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α-核蛋白制成的丝状夹杂物是一组神经退行性疾病的定义神经病理学特征，包括帕金森氏病（PD），痴呆症，具有Lewy身体（DLB）（DLB）和多个系统萎缩。在这些所谓的“α-突触核疾病”中，A-突触核蛋白在羧基末端区域的Ser129中以高磷酸化形式沉积。在这项研究中，我们尝试使用单克隆抗体P-SER129鉴定蛋白激酶在脑中磷酸化的α-突触核蛋白磷酸化，该抗体p-ser129特异性地识别了α-核蛋白的磷酸化Ser129。在ATP存在下，将重组人α-突触核蛋白与6周大鼠的脑提取物一起孵育，并通过用P-SER129抗体对磷酸化的α-核蛋白的免疫印迹检测到蛋白激酶活性。蛋白激酶活性通过硫酸铵分馏和阴离子交换柱色谱法部分纯化，CHA……更受免疫印迹和一些抑制剂进行了修饰。将蛋白激酶活性用33％硫酸铵沉淀，并在Q-塞语柱色谱法上用0.4-0.5m NaCl洗脱。用抗casein激酶2（CK2）抗体对这些馏分进行免疫印迹分析表明，通过这些步骤，CK2催化亚基富含。肝素（一种众所周知的CK2特异性抑制剂）极大地抑制了部分纯化的部分和粗脑提取物中的蛋白激酶活性。这些发现强烈表明，CK2可能是大脑中的主要蛋白激酶，可磷酸化α-核蛋白的SERL29。在DLB的大脑中，在sarkosyl-nos-n-saint中也观察到异常的CK2免疫反应性，其中富集了热磷酸化的α-突触核蛋白，而在对照组中的分数中未检测到这种免疫反应性。这些结果表明，CK2可能参与α-核蛋白在α-核核病中α-核蛋白的异常磷酸化。较少的

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
• 期刊: Acta Neuropathol. (Berl) 110
• 作者: Motoi;Y.;Hasegawa;M.;Yasuda;M.;Mizuno;Y.;Mori;H
• 通讯作者: H

Stress-and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD95 by activation of SAPK3/p38g and ERKI1/ERK2.

通过激活 SAPK3/p38g 和 ERKI1/ERK2，应激和丝裂原诱导突触相关蛋白 SAP90/PSD95 磷酸化。

• 发表时间: 2004
• 期刊: Biochem J 380
• 作者: Sabio G;Hasegawa M;Goedert M;Garner C;Cuenda A et al.
• 通讯作者: Cuenda A et al.

Accumulation of phosphorylated alpha-synuclein in the brain and peripheral ganglia of patients with multiple system atrophy.

• 发表时间: 2004
• 期刊: Acta neuropathologica
• 影响因子: 12.7
• 作者: M. Nishie;F. Mori;Hideo Fujiwara;M. Hasegawa;M. Yoshimoto;T. Iwatsubo;H. Takahashi;K. Wakabayashi

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蛋白阳性细颗粒：关岛tau蛋白病中帕金森病-痴呆症所特有的新发现

• 发表时间: 2005
• 期刊: J.Neuropathol.Exp.Neurol 64
• 作者: Yamazaki;M.;Hasegawa;M.;Mori;O.;et al.
• 通讯作者: et al.

Inhibition of heparin-induced tau filament formation by phenothiazines, polvphenols and porphyrins.

吩噻嗪、多酚和卟啉抑制肝素诱导的 tau 丝形成。

• 发表时间: 2005
• 期刊: J Biol Chem 280
• 作者: Taniguchi S;Suzuki N;Masuda M;Hisanaga S;Iwatsubo T;Goedert M;Hasegawa M
• 通讯作者: Hasegawa M