Study of the expression and function of cyclin-dependent kinases in the nervous system

神经系统中细胞周期蛋白依赖性激酶的表达和功能研究

基本信息

批准号：
08680834
负责人：
INO Hidetoshi
金额：
$ 1.6万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680834/
关键词：
CYCLIN-DEPENDENT KINASE CDK5 PCNA NERVOUS SYSTEM NEURON GLIA IMMUNOHISTOCHEMISTRY CELL CYCLE サイクリンE CDK サイクリン

项目摘要

Cyclin-dependent kinase 5 (CDK5) is one of the cyclin-dependent kinases, and is expressed in mature neurons. CDK5 has been postulated to be a neurofilament or tau protein kinase, because it phosphorylates neurofilaments and tau protein in vitro. It has been reported that CDK5 was immunohistochemically detected only in axons of neurons. We here report the immunohistochemical study of CDK5 using two distinct antibodies, one recognizing the N-terminal of CDK5 and the other the C-terminal. Immunoreactivity of CDK5 was found not only in axons, but also intensively in nuclei, though not in nucleoli, of neurons in the mouse central and peripheral nervous systems. The nuclear CDK5 possibly has a physiological function distinct from the neurofilament or tau protein kinase.Proliferating cell nuclear antigen (PCNA), the DNA polymerase auxiliary factor, is required for DNA replication and repair ; thus, PCNA is abundantly expressed in proliferating cells. Interestingly, PCNA mRNA was also detected in adult mouse brain by northern blot analysis. Two monoclonal antibodies against PCNA were used for western blot analysis, and one of them (clone PC10) detected a band in mouse brain extract, but the other (clone 19F4) did not. Immunohisto-chemistry of PCNA was also performed using these antibodies, and immunostaining was found only with PC10 in nuclei of cells, neurons and glia, over a wide area of the adult mouse central and peripheral nervous systems. On the other hand, nuclei of cells in the subventricular zone (SVZ) were stained with both PC10 and 19F4. These data suggest that the PCNA-like protein in mouse brain except for the SVZ is not PCNA but a structurally related PCNA analogue. However, the possibility also exists that the wide-spread PC10-immunoreactivity is due to the modification of amino acid residues of PCNA,such as by the phosphorylation.

依赖细胞周期蛋白的激酶5（CDK5）是细胞周期蛋白依赖性激酶之一，在成熟的神经元中表达。 CDK5被认为是神经丝或tau蛋白激酶，因为它在体外磷酸化神经丝和tau蛋白。据报道，CDK5仅在神经元的轴突中被免疫组织化学检测到。在这里，我们使用两种不同的抗体对CDK5进行了免疫组织化学研究，一种识别CDK5的N末端，另一种识别C端。 CDK5的免疫反应性不仅在轴突中，而且在小鼠中心和周围神经系统中的神经元中的核中，虽然不是在核中发现的。核CDK5可能具有与神经丝或Tau蛋白激酶不同的生理功能。增殖细胞核抗原（PCNA）（DNA聚合酶辅助因子）是DNA复制和修复所必需的。因此，PCNA在增殖细胞中大量表达。有趣的是，还通过Northern印迹分析在成年小鼠脑中也检测到PCNA mRNA。使用两种针对PCNA的单克隆抗体进行蛋白质印迹分析，其中一种（克隆PC10）检测到小鼠脑提取物中的条带，但另一种（克隆19f4）没有。还使用这些抗体进行了PCNA的免疫史学化学，并且在细胞，神经元和神经胶质细胞核中仅在成年小鼠中央和周围神经系统的广泛区域中发现免疫染色。另一方面，对室内区域（SVZ）中细胞的核被PC10和19F4染色。这些数据表明，除SVZ外，小鼠脑中的PCNA样蛋白不是PCNA，而是与结构相关的PCNA类似物。然而，可能性也存在广泛的PC10-免疫反应性是由于PCNA的氨基酸残基的修饰，例如通过磷酸化。