Ono T, Yamamoto H, Tashima K, Nakashima H, Okumura E, Yamada K, Hisanaga S, Kishimoto T, Miyakawa T, Miyamoto E
Department of Pharmacology, Kumamoto University School of Medicine, Japan.
Neurochem Int. 1995 Mar;26(3):205-15. doi: 10.1016/0197-0186(94)00135-h.
The abnormally phosphorylated forms of tau factor are major constituents of neurofibrillary tangles in Alzheimer's disease brain. In order to investigate protein phosphatases which are related to dephosphorylation of abnormal phosphorylation sites, we examined the dephosphorylation of tau factor phosphorylated by three proline-directed type protein kinases. Tau factor phosphorylated by cdc2 kinase and tau protein kinase II was dephosphorylated by the holoenzyme of protein phosphatase 2A and calcineurin, while either the catalytic subunit of protein phosphatase 2A or protein phosphatase 2C could not catalyze the dephosphorylation. From the kinetic analysis, we concluded that tau factors phosphorylated by the protein kinases serve as good substrates for protein phosphatase 2A and calcineurin. On the other hand, tau factor phosphorylated by glycogen synthase kinase 3 alpha was dephosphorylated by the catalytic subunit of protein phosphatases 2A as well as the holoenzyme of protein phosphatase 2A and calcineurin. It has been reported that serines 199, 202 and 396 according to the numbering of the longest human tau isoform are among the major abnormal phosphorylation sites of tau factor. We synthesized two phosphopeptides which contained phosphoserines 199 and 202 or phosphoserine 396 and prepared the polyclonal antibodies specific for the phosphopeptides. Using these antibodies, we confirmed that the holoenzyme of protein phosphatase 2A and calcineurin could dephosphorylate phosphoserines 199, 202 and 396 in tau factor. The catalytic subunit of protein phosphatase 2A could dephosphorylate phosphoserine 396 but not phosphoserines 199 and 202. Neurofibrillary tangles in Alzheimer's disease brain were immunostained with both antibodies but the normal neurons in the normal aged brains were not. The results suggest that protein phosphatase 2A and calcineurin can be involved in the dephosphorylation of abnormal phosphorylation sites in tau factor and that the dephosphorylation of phosphoserine 396 is differently regulated from phosphoserines 199 and 202.
异常磷酸化形式的tau因子是阿尔茨海默病大脑中神经原纤维缠结的主要成分。为了研究与异常磷酸化位点去磷酸化相关的蛋白磷酸酶，我们检测了由三种脯氨酸导向型蛋白激酶磷酸化的tau因子的去磷酸化情况。由细胞周期蛋白依赖性激酶2（cdc2激酶）和tau蛋白激酶II磷酸化的tau因子可被蛋白磷酸酶2A全酶和钙调神经磷酸酶去磷酸化，而蛋白磷酸酶2A的催化亚基或蛋白磷酸酶2C均不能催化这种去磷酸化反应。通过动力学分析，我们得出结论，被蛋白激酶磷酸化的tau因子是蛋白磷酸酶2A和钙调神经磷酸酶的良好底物。另一方面，由糖原合酶激酶3α磷酸化的tau因子可被蛋白磷酸酶2A的催化亚基以及蛋白磷酸酶2A全酶和钙调神经磷酸酶去磷酸化。据报道，按照最长的人类tau异构体编号，丝氨酸199、202和396是tau因子主要的异常磷酸化位点。我们合成了两种分别包含磷酸化丝氨酸199和202或磷酸化丝氨酸396的磷酸肽，并制备了针对这些磷酸肽的多克隆抗体。使用这些抗体，我们证实蛋白磷酸酶2A全酶和钙调神经磷酸酶可使tau因子中的磷酸化丝氨酸199、202和396去磷酸化。蛋白磷酸酶2A的催化亚基可使磷酸化丝氨酸396去磷酸化，但不能使磷酸化丝氨酸199和202去磷酸化。阿尔茨海默病大脑中的神经原纤维缠结用这两种抗体进行免疫染色均呈阳性，但正常老年大脑中的正常神经元则没有。结果表明，蛋白磷酸酶2A和钙调神经磷酸酶可能参与tau因子异常磷酸化位点的去磷酸化过程，并且磷酸化丝氨酸396的去磷酸化调控方式与磷酸化丝氨酸199和202不同。
