Skurat A V, Dietrich A D, Roach P J
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202. USA.
Diabetes. 2000 Jul;49(7):1096-100. doi: 10.2337/diabetes.49.7.1096.
In skeletal muscle, insulin activates glycogen synthase by reducing phosphorylation at both NH2- and COOH-terminal sites of the enzyme and by elevating the levels of glucose-6-phosphate, an allosteric activator of glycogen synthase. To study the mechanism of regulation of glycogen synthase by insulin and glucose-6-phosphate, we generated stable Rat-1 fibroblast clones expressing rabbit muscle glycogen synthase with Ser-->Ala substitutions at key phosphorylation sites. We found that 1) elimination of the phosphorylation of either NH2- or COOH-terminal sites did not abolish insulin stimulation of glycogen synthase; 2) mutations at both Ser-7 and Ser-640 were necessary to bypass insulin activation; 3) mutation at Ser-7, coupled with the disruption of the motif for recognition by glycogen synthase kinase-3 (GSK-3), did not eliminate the insulin effect; and 4) mutation of either Ser-7 or Ser-640 increased the sensitivity of glycogen synthase to glucose 6-phosphate >10-fold. We conclude that Ser-7 and Ser-640 are both involved in mediating the response of glycogen synthase to insulin and activation by glucose 6-phosphate. In Rat-1 fibroblasts, GSK-3 action is not essential for glycogen synthase activation by insulin, and GSK-3-independent mechanisms also operate.
在骨骼肌中，胰岛素通过减少糖原合酶氨基端和羧基端位点的磷酸化以及提高糖原合酶的变构激活剂6-磷酸葡萄糖的水平来激活糖原合酶。为了研究胰岛素和6-磷酸葡萄糖对糖原合酶的调节机制，我们构建了稳定表达兔肌肉糖原合酶的大鼠-1成纤维细胞克隆，其中关键磷酸化位点的丝氨酸被丙氨酸取代。我们发现：1）消除氨基端或羧基端位点的磷酸化并不会消除胰岛素对糖原合酶的刺激作用；2）丝氨酸-7和丝氨酸-640同时发生突变才能绕过胰岛素的激活作用；3）丝氨酸-7发生突变，同时破坏糖原合酶激酶-3（GSK-3）识别基序，并不会消除胰岛素的作用；4）丝氨酸-7或丝氨酸-640发生突变会使糖原合酶对6-磷酸葡萄糖的敏感性增加10倍以上。我们得出结论，丝氨酸-7和丝氨酸-640均参与介导糖原合酶对胰岛素的反应以及被6-磷酸葡萄糖激活的过程。在大鼠-1成纤维细胞中，GSK-3的作用对于胰岛素激活糖原合酶并非必不可少，并且还存在不依赖GSK-3的机制。
