Phosphorylation-dependent Monoclonal Tau Antibodies Do Not Reliably Report Phosphorylation by Extracellular Signal-regulated Kinase 2 at Specific Sites*
Phosphorylation-dependent Monoclonal Tau Antibodies Do Not Reliably Report Phosphorylation by Extracellular Signal-regulated Kinase 2 at Specific Sites*
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DOI：
10.1074/jbc.272.7.4509
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发表时间：
1997-02
期刊：
影响因子：
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通讯作者：
H. Roder;R. Fracasso;F. Hoffman;James A. Witowsky;G. Davis;C. Pellegrino
中科院分区：
文献类型：
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作者：
H. Roder;R. Fracasso;F. Hoffman;James A. Witowsky;G. Davis;C. Pellegrino
tau 蛋白（阿尔茨海默病中过度磷酸化的微管相关蛋白）的磷酸化分析通常使用磷酸化敏感的单克隆抗体进行，这些抗体被认为可以报告一或两个特定磷酸化（同源位点）的存在或不存在。使用几种这样的抗体，我们发现特定位点的磷酸化（通过二维磷酸肽图谱监测）与这些位点的抗体识别之间存在更加复杂的关系。脑细胞外信号调节激酶 2 同种型 PK40 在多个阶段对 tau 蛋白进行多重磷酸化，表明同源位点的磷酸化有时对于诱导抗体反应性的变化是必要的（但不充分），并且在某些情况下，在其他位点多重磷酸化的背景下甚至是不必要的。没有发现任何单个磷酸化位点导致与磷酸化相关的任何水平的凝胶迁移率变化。 Tau 在大多数位点的亚化学计量磷酸化时获得了最大的凝胶迁移率延迟和最终的免疫化学特征。这表明许多替代的磷酸化模式可以在十二烷基硫酸钠凝胶电泳上产生相同的构象和免疫化学呈现。尽管 PK40erk2 更喜欢某些磷酸化位点，最显着的是 Ser235，其次是 Ser199 或 Ser202 和 Thr205，但多个 Ser/Thr-Pro 位点的磷酸化不是高度连续的。 Ser396 是最不优选的位点之一，似乎需要在 Ser404 处预先磷酸化。
Analysis of phosphorylation of tau, the microtubule-associated proteins hyperphosphorylated in Alzheimer's disease, is often performed using phosphorylation-sensitive monoclonal antibodies thought to report the presence or absence of one or two specific phosphorylations (cognate sites). Using several such antibodies we found a much more complicated relationship between phosphorylation at specific sites, as monitored by two-dimensional phosphopeptide mapping, and antibody recognition of these sites. Multiple phosphorylation of tau in several stages by the brain extracellular signal-regulated kinase 2 isoform PK40 suggested that phosphorylation at cognate sites is sometimes necessary (but not sufficient) to induce a change of antibody reactivity and in some cases is not even necessary in the background of multiple phosphorylation at other sites. No single phosphorylation site was found to be responsible for any level of gel mobility shift associated with phosphorylation. Tau acquired its maximal gel mobility retardation and final immunochemical profile at substoichiometric phosphorylation of most sites. This suggests that many alternate phosphorylation patterns can produce the same conformational and immunochemical presentation on sodium dodecyl sulfate-gel electrophoresis. Although PK40erk2 prefers some phosphorylation sites, most notably Ser235, followed by Ser199 or Ser202 and Thr205, the phosphorylation of multiple Ser/Thr-Pro sites is not highly sequential. Ser396 is one of the least preferred sites and seems to require prior phosphorylation at Ser404.