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Activation or inactivation mechanism of brain cdc2-like kinase, cdk5

脑cdc2样激酶cdk5的激活或失活机制

基本信息

批准号：
09480191
负责人：
HISANAGA Shin-ichi
金额：
$ 8.45万
依托单位：
TOKYO METROPOLITAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Cyclin-dependent kinases are key factors in progression of cell cycle in eukaryotic cells. CDK5 is an unique CDK whosc kinase activity is detected only in post-mitotic neurons. Several lines of evidence suggest that CDK5 involves in neuronal migration or positioning during development via phosphorylation of axonal cytoskeletons such as microtubule-associated protein tau and neurofilaments. However, it is not known how its kinase activity is regulated. We investigated here the activation or inactivation mechanism of CDK5.1. We studied the activation mechanism of CDK5 in primary cultured neurons using the phosphorylation of neurofilament-H subunit as a marker. CDK5 was activated at 4 to 5 days after plating when phosphorylated form of neurofilament-H subunit appeared and synapse formation occurred. Addition of BDNF into culture medium induced activation of CDK5. These results suggest that activation of CDK5 was induced by increased secretion of BDNF after synapse formation.2. CDK5 is activated by binding to activating subunit p35 which is expressed in neurons exclusively. p35 is a short life protein degraded in a few hours. CDK5 decreased its activity along with degradation of p35. p35 was degraded by proteasome as well as cyclins in proliferating cells. Phosphorylation was suggested to be a signal for degradation.3. p35 is proteolysed to p25 during purification of CDK5 from bovine or porcine brains. Proteases involving in this limited proteolysis was shown to be calpain. Addition of CaィイD12+ィエD1 to porcine brain extract induced proteolysis of p35 to p25. This proteolysis was suppressed by calpain inhibitor. The proteolysis changed the solubility of CDK5. P35/CDK5 precipitated by brief centrifugation became to be soluble after proteolytic conversion to p25. Limited proteolysis of p35 to p25 was enhanced at the time of neuronal cell death.

细胞周期蛋白依赖性激酶是真核细胞中细胞周期进程的关键因子。CDK 5是一种独特的CDK，其激酶活性仅在有丝分裂后的神经元中检测到。一些证据表明，CDK 5通过轴突细胞骨架如微管相关蛋白tau和神经丝的磷酸化参与发育过程中的神经元迁移或定位。然而，尚不清楚其激酶活性如何调节。我们研究了CDK5.1的激活或失活机制。本研究以原代培养的神经元为研究对象，以神经递质-H亚基的磷酸化为标志，研究了CDK 5在神经元中的激活机制。接种后4至5天，CDK 5被激活，此时出现磷酸化形式的神经丝H亚基并发生突触形成。向培养基中添加BDNF诱导CDK 5活化。这些结果提示，突触形成后BDNF分泌增加，诱导CDK 5活化. CDK 5通过与仅在神经元中表达的活化亚基p35结合而活化。p35是一种寿命短的蛋白质，在几小时内降解。CDK 5随着p35的降解而沿着降低其活性。p35在增殖细胞中被蛋白酶体和细胞周期蛋白降解。磷酸化被认为是降解的信号.在从牛或猪脑中纯化CDK 5期间，p35被蛋白水解为p25。参与这种有限的蛋白水解的蛋白酶被证明是钙蛋白酶。向猪脑提取物中加入Ca ~（2+）D_12 + Ca ~（2+）D_1诱导p35蛋白水解为p25。钙蛋白酶抑制剂抑制这种蛋白水解。蛋白水解改变了CDK 5的溶解度。通过短暂离心沉淀的P35/CDK 5在蛋白水解转化为p25后变得可溶。在神经元细胞死亡时，p35到p25的有限蛋白水解增强。