Pope W B, Lambert M P, Leypold B, Seupaul R, Sletten L, Krafft G, Klein W L
Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208.
Exp Neurol. 1994 Apr;126(2):185-94. doi: 10.1006/exnr.1994.1057.
A phosphorylated tau epitope specific for paired helical filaments in Alzheimer's disease is recognized by monoclonal antibody PHF-1. Healthy adult brains lack the PHF-1 epitope (PHF-1 tau), but it is transiently expressed by immature neurons during development. We have found that proliferating SH-SY5Y human neuroblastoma cells also express PHF-1 tau. Consistent with the recent finding that cell-cycle-dependent kinases can phosphorylate tau in vitro, flow cytometry showed that mitotic SH-SY5Y cells were up to 18-fold more PHF-1 immunoreactive than nonmitotic cells. On immunoblots, PHF-1 tau in mitotic and nonmitotic cells also was strikingly different. First, mitosis induced a prominent PHF-1 reactive band at 120 kDa, which likely accounted for the large increase in PHF-1 signal seen at mitosis. Although the size of the 120-kDa band is consistent with it being the high-molecular-weight form of tau, other antibodies to tau did not recognize it. Second, mitosis caused a hyperphosphorylation of the PHF-1 immunoreactive tau band normally seen at 50 kDa. In mitotic cells this band had an increased intensity and molecular weight. Alkaline phosphatase treatment abolished tau M(r) heterogeneity, verifying that the variations in mobility were due to phosphorylation. These data show that cell-cycle-dependent hyperphosphorylation of tau occurs in intact cells, and they support the hypothesis that aberrant activity of cell-cycle-dependent kinases may contribute to tau phosphorylation and PHF formation in Alzheimer's disease.
阿尔茨海默病中对双螺旋丝特异的磷酸化tau表位可被单克隆抗体PHF-1识别。健康成人大脑缺乏PHF-1表位（PHF-1 tau），但在发育过程中未成熟神经元会短暂表达。我们发现增殖的SH-SY5Y人神经母细胞瘤细胞也表达PHF-1 tau。与最近细胞周期依赖性激酶可在体外使tau磷酸化的发现一致，流式细胞术显示有丝分裂的SH-SY5Y细胞的PHF-1免疫反应性比非有丝分裂细胞高18倍。在免疫印迹上，有丝分裂和非有丝分裂细胞中的PHF-1 tau也显著不同。首先，有丝分裂诱导出一条120 kDa的明显的PHF-1反应带，这可能是有丝分裂时观察到的PHF-1信号大幅增加的原因。尽管120 kDa条带的大小与tau的高分子量形式一致，但其他tau抗体不能识别它。其次，有丝分裂导致通常在50 kDa处可见的PHF-1免疫反应性tau条带发生过度磷酸化。在有丝分裂细胞中，这条带的强度和分子量增加。碱性磷酸酶处理消除了tau分子量的异质性，证实迁移率的变化是由于磷酸化所致。这些数据表明tau的细胞周期依赖性过度磷酸化发生在完整细胞中，并且支持细胞周期依赖性激酶的异常活性可能导致阿尔茨海默病中tau磷酸化和PHF形成这一假说。
