title: Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro.
abstract: OBJECTIVE
  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 pulldown 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 vitro 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 significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2.
  
  
  CONCLUSIONS
  p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.
authors.affiliation: Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science & Technology of China, Chinese Academy of Sciences, Hefei 230027, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhou
authors.forename: Hong-feng
authors.initials: HF
authors.lastname: Wang
authors.forename: Hai-gang
authors.initials: HG
authors.lastname: Ren
authors.forename: Dong
authors.initials: D
authors.lastname: Chen
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Qing-song
authors.initials: QS
authors.lastname: Hu
authors.forename: Chen
authors.initials: C
authors.lastname: Fu
authors.forename: Ran-jie
authors.initials: RJ
authors.lastname: Xu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Ying
authors.forename: Guang-hui
authors.initials: GH
authors.lastname: Wang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy; D000071186:Beclin-1; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D060066:Protein Interaction Maps; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D000071456:Sequestosome-1 Protein; D014162:Transfection; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein
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