糖原合成酶激酶-3对τ蛋白磷酸化作用的研究

Study on Phosphorylation of τ Protein by Glycogen Synthetase Kinase 3

糖原合成酶激酶３（ Ｇ Ｓ Ｋ３）在 ３０℃与 τ蛋白保温 ４ ｈ 可催化 １７±０４ ｍ ｏｌ磷酸参入 １ ｍ ｏｌτ蛋白 将磷酸化的 τ蛋白经胰蛋白酶消化， Ｆｅ Ｃｌ３ 亲和柱分离及 Ｃ１８反相高压液相层析纯化后，再用高压电泳，手工 Ｅｄｍ ａｎ 降解及自动氨基酸序列分析等检测技术，对其磷酸化位点进行鉴定 结果发现： Ｇ Ｓ Ｋ３ 可使 τ蛋白 Ｔｈｒ１８１， Ｓｅｒ１８４， Ｓｅｒ２６２， Ｓｅｒ３５６ 和 Ｓｅｒ４００ 发生磷酸化 其中 Ｓｅｒ２６２ 和 Ｓｅｒ４００ 为 Ａｌｚｈｅｉｍ ｅｒ 病（ Ａ Ｄ）τ蛋白的异常磷酸化位点根据上述磷酸化作用仅轻度抑制τ蛋白生物学活性，推测： Ａ Ｄ τ蛋白 Ｓｅｒ２６２ 和 Ｓｅｒ４００ 的磷酸化可能不是决定其生物功能的关键性位点，单纯 Ｇ Ｓ Ｋ３ 不能复制 Ａ Ｄ 样 τ蛋白的病理改变

Glycogen synthetase kinase 3(GSK 3)incorporated 1 7±0 4 mol 32 P to 1 mol of τ protein in 4 h at 30℃ After digestion of phosphorylated τ protein with trypsin, 32 P labeled τ peptides were purified by a sequential FeCl 3 affinity column and a C 18 reverse phase high performance liquid chromatography The results from a combined technique of high voltage electrophoresis,manual Edman degradation and auto gas phase amino acid sequence analysis demonstrated that the phosphorylation sites of τ protein by GSK 3 were Thr 181,Ser 184,Ser 262,Ser 356 and Ser 400 Among them,Ser 262 and Ser 400 were abnormal phosphorylation sites of τ protein found in Alzheimer disease As the above phosphorylation only slightly inhibited the biological activity of τ protein,it was concluded that phosphorylation of τ protein at Ser 262 and Ser 400 might not be critical to its biofunction,and that GSK 3 alone was not enough to reproduce Alzheimer pathology of τ protein