摘要
目的 探讨 Alzheimer病 (AD) τ蛋白异常磷酸化位点与其促微管组装及与微管结合活性抑制的关系。方法 采用超速离心和离子交换柱层析法分离制备重组 τ蛋白及 τ蛋白的体外磷酸化 ,以 Fe Cl3亲和柱分离纯化 32 Pτ肽 ,手工 Edman降解和氨基酸自动分析仪鉴定 32 Pτ肽磷酸氨基酸位点。结果 (1)酪蛋白激酶 - 1(CK- 1)、c AMP依赖性蛋白激酶 (PKA )和糖元合成酶激酶 - 3(GSK- 3)的磷酸化作用可不同程度地抑制τ蛋白的功能 ,CK - 1或 PKA的预处理可明显增强 GSK - 3对τ促微管组装活性的抑制 ,其中 PKA加 GSK- 3的抑制作用最强。 (2 )单纯GSK- 3磷酸化τ蛋白的位点为 :苏氨酸 (Thr) - 181,丝氨酸 (Ser) - 184,Ser- 2 6 2 ,Ser- 35 6和 Ser- 40 0 ,而 GSK- 3对PKA预处理τ蛋白的磷酸化位点为 :Ser- 195 ,Ser- 198,Ser- 199,Ser- 2 0 2 ,Ser- 2 35 ,Ser- 2 6 2 ,Ser- 35 6 ,Ser- 40 4,Thr-2 0 5和 Thr- 2 31。其中 Ser- 198,Ser- 199,Ser- 2 0 2 ,Thr- 2 0 5 ,Thr- 2 31,Ser- 2 35 ,Ser- 2 6 2 ,Ser- 40 0和 Ser- 40 4为 AD患者τ蛋白异常磷酸化位点。此外 ,Ser- 2 6 2磷酸化仅轻度抑制τ蛋白活性 ,而 Ser- 198,Ser- 199,Ser- 2 0 2 ,Thr- 2 31,Ser- 2 35 ,Ser- 40 0和 Ser- 40
Objective To explore the association between the abnormal phosphorylation sites found in Alzheimer disease(AD) τ and the inhibition of its biological activity. Methods Ultracentrifugation、chromatography、manual Edman degradation and autosequence techniques were used to prepare and phosphorylate human recombinant τ, isolate and purify 32 P τ peptides and determine phosphorylation sites. Results (1) Phosphorylation of τ by casein kinase1 (CK1), cyclic AMPdependent protein kinase (PKA) and glycogen synthetase kinase3 (GSK3) differentially inhibited its biological activity, and the inhibition of this activity of τ by GSK3 was significantly increased if τ was prephosphorylated by CK1 or PKA. The most potent inhibition was seen by a combined phosphorylation of τ with PKA and GSK3. (2) The treatment of τ by PKA and GSK3 combination induced phosphorylation of τ at Ser195,Ser198,Ser199,Ser202,Thr205, Thr231, Ser235, Ser262, Ser356, Ser404, whereas only Thr181,Ser184,Ser262,Ser356 and Ser400 were phosphorylated by GSK3 alone under the same conditions. Among the abovementioned phosphorylation sites, Ser198, Ser199,Ser202,Thr205,Thr231,Ser235, Ser262, Ser400 and Ser404 were seen in Alzheimer τ. The phosphorylation of Ser262 only slightly inhibited its biological activity, and Ser198,Ser199,Ser202,Thr231,Ser235, Ser400 and Ser404 also presented in fetal τ which was highly active, suggesting that Thr205 was the unique site which both caused the potent inhibition of biological activity and specifically presented in AD abnormally phosphorylated τ. Conclusions Phosphorylation of Thr205 might play a key role in τ pathology in AD.
出处
《中国医学科学院学报》
CSCD
北大核心
2000年第2期120-123,共4页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金!(39770175)资助
