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

细胞周期蛋白依赖性激酶是真核细胞细胞周期进展的关键因素。 CDK5 是一种独特的 CDK，其激酶活性仅在有丝分裂后神经元中检测到。多项证据表明，CDK5 通过轴突细胞骨架（例如微管相关蛋白 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是如何调节的。我们在这里研究了CDK5.1的激活或失活机制。我们以神经丝-H亚基的磷酸化为标记，研究了原代培养神经元中CDK5的激活机制。当磷酸化形式的神经丝-H亚基出现并发生突触形成时，CDK5在铺板后4至5天被激活。将 BDNF 添加到培养基中诱导 CDK5 的激活。这些结果表明CDK5的激活是由突触形成后BDNF分泌增加诱导的。2． CDK5 通过与仅在神经元中表达的激活亚基 p35 结合而被激活。 p35是一种寿命较短的蛋白质，在几个小时内就会降解减少。牛脑或猪脑。参与这种有限蛋白水解的蛋白酶被证明是钙蛋白酶。向猪脑提取物中添加 CaiD12 可诱导 p35 蛋白水解为 p25。这种蛋白水解作用被钙蛋白酶抑制剂抑制。蛋白水解改变了CDK5的溶解度。通过短暂离心沉淀的 P35/CDK5 在蛋白水解转化为 p25 后变得可溶。 p35 到 p25 的有限蛋白水解在神经元细胞死亡时增强。

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.