OVEREXPRESSED TAU-PROTEIN IN CULTURED-CELLS IS PHOSPHORYLATED WITHOUT FORMATION OF PHF - IMPLICATION OF PHOSPHOPROTEIN PHOSPHATASE INVOLVEMENT
OVEREXPRESSED TAU-PROTEIN IN CULTURED-CELLS IS PHOSPHORYLATED WITHOUT FORMATION OF PHF - IMPLICATION OF PHOSPHOPROTEIN PHOSPHATASE INVOLVEMENT
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DOI：
10.1016/0169-328x(95)00111-5
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发表时间：
1995-12-01
期刊：
影响因子：
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通讯作者：
SAITOH, T
中科院分区：
文献类型：
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作者：
BAUM, L;SEGER, R;SAITOH, T
阿尔茨海默病(AD)脑内受影响区域的锥体神经元含有神经纤维缠结(NFT)，即主要由磷酸化微管相关蛋白tau组成的成对螺旋细丝(PHF)的聚集体。为了探索tau磷酸化在tau聚集成PHF中的作用，我们构建了哺乳动物细胞培养体系，产生高水平的细胞内磷酸化tau。瞬时转染COS-1成纤维样细胞，同时表达tau、MAPK和MAPKK，或交替表达tau和糖原合成酶3(GSK3)。B103神经元样细胞(含MAPK，但不含tau和GSK3)稳定表达tau或tau和GSK3。在这两个系统中，GSK3转基因细胞都含有tau(AT8/M)(由AT8染色和tau(PHF)样迁移率定义)，但MAPK转基因细胞需要磷酸酶抑制剂，如冈田酸(OKA)或花蕾蛋白(CAL)，才能产生tau(AT8/M)。在体外，相同浓度的CAL和OKA抑制磷酸酶1和2A(PP1和PP2A)，但需要100-1000倍的OKA来抑制PP1。在MAPK转基因细胞中诱导AT8位点的tau磷酸化需要比CAL多2-10倍的OKA，这表明PPI和PP2A都有助于阻断这种磷酸化。虽然在COS-1细胞中tau(AT8/M)水平达到细胞总蛋白的2-8%，但颗粒与上清液tau水平的比率并没有增加，也没有观察到缠结；可能需要翻译后修饰或共聚集蛋白来诱导PHF。
Pyramidal neurons in affected regions of Alzheimer's disease (AD) brain contain neurofibrillary tangles (NFT), aggregates of paired helical filaments (PHF) composed mainly of phosphorylated microtubule-associated protein tau. To explore the role of tau phosphorylation in the aggregation of tau into PHF, we constructed mammalian cell culture systems producing high levels of intracellular phosphorylated tau. COS-1 fibroblast-like cells were transiently transfected to simultaneously express tau, MAP kinase (MAPK), and MAP kinase kinase (MAPKK), or alternatively to express tau and glycogen synthase kinase 3 (GSK3). B103 neuron-like cells (which contain MAPK but little tau or GSK3) were stably transfected to express tau or tau and GSK3. In both systems, GSK3-transfected cells contained tau(AT8/M) (defined by AT8 staining and tau(PHF)-like mobility), but MAPK-transfected cells required phosphatase inhibitors, such as okadaic acid (OKA) or calyculin (CAL), to produce tau(AT8/M). In vitro, the same concentrations of CAL and OKA inhibit phosphatases 1 and 2A (PP1 and PP2A), except that 100-1000 times as much OKA is needed to inhibit PP1. Inducing tau phosphorylation at the AT8 site in MAPK-transfected cells required 2-10 times more OKA than CAL, suggesting both PPI and PP2A helped block the phosphorylation. Though levels of tau(AT8/M) reached 2-8% of total cellular proteins in COS-1 cells, the ratio of particulate to supernatant tau levels did not increase, and no tangles were observed; perhaps post-translational modifications or co-aggregating proteins are needed to induce PHF.