Global analysis of phosphorylation of tau by the checkpoint kinases Chk1 and Chk2 in vitro.
Global analysis of phosphorylation of tau by the checkpoint kinases Chk1 and Chk2 in vitro.
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DOI：
10.1021/pr400008f
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发表时间：
2013-06-07
影响因子：
4.4
通讯作者：
Ando K
中科院分区：
文献类型：
--
作者：
Mendoza J;Sekiya M;Taniguchi T;Iijima KM;Wang R;Ando K
微管相关蛋白 tau 的过度磷酸化被认为与阿尔茨海默病 (AD) 的发病机制有关。我们之前表明 DNA 损伤激活的细胞周期检查点激酶 Chk1 和 Chk2 在 AD 相关位点磷酸化 tau 并增强 tau 毒性，表明这些激酶在 AD 中的潜在作用。本研究的目的是系统地鉴定 tau 蛋白中的哪些位点直接被 Chk1 和 Chk2 磷酸化。使用体外被 Chk1 和 Chk2 磷酸化的重组人 tau，我们首先使用磷酸 tau 特异性抗体通过蛋白质印迹分析 AD 相关位点的 tau 磷酸化。其次，为了全面鉴定 tau 蛋白的磷酸化位点，采用了液相色谱-串联质谱 (LC-MS/MS)。这些系统分析总共确定了 27 个 Ser/Thr 残基作为 Chk1 或 Chk2 靶位点。它们都不是脯氨酸导向的激酶靶标。其中许多位点位于微管结合域和 C 端域内，其磷酸化已被证明可减少 tau 与微管的结合和/或与 tau 毒性有关。在这 27 个位点中，有 13 个位点已被确定在 AD 大脑中被磷酸化。由于 DNA 损伤在患病大脑中累积，Chk1 和 Chk2 可能参与 AD 发病机制中的 tau 磷酸化和毒性。
Hyperphosphorylation of microtubule-associated protein tau is thought to contribute to Alzheimer’s disease (AD) pathogenesis. We previously showed that DNA damage-activated cell cycle checkpoint kinases Chk1 and Chk2 phosphorylate tau at an AD-related site and enhance tau toxicity, suggesting potential roles of these kinases in AD. The purpose of this study is to systematically identify which sites in tau are directly phosphorylated by Chk1 and Chk2. Using recombinant human tau phosphorylated by Chk1 and Chk2 in vitro, we firstly analyzed tau phosphorylation at the AD-related sites by Western blot with phospho-tau-specific antibodies. Secondly, to globally identify phosphorylated sites in tau, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed. These systematic analyses identified a total of 27 Ser/Thr residues as Chk1- or Chk2- target sites. None of them were proline-directed kinase targets. Many of these sites are located within the microtubule-binding domain and C-terminal domain, whose phosphorylation has been shown to reduce tau binding to microtubules and/or has been implicated in tau toxicity. Among these 27 sites, 13 sites have been identified to be phosphorylated in AD brains. Since DNA damage is accumulated in diseased brains, Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in AD pathogenesis.
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影响因子：
12.3
作者：
Blasius M;Forment JV;Thakkar N;Wagner SA;Choudhary C;Jackson SP
通讯作者：
Jackson SP
影响因子：
4.8
作者：
DREWES, G;TRINCZEK, B;MANDELKOW, E
通讯作者：
MANDELKOW, E
影响因子：
12.8
作者：
Kim, Myoung-Ae;Kim, Hyun-Ju;Yun, Jeanho
通讯作者：
Yun, Jeanho
影响因子：
3.5
作者：
Iijima-Ando, Kanae;Zhao, LiJuan;Iijima, Koichi
通讯作者：
Iijima, Koichi
影响因子：
3.5
作者：
BAUMANN, K;MANDELKOW, EM;MANDELKOW, E
通讯作者：
MANDELKOW, E