CK2β促进PINK1S自我磷酸化

Promotion of Pink1S Auto-phosphorylation with CK2β

本研究旨在探究PTEN诱导激酶1短亚型(PINK1S)在细胞质中激酶活性调节机制。首先,通过免疫共沉淀筛选的方法证明PINK1S能够和酪蛋白激酶2(CK2)蛋白复合体中的β亚基(CK2β)相互结合,但不与其他两个催化亚基α1(CK2α1)和α2(CK2α2)结合。其次,同时过表达CK2β和PINK1S,利用免疫共沉淀的方法纯化PINK1S,将得到的PINK1S用生物素标记的磷酸化蛋白检测标签(Phos-tagTM Biotin)检测其磷酸化水平,发现CK2β能够促进PINK1S的自我磷酸化。最后,利用RNA干扰技术,建立干扰CK2β的细胞株;在对照组和干扰CK2β的实验组细胞内表达PINK1S,发现缺少CK2β表达导致PINK1S的磷酸化水平降低。本文研究表明:CK2β作为胞质PINK1S自我磷酸化的辅助亚基,对其活性起到正调节作用。

The aim of this study is to determine the regulatory mechanism of PTEN-induced putative kinase protein 1 short isoform （PINK1S） in cytoplasm. By α-immunoprecipitation （Co-IP） assay, we identified that PINK1S inter- acted with the beta regulatory subunit of Casein Kinase 2 （CK2β）, but not with the catalytic subunits CK2αl and CK2α2. Furthermore, cells were transfeeted with PINKIS and CK2β and then PINK1S was purified by immunopre- cipitation. After detecting the phosphorylated proteins by Phos-tagTM Biotin, we found that CK2β overexpression in- creased auto-phosphorylation of PINK1S. Finally, we generated CK2β knockdown cell lines by RNA interference. Purified PINK1S from CK2β knockdown cells significantly reduced its auto-phosphorylation compared with control cells. These results suggested that CK2β functions as a regulatory subunit of PINK1S kinase complex promoted its activation by self-phosphorylation.