Straightforward and rapid method for detection of cyclin-dependent kinase-like 5 activity
Straightforward and rapid method for detection of cyclin-dependent kinase-like 5 activity
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DOI：
10.1016/j.ab.2018.11.013
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发表时间：
2019-02-01
影响因子：
2.9
通讯作者：
Inazu, Tetsuya
中科院分区：
文献类型：
--
作者：
Katayama, Syouichi;Inazu, Tetsuya
细胞周期蛋白依赖性激酶样5（CDKL 5）是一种丝氨酸/苏氨酸蛋白激酶，其基因突变导致神经发育障碍。致病性点突变主要在CDKL 5的催化结构域内观察到，因此催化活性的丧失可能与疾病发作有关。然而，这一假设很少得到证实。在这里，我们报告了一种检测CDKL 5活性的有效方法。适当地，CDKL 5在大肠杆菌中表达后经历了自磷酸化，其中自磷酸化的CDKL 5通过SDS-PAGE检测为条带移位，无需酶纯化。因此，该方案可用于检查致病突变与其活性之间的关系。
Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase, with its gene mutation leading to a neurodevelopmental disorder. Pathogenic point mutations are mostly observed within the catalytic domain of CDKL5, therefore loss of catalytic activity may be related to disease onset. However, this hypothesis has rarely been demonstrated. Here, we report an efficient method for detecting CDKL5 activity. Appropriately, CDKL5 underwent autophosphorylation following expression in Escherichia coil, with autophosphorylated CDKL5 detected as a band shift by phos-tag SDS-PAGE, without enzyme purification. Thus, this protocol is useful for examining the relationship between disease-causing mutations and their activity.