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

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，包括帕金森病（PD）、路易体痴呆（DLB）和多系统萎缩。在这些所谓的“α-突触核蛋白病”中，α-突触核蛋白以高度磷酸化的形式沉积在羧基末端区域的Ser 129处。在这项研究中，我们试图确定一个蛋白激酶，磷酸化丝氨酸129的α-突触核蛋白在大脑中，使用单克隆抗体P-Ser 129特异性识别磷酸化丝氨酸129的α-突触核蛋白。将重组人α-突触核蛋白与6周龄大鼠脑提取物在ATP存在下孵育，并通过磷酸化α-突触核蛋白与P-Ser 129抗体的免疫印迹检测蛋白激酶活性。用硫酸铵分级沉淀和阴离子交换柱层析法部分纯化蛋白激酶活性， ...更多信息 通过免疫印迹和一些抑制剂表征。蛋白激酶活性用33%硫酸铵沉淀，并在Q-Sepharose柱层析上用0.4-0.5M NaCl洗脱。用抗酪蛋白激酶2（CK 2）抗体对这些组分进行免疫印迹分析，结果显示通过这些步骤富集了CK 2催化亚基。在部分纯化的馏分和粗脑提取物中的蛋白激酶活性显着抑制肝素，一个众所周知的CK 2的特异性抑制剂。这些发现有力地表明，CK 2可能是脑中使α-突触核蛋白的Ser 129磷酸化的主要蛋白激酶。在DLB脑中，异常CK 2免疫反应性也观察到在肌氨酸不溶性部分，其中高度磷酸化的α-突触核蛋白富集，而没有这样的免疫反应性检测到在对照组的脑部分。这些结果表明，CK 2可能参与α-突触核蛋白病中α-突触核蛋白的异常磷酸化。少

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