表达的组成型活性突变体的AMPK，即使在2-DG的情况下。随后的分析显示，PAS抗体识别在Ser 243处磷酸化的GFAT 1，这在不同物种中是保守的。细胞裂解物中GFAT酶活性的测定表明，2-DG处理抑制酶活性，化合物C预孵育部分防止了2-DG诱导的活性降低。此外，用丙氨酸取代Ser 243的突变体部分阻止了2-DG处理引起的GFAT活性下降。这些结果表明AMPK对GFAT 1的Ser 243磷酸化在调节GFAT 1酶活性中具有重要作用。
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.