上海交通大学学报(医学版) ›› 2019, Vol. 39 ›› Issue (11): 1243-.doi: 10.3969/j.issn.1674-8115.2019.11.005

• 论著·基础研究 • 上一篇    下一篇

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

李 杏,魏佳乐,汤在明,钟 清,留筱厦   

  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,上海 200025
  • 出版日期:2019-11-28 发布日期:2019-12-16
  • 通讯作者: 留筱厦,电子信箱:xxliu@shsmu.edu.cn。
  • 作者简介:李 杏(1994—),女,硕士生;电子信箱:15720625797@163.com。
  • 基金资助:
    国家自然科学基金(31771523,31870830)

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

LI Xing, WEI Jia-le, TANG Zai-ming, ZHONG Qing, LIU Xiao-xia   

  1. 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
  • Online:2019-11-28 Published:2019-12-16
  • Supported by:
    National Natural Science Foundation of China, 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(class Ⅲ phosphatidylinositol 3-kinase complex Ⅰ,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。

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

Abstract: Objective · To study the effects of different phosphorylation levels of nuclear receptor-binding factor 2 (NRBF2) on the activity of class Ⅲ phosphatidylinositol 3-kinase complex Ⅰ (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 determinedgel 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.

Key words: nuclear receptor-binding factor 2 (NRBF2), class Ⅲ phosphatidylinositol 3-kinase complex Ⅰ (PI3KC3-C1), kinase activity detection, autophagy regulation

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