Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro  

作　　者：Liang ZHOU Hong-feng WANG Hai-gang REN Dong CHEN Feng GAO Qing-song HU Chen FU Ran-jie XU ZhengYING Guang-hui WANG 

机构地区：[1]Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science & Technology of China, Chinese Acad-emy of Sciences, Hefei 230027, China [2]Laboratory of Molecular Neuropathology, Department of Pharmacology, Soochow UniversityCollege of Pharmaceutical Sciences, Suzhou 215123, China

出　　处：《Acta Pharmacologica Sinica》2013年第5期651-656,共6页中国药理学报（英文版）

摘　　要：To investigate whether sequestosome 1/p62 （p62）, a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro. Methods： HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pull- down assay was used to study the interaction of p62 with Bcl-2. Results： Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vffro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells signifi- cantly increased the amount of Beclin I that co-immunoprecipitated with Bcl-2. Conclusion： p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.To investigate whether sequestosome 1/p62 （p62）, a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro. Methods： HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pull- down assay was used to study the interaction of p62 with Bcl-2. Results： Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vffro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells signifi- cantly increased the amount of Beclin I that co-immunoprecipitated with Bcl-2. Conclusion： p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.

关 键 词：AUTOPHAGY sequestosome 1/p62 （p62） LC3 BCL-2 Beclin 1 HEK 293 cell HeLa cell in vitro pulldown assay 

分 类 号：Q786[生物学—分子生物学] Q78