Buajeeb Waranun, Zhang Xue, Ohyama Hiroe, Han David, Surarit Rudee, Kim Yong, Wong David T W
Department of Oral Medicine, Faculty of Dentistry, Mahidol University, Bangkok, Thailand.
Biochem Biophys Res Commun. 2004 Mar 19;315(4):998-1003. doi: 10.1016/j.bbrc.2004.02.003.
Human DOC-1/CDK2AP1 gene encodes a growth suppressor protein of 12kDa (p12(DOC-1/CDK2AP1)). Recently, p12(DOC-1/CDK2AP1) has been shown to associate with cell cycle proteins including CDK2 and DNA polymerase alpha/primase. It negatively regulates CDK2 activities and suppresses DNA replication. Therefore, identification of other p12(DOC-1/CDK2AP1) interacting proteins might clarify its role in the cell cycle regulation and carcinogenesis. The purpose of this study was to identify additional p12(DOC-1/CDK2AP1) interacting proteins using the yeast two-hybrid system. Using human p12(DOC-1/CDK2AP1) as a bait in a liver cDNA library screening, cDNA clones identical to human DOC-1R transcript were identified. The interaction between p12(DOC-1/CDK2AP1) and p14(DOC-1R) was verified in vitro and in cells. GST pull-down assay and immunoprecipitation experiments confirmed the interaction between the two proteins. The critical region for p12(DOC-1/CDK2AP1)'s interaction with p14(DOC-1R) was defined to amino acids 20-25 by using a series of deletion mutants as baits in the yeast two-hybrid system. Our data indicated that p12(DOC-1/CDK2AP1) could associate with its homologous protein, p14(DOC-1R).
人类DOC-1/CDK2AP1基因编码一种12kDa的生长抑制蛋白（p12(DOC-1/CDK2AP1)）。最近研究表明，p12(DOC-1/CDK2AP1)与包括CDK2和DNA聚合酶α/引发酶在内的细胞周期蛋白相关。它对CDK2活性起负调控作用并抑制DNA复制。因此，鉴定其他与p12(DOC-1/CDK2AP1)相互作用的蛋白可能会阐明其在细胞周期调控和致癌作用中的角色。本研究的目的是利用酵母双杂交系统鉴定其他与p12(DOC-1/CDK2AP1)相互作用的蛋白。以人类p12(DOC-1/CDK2AP1)作为诱饵进行肝脏cDNA文库筛选，鉴定出与人类DOC-1R转录本相同的cDNA克隆。在体外和细胞内验证了p12(DOC-1/CDK2AP1)与p14(DOC-1R)之间的相互作用。GST沉降分析和免疫沉淀实验证实了这两种蛋白之间的相互作用。通过在酵母双杂交系统中使用一系列缺失突变体作为诱饵，将p12(DOC-1/CDK2AP1)与p14(DOC-1R)相互作用的关键区域确定为氨基酸20 - 25。我们的数据表明p12(DOC-1/CDK2AP1)可与其同源蛋白p14(DOC-1R)相关联。
