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

神经细胞或胶质细胞中由α -突触核蛋白组成的丝状包涵体是帕金森病（PD）、路易体痴呆（DLB）和多系统萎缩等一系列神经退行性疾病的决定性神经病理学特征。在这些所谓的“α -突触核蛋白病”中，a-突触核蛋白以羧基末端Ser129的过度磷酸化形式沉积。在本研究中，我们利用特异性识别α -突触核蛋白磷酸化Ser129的单克隆抗体P-Ser129，试图鉴定一种磷酸化α -突触核蛋白Ser129的蛋白激酶。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，用P-Ser129抗体免疫印迹法检测磷酸化α -突触核蛋白的蛋白激酶活性。通过硫酸铵分离和阴离子交换柱层析纯化了部分蛋白激酶活性，并通过免疫印迹和一些抑制剂对其活性进行了进一步鉴定。蛋白激酶活性用33%硫酸铵沉淀，用Q-Sepharose柱层析，用0.4 ~ 0.5 m NaCl洗脱。用抗酪蛋白激酶2 （CK2）抗体对这些组分进行免疫印迹分析，发现通过这些步骤富集了CK2催化亚基。部分纯化部分和粗脑提取物中的蛋白激酶活性被肝素显著抑制，肝素是一种众所周知的CK2特异性抑制剂。这些发现强烈提示CK2可能是脑内磷酸化α -突触核蛋白Serl29的主要蛋白激酶。在DLB大脑中，在萨科齐不溶性部分中也观察到异常的CK2免疫反应，其中富含过度磷酸化的α -突触核蛋白，而在对照大脑的部分中没有检测到这种免疫反应。这些结果提示CK2可能参与了α -突触核蛋白在α -突触核蛋白病中的异常磷酸化。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