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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 purpose of this study was to investigate the mechanism by which PTEN induces the regulation of kinase activity in the cytoplasm of PINK1S. First, the co-immunoprecipitation method was used to confirm that PINK1S binds to the β subunit (CK2β) in the protein complex of casein kinase 2 (CK2) but not to the other two catalytic subunits α1 (CK2α1) and α2 CK2α2). Secondly, while over-expressing CK2βand PINK1S, PINK1S was purified by co-immunoprecipitation. The phosphorylation of PINK1S was detected by Phos-tagTM Biotin and found that CK2βwas able to promote PINK1S Self-phosphorylation. Finally, the RNA interference technique was used to establish a cell line that interfered with CK2β. PINK1S was expressed in the control group and the experimental group interfering with CK2β. The lack of CK2β expression led to the decrease of PINK1S phosphorylation. This study shows that: CK2β as a cytoplasmic PINK1S autophosphorylation of ancillary subunits play a positive regulatory effect on its activity.