Phosphorylation of human glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase at serine 205 blocks the enzyme activity
Phosphorylation of human glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase at serine 205 blocks the enzyme activity
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DOI：
10.1074/jbc.m001049200
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发表时间：
2000-07-21
影响因子：
4.8
通讯作者：
Kudlow, JE
中科院分区：
文献类型：
--
作者：
Chang, Q;Su, KH;Kudlow, JE
谷氨酰胺：果糖-6-磷酸酰胺转移酶（GFAT）是葡萄糖胺合成的限速酶。我们实验室先前的研究表明，腺苷酸环化酶的激活与O-GlcNAc修饰的耗竭有关。这一发现和证据表明，人GFAT（hGFAT）可能是由cAMP依赖性蛋白激酶（PKA）的调节，使我们调查PKA在hGFAT功能的作用。我们证实，通过毛喉素激活腺苷酸环化酶导致几种细胞蛋白质的O-GlcNAc修饰减少，这可以通过将细胞暴露于葡糖胺而不是葡萄糖来克服，这表明PKA激活导致用于O-糖基化的UDP-GlcNAc耗尽。为了确定GFAT是否确实受PKA调节，我们使用牛痘病毒表达系统表达了该酶的活性形式，并显示该酶的活性通过PKA磷酸化降低到不可检测的水平。我们借助基质辅助激光解吸电离质谱绘制了PKA磷酸化位点，并表明该蛋白质在丝氨酸205处化学计量磷酸化，在丝氨酸235处也磷酸化，程度较低，突变研究表明，PKA对丝氨酸205的磷酸化是所观察到的酶活性抑制所必需的，而丝氨酸235的磷酸化则不起作用。可观察的作用。GFAT的活性被cAMP下调，从而对与生物体的能量需求一致的己糖胺途径进行调节。在饥饿期间，通过腺苷酸环化酶起作用的激素可以将葡萄糖代谢流引导到能量生产中，而不是进入需要己糖胺的合成途径。
Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme in glucosamine synthesis. Prior studies from our laboratory indicated that activation of adenylate cyclase was associated with depletion of O-GlcNAc modification. This finding and evidence that human GFAT (hGFAT) might be regulated by cAMP-dependent protein kinase (PKA) led us to investigate the role of PKA in hGFAT function. We confirmed that adenylate cyclase activation by forskolin results in diminished O-GlcNAc modification of several cellular proteins which can be overcome by exposure of the cells to glucosamine but not glucose, suggesting the PKA activation results in depletion of UDP-GlcNAc for O-glycosylation, To determine if GFAT is indeed regulated by PKA, we expressed the active form of the enzyme using a vaccinia virus expression system and showed that the activity of the enzyme was to decrease to undetectable levels by PKA phosphorylation. We mapped the PKA phosphorylation sites with the aid of matrix-assisted laser desorption ionization mass spectroscopy and showed that the protein was stoichiometrically phosphorylated at serine 205 and also phosphorylated, to a lesser extent at serine 235, Mutagenesis studies indicated that the phosphorylation of serine 205 by PKA was necessary for the observed inhibition of enzyme activity while serine 235 phosphorylation played no observable role. The activity of GFAT is down-regulated by cAMP, thus placing regulation on the hexosamine pathway that is in concert with the energy requirements of the organism. During starvation, hormones acting through adenylate cyclase could direct the flux of glucose metabolism into energy production rather than into synthetic pathways that require hexosamines.