HDAC2磷酸化区域突变体的构建及其对小鼠成纤维细胞增殖的影响

Construction of phosphorylation site mutant of HDAC2 and its impact on proliferation of mouse fibroblasts

目的应用片段缺失突变技术鉴定HDAC2自身磷酸化对其Sumo-E3连接酶活性及蛋白质翻译的影响。方法以前期获得的鼠源性HDAC2Sumo-E3连接酶结构域基因片段为模板,设计片段缺失突变引物,经长片段重叠延伸PCR技术扩增获得片段缺失突变基因片段,后于Top10大肠杆菌中自然连接扩增获得pcDNA3.1/HDAC2-Sumo-E3连接酶磷酸化结构域缺失突变的真核表达载体。然后将载体转染于L929成纤维细胞瞬时过表达突变基因,再经翻译反应性荧光素酶报告基因实验和Western-blot鉴定HDAC2自身磷酸化修饰对Sumo-E3连接酶介导的报告基因和靶蛋白质表达的影响。最后通过MTT实验检测HDAC2自身磷酸化修饰对其Sumo-E3连接酶介导的L929细胞增殖的影响。结果成功构建获得磷酸化修饰完全缺失的HDAC2片段缺失突变体pcDNA3.1/HDAC2-Sumo-E3(DEL 394-424AA)。LUC报告基因检测结果显示,DEL 394-424AA片段缺失突变体促进LUC翻译是对照组的4.24倍,且能显著促进靶蛋白ODC和c-Myc的表达,以及L929小鼠成纤维细胞的增殖效应。结论 HDAC2自身磷酸化修饰对HDAC2Sumo-E3连接酶活性及其调节的蛋白质翻译和细胞增殖作用起负性调节作用。

Objective To determine the effect of autophosphorylation of HDAC2 on the Sumo-E3 ligase activity and cap-de- pendent protein translation by the segment-deletion mutation technique. Methods＇ By using the pcDNA3.1/HDAC2-Sumo-E3 vec- tor expressing ligase domain of mouse HDAC2 as template, primers were designed to construct the segment-deletion mutation by SOE PCR （gene splicing by overlap extension PCR） ,then the segment was ligated and amplified in Top10 strain of E. eoli tO get the pcDNA3.1/HDAC2-Sumo-E3（DEL394-424AA） vector that expressing the ligase domain of mouse HDAC2 with 394-424AA dele- tion. The pcDNA3.1/HDAC2-Sumo-E3（DEL394-424AA）vector was then transiently over-expressed in L929 fibroblast, followed by assessment of cap-dependent translation by luciferase reporter gene and target protein expression by Western blot. At last, the effect of HDAC2 phosphorylation on Sumo-E3 ligase activity-induced proliferation of L929 cell line was determined by MTT assay. Results the peDNA3.1/HDAC2-Sumo-E3 （DEL394-424AA） vector that completely abolished phosphorylation of this segment of HDAC2 was successfully constructed. And when transiently over-expressed in L929 mouse fibroblast, the luciferase activity was in- creased to 4.24 fold that of control,and expression of the target gene ODC and c-Myc were significantly elevated,and cell prolifera- tion was significantly accelerated. Conclusion Autophosphorylation of HDAC has the negative regulation effect on the Sumo-E3 activity of HDAC2 and downstream cap-dependent protein translation and cell proliferation.