国家自然科学基金(30572076),江苏省自然科学基金(BK2004047)和江苏省六大人才高峰资助项目.
This work was supported by grants from The National Natural Science Foundation of China (30572076), The Natural Science Foundation of Jiangsu Province (BK2004047)and Six Talented-man Peak of Jiangsu Province.
tau蛋白是中枢神经系统中重要的微管相关蛋白,其功能受磷酸化调节. 异常过度磷酸化的tau蛋白是阿尔茨海默病患者脑中神经纤维缠结的主要组成部分. 糖元合成酶激酶3β (glycogen synthase kinase-3β, GSK-3β) 是重要的tau蛋白激酶之一,它虽可催化tau蛋白多个位点的磷酸化,但对不同位点,其催化效率不同. 通过位点特异性、磷酸化依赖的tau蛋白抗体,用免疫印迹技术,检测GSK-3β对tau蛋白位点特异性的磷酸化作用及动力学. 用双倒数作图,计算GSK-3β催化tau磷酸化以及各个位点磷酸化的Km值,并结合培养细胞中的实验,研究GSK-3β对tau蛋白磷酸化作用的位点特异性. 结果显示,GSK-3β催化tau蛋白多个位点的磷酸化,其中包括Thr181、Ser199、Ser202、Thr205、Thr212、Thr217、Thr231、Ser396和Ser404,对不同的位点磷酸化作用,其Km值不同,GSK-3β对Ser396的Km值最低,即对Ser396位点的亲和性最高,催化其磷酸化的能力最强. 在培养的细胞中,也显示了GSK-3β的表达引起Ser396位点的磷酸化最明显.
Tau is a microtubule associated protein in neuron. The biological functions of tau are to stimulate microtubule assembly and to stabilize microtubule structure. These functions are regulated by its phosphorylation status. Abnormally phosphorylated tau is a major component of neurofibrillary tangles. As one of the major tau kinases, glycogen synthase kinase-3β (GSK-3β) can phosphorylate tau at several sites. Site-specific phosphorylation of tau by GSK-3β and the kinetics of GSK-3β on tau phosphorylation was investigated. Tau441 was phosphorylated by recombinant GSK-3β in vitro, and the site-specific phosphorylation was detected by Western blots using site-specific and phosphorylation-dependent tau antibodies. The kinetics of phosphorylation by GSK-3β to total tau and to individual site was studied by incubating GSK-3β with various concentration of tau at 30℃ for 10 min. These velocities of phosphorylation reaction were determined at various concentrations of tau by measuring 32P incorporation to tau and phosphorylation level at individual site detected with immuno-dot blots using site-specific and phosphorylation dependent anti-tau antibodies, respectively. The Km values of GSK-3β toward total tau and toward individual site were calculated by using Lineweaver-Burk double-reciprocal method. The site-specific phosphorylation of tau by GSK-3β was further confirmed in cultured CHO cells by co-transfection of tau441 with GSK-3β. It was found that GSK-3β phosphorylated tau at several sites, including Thr181, Ser199, Ser202, Thr205, Thr212, Thr 217, Thr231, Ser396, and Ser404. The Km value of GSK-3β toward total tau was 42 μmol/L, but the Km value of GSK-3β to every site was different. Among all the phosphorylation sites detected here, Ser396 phosphorylation catalyzed by GSK-3β showed the lowest Km, only 16 μmol/L. In cultured CHO cells, GSK-3β over-expression also induced tau phosphorylation at Ser396 the most dramatically. These results suggested that GSK-3β catalyzed tau phosphorylation at several sites with different efficiency, and Ser396 was the most efficient site for phosphorylation by GSK-3β .
刘飞,施建华,丁绍红,尹晓敏.糖元合成酶激酶3β对微管相关蛋白tau的磷酸化作用[J].生物化学与生物物理进展,2007,34(9):945-951
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