Quantitative and combinatory determination of in situ phosphorylation of tau and its FTDP-17 mutants.
Quantitative and combinatory determination of in situ phosphorylation of tau and its FTDP-17 mutants.
复制标题
DOI：
10.1038/srep33479
复制
发表时间：
2016-09-19
影响因子：
4.6
通讯作者：
Hisanaga S
中科院分区：
文献类型：
--
作者：
Kimura T;Hosokawa T;Taoka M;Tsutsumi K;Ando K;Ishiguro K;Hosokawa M;Hasegawa M;Hisanaga S
Tau在患有Tau蛋白病的患者的脑中过度磷酸化，所述Tau蛋白病例如阿尔茨海默病和与染色体17（FTDP-17）相关的额颞叶痴呆和帕金森综合征。然而，无论是过度磷酸化的机制，也不知道其对发病机制的贡献。我们应用Phos-tag SDS-PAGE，磷酸化亲和电泳，在体外由Cdk 5，在培养的细胞和小鼠脑中的tau磷酸化的分析。在此，我们发现Cdk 5-p25在体外依次在Ser 404、Ser 235、Thr 205和Ser 202处磷酸化tau。相反，在培养的细胞中，Ser 404被Cdk 5-p35优先磷酸化，而Thr 205不被磷酸化。Ser 202和Ser 235被内源性激酶磷酸化。Tau在COS-7细胞中表现出约12种磷酸化同种型，在Thr 181、Ser 202、Thr 231、Ser 235和Ser 404处具有不同的磷酸化组合。这些磷酸化位点类似于小鼠脑中的tau磷酸化。FTDP-17 tau在C-末端区域具有突变，具有不同的带型，表明不同的磷酸化模式。特别是，很明显R406 W突变导致Ser 404磷酸化的丧失。这些结果证明了Phos-标签技术在定量分析tau的位点特异性体内磷酸化中的有用性，并提供了关于tau的原位组合磷酸化的详细信息。
Tau is hyperphosphorylated in the brains of patients with tauopathies, such as Alzheimer’s disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). However, neither the mechanism of hyperphosphorylation nor its contribution to pathogenesis is known. We applied Phos-tag SDS-PAGE, a phosphoaffinity electrophoresis, to the analysis of tau phosphorylation in vitro by Cdk5, in cultured cells and in mouse brain. Here, we found that Cdk5-p25 phosphorylated tau in vitro at Ser404, Ser235, Thr205 and Ser202 in this order. In contrast in cultured cells, Ser404 was preferentially phosphorylated by Cdk5-p35, whereas Thr205 was not phosphorylated. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau exhibited ~12 phosphorylation isotypes in COS-7 cells with different combinations of phosphorylation at Thr181, Ser202, Thr231, Ser235 and Ser404. These phosphorylation sites were similar to tau phosphorylated in mouse brains. FTDP-17 tau with a mutation in the C-terminal region had different banding patterns, indicating a different phosphorylation pattern. In particular, it was clear that the R406W mutation causes loss of Ser404 phosphorylation. These results demonstrate the usefulness of the Phos-tag technique in the quantitative analysis of site-specific in vivo phosphorylation of tau and provide detailed information on in situ combinatory phosphorylation of tau.
登录
查看更多内容
DOI：
10.1083/jcb.201007161
发表时间：
2011-02-21
期刊：
The Journal of cell biology
影响因子：
--
作者：
Gauthier-Kemper A;Weissmann C;Golovyashkina N;Sebö-Lemke Z;Drewes G;Gerke V;Heinisch JJ;Brandt R
通讯作者：
Brandt R
影响因子：
6
作者：
DeTure, M;Ko, LW;Yen, SH
通讯作者：
Yen, SH
影响因子：
14.5
作者：
Bird, TD;Nochlin, D;Schellenberg, GD
通讯作者：
Schellenberg, GD
影响因子：
7
作者：
Hosokawa, Tomohisa;Saito, Taro;Hisanaga, Shin-ichi
通讯作者：
Hisanaga, Shin-ichi
影响因子：
3.5
作者：
Hasegawa, M;Smith, MJ;Goedert, M
通讯作者：
Goedert, M