Garver T D, Lehman R A, Billingsley M L
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033, USA.
J Neurosci Res. 1996 Apr 1;44(1):12-20. doi: 10.1002/(SICI)1097-4547(19960401)44:1<12::AID-JNR2>3.0.CO;2-L.
Phosphorylation of the microtubule-associated protein tau regulates its binding to microtubules; highly phosphorylated tau is also a prime component of paired helical filaments (PHFs) of Alzheimer's disease (AD). Tau from freshly biopsied human, monkey, and rat brain share similar electrophoretic mobility patterns and overlapping phosphorylated epitopes when compared to AD tau isolated from AD brain. We compared the microtubule reassembly competence of fresh isolates of phosphorylated tau to that of maximally dephosphorylated tau and tau from AD brain. A rapid procedure was developed which permitted the enrichment of phosphorylated and dephosphorylated tau from human biopsies in the absence of protein kinase and phosphatase activity. Microtubule assembly assays, using a spectrophotometric measure and purified bovine brain tubulin, were used to correlate assembly competence with states of tau electrophoretic mobility. Maximally dephosphorylated human biopsy-derived tau and monkey tau were assembly competent; tau from AD brain was virtually unable to direct microtubule assembly. Unmodified, biopsy-derived tau from non-AD brain was intermediate in assembly competence relative to AD tau and dephosphorylated tau. Several lines of evidence were used to correlate phosphorylation states of tau with microtubule assembly. First, in vitro dephosphorylation of human biopsy-derived tau with either PP2A or PP2B alone or in combination led to increasing assembly competence as the electrophoretic mobility of tau increased. Second, addition of the protein phosphatase inhibitor okadaic acid (10 microM) to brain-slice preparations slowed electrophoretic mobility of tau and decreased binding competence. We suggest that tau derived from freshly-biopsied brain exists in a range of phosphorylated states, and that dephosphorylation by PP2A and/or PP2B increases microtubule assembly competence.
微管相关蛋白tau的磷酸化作用调节其与微管的结合；高度磷酸化的tau蛋白也是阿尔茨海默病（AD）成对螺旋丝（PHF）的主要成分。与从AD脑分离出的AD tau相比，来自新鲜活检的人、猴和大鼠脑的tau具有相似的电泳迁移模式和重叠的磷酸化表位。我们将磷酸化tau新鲜分离物的微管重装配能力与最大程度去磷酸化的tau以及AD脑tau的微管重装配能力进行了比较。开发了一种快速方法，可在不存在蛋白激酶和磷酸酶活性的情况下，从人活检组织中富集磷酸化和去磷酸化的tau。使用分光光度法测量和纯化的牛脑微管蛋白进行微管装配测定，以将装配能力与tau电泳迁移状态相关联。最大程度去磷酸化的人活检来源的tau和猴tau具有装配能力；AD脑的tau实际上无法指导微管装配。来自非AD脑的未修饰活检来源的tau在装配能力上相对于AD tau和去磷酸化tau处于中间水平。使用了几条证据将tau的磷酸化状态与微管装配相关联。首先，单独或联合使用PP2A或PP2B对人活检来源的tau进行体外去磷酸化，随着tau电泳迁移率的增加，装配能力增强。其次，向脑片制剂中添加蛋白磷酸酶抑制剂冈田酸（10 microM）会减缓tau的电泳迁移率并降低结合能力。我们认为，新鲜活检脑来源的tau以一系列磷酸化状态存在，并且PP2A和/或PP2B的去磷酸化会增加微管装配能力。
