Identification of protein kinase(s) involved in abnormal phosphorylation of alpha-synuclein

鉴定参与 α-突触核蛋白异常磷酸化的蛋白激酶

• 批准号: 15300119
• 负责人: HASEGAWA Masato
• 金额: $ 8.96万
• 依托单位: Tokyo Metropolitan Organization for Medical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15300119/
• 关键词: alpha-synuclein; phosphorylation; ubiquitination; filaments; casein kinase 2; Parkinson's disease; Lewy body; レヴィー小体; レヴィー小体型痴呆; 多系統萎縮症

Filamentous inclusions made of alpha-synuclein in nerve cells or glial cells are the defining neuropathological feature of a group of neurodegenerative diseases which include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. In these so-called "alpha-synucleinopathies", a-synuclein is deposited in a hyperphosphorylated form at Ser129 in the carboxyl-terminal region. In this study, we have attempted to identify a protein kinase that phosphorylates Ser129 of alpha-synuclein in brain, using a monoclonal antibody P-Ser129 which specifically recognizes phosphorylated Ser129 of alpha-synuclein. Recombinant human alpha-synuclein was incubated with brain extracts from 6-week rat in the presence of ATP, and the protein kinase activity was detected by immunoblotting of phosphorylated alpha-synuclein with the P-Ser129 antibody. The protein kinase activity was partially purified by ammonium sulfate fractionation and anion exchange column chromatography, and cha … More racterized by immunoblotting and some inhibitors. The protein kinase activity was precipitated with 33% ammonium sulfate and eluted with 0.4-0.5M NaCl on a Q-Sepharose column chromatography. Immunoblot analysis of these fractions with anti-casein kinase 2 (CK2) antibody revealed that CK2 catalytic subunit was enriched through these steps. The protein kinase activities in the partially purified fraction and crude brain extracts were dramatically inhibited by heparin, a well-known specific inhibitor of CK2. These findings strongly suggest that CK2 may be the major protein kinase in brain that phosphorylates Serl29 of alpha-synuclein. In DLB brains, aberrant CK2 immunoreactivities were also observed in the Sarkosyl-insoluble fraction where the hyperphosphorylated alpha-synuclein was enriched, whereas no such immunoreactivity was detected in the fraction from control brains. These results suggest that CK2 may be involved in the abnormal phosphorylation of alpha-synuclein in alpha-synucleinopathies. Less

神经细胞或神经胶质细胞中由α-突触核蛋白构成的丝状包涵体是一组神经退行性疾病的定义性神经病理学特征，这些神经退行性疾病包括帕金森病（PD）、路易体痴呆（DLB）和多系统萎缩症。 α-突触核蛋白病”，α-突触核蛋白以过度磷酸化的形式沉积在羧基末端区域的 Ser129 处。在这项研究中，我们尝试使用特异性识别 α-突触核蛋白磷酸化 Ser129 的单克隆抗体 P-Ser129 来鉴定脑中磷酸化 α-突触核蛋白 Ser129 的蛋白激酶。将重组人 α-突触核蛋白与来自 6 周大鼠的脑提取物一起孵育。 ATP 的存在，并通过磷酸化 α-突触核蛋白的免疫印迹检测蛋白激酶活性P-Ser129 抗体通过硫酸铵分级和阴离子交换柱层析进行部分纯化，并通过免疫印迹和一些抑制剂进行表征。蛋白激酶活性用 33% 硫酸铵沉淀并用 0.4-0.5 洗脱。使用抗酪蛋白对这些级分进行 Q-Sepharose 柱色谱免疫印迹分析。激酶 2 (CK2) 抗体显示，通过这些步骤，部分纯化的级分和粗脑提取物中的蛋白激酶活性被肝素（一种众所周知的 CK2 特异性抑制剂）显着抑制。 CK2 可能是大脑中磷酸化 α-突触核蛋白 Ser129 的主要蛋白激酶。在 DLB 大脑中，也观察到异常的 CK2 免疫反应性。肌酰基不溶性部分富含高度磷酸化的 α-突触核蛋白，而在对照大脑的部分中未检测到此类免疫反应性。这些结果表明 CK2 可能参与 α-突触核蛋白病中的 α-突触核蛋白的异常磷酸化。

项目成果

Four-repeat tau-positive Pick body-like inclusions are distinct from classic Pick bodies.

四重复 tau 阳性 Pick 体样内含物与经典的 Pick 体不同。

• 发表时间: 2005
• 作者: Motoi; Y.; Hasegawa; M.; Mizumo; Y.; Mori; H
• 通讯作者: H

Tau-positive fine granules in the cerebral white matter : a novel finding exclusive to parkinsonism-dementia complex of Guam among the tauopathies.

大脑白质中的 tau 阳性细颗粒：关岛 tau 病中帕金森病-痴呆症复合体独有的新发现。

• 发表时间: 2005
• 作者: Yamazaki M; Hasegawa M; Mori O; Murayama S; Tsuchiya K; Ikeda K; Chen K
• 通讯作者: Chen K

Phosphorylated alpha-synuclein in normal mouse brain.

正常小鼠大脑中磷酸化的α-突触核蛋白。

• 发表时间: 2004
• 作者: Hirai Y; Fujita SC; Iwatsubo T; Hasegawa M
• 通讯作者: Hasegawa M

Frontopemporal dementia with Pick-type histology associated with Q336R mutation in tau gene.

组织学类型为 Pick 型的额颞叶痴呆与 tau 基因 Q336R 突变相关。

• 发表时间: 2004
• 作者: Pickering

Identification of amino-terminal cleaved tau fragments that distinguish progressive supranuclear palsy from corticobasal degeneration.

鉴定氨基末端切割的 tau 片段，以区分进行性核上性麻痹和皮质基底节变性。

• 发表时间: 2004
• 作者: Arai T; Ikeda K; Akiyama H; Nonaka T; Hasegawa M et al.
• 通讯作者: Hasegawa M et al.