Cdk5–p39 is a labile complex with the similar substrate specificity to Cdk5–p35
Cdk5–p39 is a labile complex with the similar substrate specificity to Cdk5–p35
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DOI：
10.1111/j.1471-4159.2007.04505.x
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发表时间：
2007-09
影响因子：
4.7
通讯作者：
Mari Yamada;Taro Saito;Yutaka Sato;Yusei Kawai;A. Sekigawa;Y. Hamazumi;A. Asada;M. Wada;H. Doi-H.
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文献类型：
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作者：
Mari Yamada;Taro Saito;Yutaka Sato;Yusei Kawai;A. Sekigawa;Y. Hamazumi;A. Asada;M. Wada;H. Doi-H.
细胞周期蛋白依赖性激酶5 （Cdk5）是一种脯氨酸导向的丝氨酸/苏氨酸激酶，在各种神经元活动中起重要作用，包括神经元迁移、突触活性和神经元细胞死亡。Cdk5通过与神经元特异性激活因子p35或其异构体p39结合而激活，但对Cdk5 - p39的激酶活性知之甚少。事实上，激酶活性Cdk5 - p39不是从大鼠脑提取物中制备的，也不是在非离子洗涤剂存在的情况下通过免疫沉淀从表达Cdk5和p39的HEK293细胞中制备的，在这种条件下可以分离活性Cdk5 - p35。p39在去污剂存在下与Cdk5解离，表明p39与Cdk5的结合亲和力低于p35。我们开发了一种从编码Cdk5和p39的杆状病毒感染的Sf9细胞中纯化激酶活性Cdk5 - p39的方法。纯化的Cdk5-p39复合物显示出与Cdk5-p35相似的底物特异性，但对洗涤剂的敏感性相反。Triton X‐100灭活了Cdk5-p39，而激活了Cdk5-p35。p35和p39的N端缺失（氨基酸序列不同）不会改变Cdk5复合物的稳定性或底物特异性。Cdk5-p35和Cdk5-p39的稳定性差异表明它们在神经元中不同的调控机制下具有不同的作用。
Cyclin‐dependent kinase 5 (Cdk5) is a proline‐directed Ser/Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron‐specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5–p39. In fact, kinase‐active Cdk5–p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non‐ionic detergent, under conditions with which active Cdk5–p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase‐active Cdk5–p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5–p39 complex showed similar substrate specificity to that of Cdk5–p35, but with opposite sensitivity to detergent. Cdk5–p39 was inactivated by Triton X‐100, whereas Cdk5–p35 was activated. The N‐terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5–p35 and Cdk5–p39 suggests their distinct roles under different regulation mechanisms in neurons.