核受体结合因子2对自噬起始复合物PI3KC3-C1活性的调节作用  

Modulation of nuclear receptor-binding factor 2 on the activity of autophagy initiation complex PI3KC3-C1

作　　者：李杏[1] 魏佳乐 汤在明 钟清 留筱厦 LI Xing;WEI Jia-le;TANG Zai-ming;ZHONG Qing;LIU Xiao-xia(Department of Pathophysiology,Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education,Shanghai Jiao Tong University College of Basic Medical Sciences,Shanghai 200025,China)

机构地区：[1]上海交通大学基础医学院病理生理学系细胞分化与凋亡教育部重点实验室

出　　处：《上海交通大学学报（医学版）》2019年第11期1243-1247,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(31771523,31870830)~~

摘　　要：目的·研究核受体结合因子2(nuclear receptor-binding factor 2,NRBF2)的不同磷酸化水平对Ⅲ型磷脂酰肌醇-3-激酶复合物Ⅰ(classⅢphosphatidylinositol 3-kinase complexⅠ,PI3KC3-C1)的活性影响,寻找一种活性最高的PI3KC3-C1作为自噬特异性小分子抑制剂筛选的靶标。方法·首先通过建立体外蛋白纯化体系,分别获得四元不含NRBF2的PI3KC3-C1和五元包含不同磷酸化水平NRBF2的PI3KC3-C1,通过凝胶过滤色谱检测复合物的完整性,然后体外测定纯化的不同PI3KC3-C1的激酶活性差异,观察NRBF2的磷酸化对PI3KC3-C1活性的影响。结果·纯化的PI3KC3-C1具有良好的纯度和产量。包含NRBF2的五元PI3KC3-C1表现出比四元的蛋白复合物更高的激酶活性,其中包含持续去磷酸化NBRF2的PI3KC3-C1具有最高的酶活性。结论·NRBF2确实作为PI3KC3-C1的组分之一稳定存在,NRBF2的存在可提升PI3KC3-C1的激酶活性;包含持续去磷酸化NRBF2的PI3KC3-C1可以作为一种新的自噬调控靶标,用来筛选自噬特异性小分子抑制剂。Objective·To study the effects of different phosphorylation levels of nuclear receptor-binding factor 2(NRBF2)on the activity of classⅢphosphatidylinositol 3-kinase complexⅠ(PI3KC3-C1),and find a type of PI3KC3-C1 with the highest kinase activity as a target for the screening of autophagy-specific inhibitors.Methods·First,in vitro protein purification system was established to obtain quaternary NRBF2-free PI3KC3-C1 and quinary PI3KC3-C1 containing different phosphorylation levels of NRBF2.The integrity of the complex was determined by gel filtration chromatography.The different kinase activity of purified PI3KC3-C1 was detected in vitro,and thus the effect of phosphorylation of NRBF2 on PI3KC3-C1 activity was observed.Results·The purified PI3KC3-C1 had good purity and yield.The five-membered PI3KC3-C1 containing NRBF2 showed higher kinase activity than the quaternary protein complex,and PI3KC3-C1 containing the continuous dephosphorylated NBRF2 had the highest kinase activity.Conclusion·NRBF2 does exist as one of the components of PI3KC3-C1.The presence of NRBF2 promotes the kinase activity of PI3KC3-C1,and PI3KC3-C1 containing continuous dephosphorylation of NRBF2 may serve as a new regulatory target for the screening of autophagy-specific inhibitor.

关 键 词：核受体结合因子2 Ⅲ型磷脂酰肌醇-3-激酶复合物Ⅰ 激酶活性检测 自噬调控 

分 类 号：Q291[生物学—细胞生物学]