ATF1与CDK3蛋白相互作用研究

Interaction analysis of ATF1 and CDK3

研究真核细胞转录激活因子1(activating transcription factor1,ATF1)与细胞周期蛋白依赖性蛋白激酶3(cyclin-dependent kinase3,CDK3)的相互作用机制,分别构建ATF1的激酶诱导区域(kinaseinducible domain,KID)结构域、谷氨酰胺富含域(glutamine rich domain,Q2)结构域及亮氨酸拉链区(basic leucine zipper domain,bZIP)结构域缺失体与VP16的融合表达载体,在HEK293细胞过表达,应用真核细胞双杂交系统,研究ATF1结构域与CDK3的相互作用.结果表明,ATF1全蛋白与CDK3蛋白之间存在强烈的相互作用;ATF1结构域缺失体与VP16的融合蛋白在HEK293细胞中可获得高表达,但ATF1的任何一种结构缺失体与CDK3蛋白结合都会严重减弱其与CDK3蛋白的相互作用,只有完整的ATF1蛋白才能实现与上游CDK3蛋白的结合.

To study the interaction mechanism between activating transcription factor 1（ATF1）and cyclin-dependent kinase 3（CDK3）,the plasmids for expression of VP16-tagged ATF1 and the VP16-ATF1 deletions（residues 1~57,1~214,or 215~271）were constructed.HEK293 cells were transfected and proteins overexpressed were detected by Western blotting.Mammalian two-hybrid assay was conducted and luciferase activity was measured.Results show that an 11-fold increase in interacting activity between pACT-ATF1 and pBIND-CDK3 compared with the basal level（value of 1）of pG5-luc/pBIND-CDK3/pACT control.Neither of ATF1 deletion mutants show strong interaction with CDK3,suggesting that the full length of ATF1 is required for the interaction with CDK3.