Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Anal Biochem. 2019 Feb 1;566:58-61. doi: 10.1016/j.ab.2018.11.013. Epub 2018 Nov 14.
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 coli, 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.
周期素依赖性激酶样 5（CDKL5）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。致病性点突变主要发生在 CDKL5 的催化结构域内，因此催化活性的丧失可能与疾病的发生有关。然而，这一假说很少得到证实。在这里，我们报告了一种检测 CDKL5 活性的有效方法。在大肠杆菌中表达后，CDKL5 可进行自身磷酸化，利用磷酸化 SDS-PAGE 胶（phos-tag SDS-PAGE）检测到自身磷酸化的 CDKL5 发生条带位移，而无需酶的纯化。因此，该方案可用于研究致病突变与其活性之间的关系。
