体外激酶实验筛选p21活化激酶6对雄激素受体的磷酸化位点  

The screening of PAK6-mediated phosphorylated sites of AR by in vitro kinase assay

作　　者：刘彤[1] 李洋[1] 耿楠希[1] 李丰[1] 

机构地区：[1]中国医科大学基础医学院细胞生物教研室//教育部医学细胞生物学重点实验室,辽宁沈阳110001

出　　处：《解剖学研究》2013年第3期161-164,共4页Anatomy Research

基　　金：国家自然科学基金(31171360)

摘　　要：目的构建雄激素受体(AR)的点突变原核表达质粒,以体外激酶实验分析筛选p21活化激酶6(PAK6)对雄激素受体的磷酸化位点,更好的研究PAK6对AR的磷酸化所发挥的生物学功能。方法利用体外激酶实验分析PAK6对AR的较小磷酸化区域,并利用重叠延伸PCR的方法构建可能的磷酸化定点突变体;然后,体外纯化带有GST标签的突变体蛋白,及体外激酶实验筛选PAK6对AR的磷酸化位点。结果证明质粒构建成功,构建雄激素受体定点突变体,确定PAK6磷酸化雄激素受体位点。结论成功筛选PAK6对AR的磷酸化位点是578位丝氨酸。Objective To learn more biological function of PAK6-mediated phosphorylation of AR,in vitro kinase assay was used to screen out the phosphorylated sites of AR by PAK6.Methods Firstly,in vitro kinase assay was used to map the shorter region of PAK6-mediated phosphorylation of AR.Secondly,overlapping PCR was used to construct the potential phosphorylated sites of AR by PAK6.Next,we purified the proteins of GST tagged AR mutant.Results In vitro kinase assay was used to screen out the phosphorylated sites of AR by PAK6.Conclusion The phosphorylated sites of AR by PAK6 is Ser 578.

关 键 词：体外激酶实验 p21活化激酶6 雄激素变体 定点突变体 磷酸化 

分 类 号：R737.25[医药卫生—肿瘤]