An analysis of an interactome for apoptosis factor, Ei24/PIG8, using the inducible expression system and shotgun proteomics.
ei24 (etoposide-induced 2.4 kb transcript, also designated PIG8 (p53-induced gene 8)), is a DNA damage response gene involved in growth suppression and apoptosis. ei24 gene expression is specifically induced by wild type p53, and its overexpression suppresses cell growth by inducing apoptotic cell death. Generally, the protein-protein interaction is known to regulate their targets, as well as to modify cell fates. In this study, using the established NIH/3T3, oncogenic H-Ras/G12V transformed NIH/3T3, and B16F10 cells, which are expressing mouse Ei24 proteins under the tight control of expression by doxycycline, a proteomic screening was conducted to identify the binding partners for Ei24. Immunoprecipitation of mEi24 and associated proteins was performed using the mEi24 expressing cell lysates. Isolated immuno-complexes were resolved by SDS-PAGE and analyzed by liquid chromatography tandem mass spectrometry. There were 61 novel potential mEi24 interacting proteins identified, among which are NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and B16F10/mEi24 cells; however, some mEi24 interacting proteins were specific to two NIH/3T3 related cells and to B16F10/mEi24 cells. This approach led to the identification of many interacting partners, and the discovery of these associated proteins will lead to a better understanding of the mechanisms underlying the physiological and cell biological roles of Ei24.
Bahk
Young Yil
YY
Department of Integrated OMICS for Biomedical Sciences, Graduate School, Yonsei University, Seoul, Korea. bahk12@empal.com
Lee
Jaehoon
J
Cho
Ick-Hyun
IH
Lee
Han-Woong
HW
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004318:Doxycycline / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C526185:EI24 protein, mouse; D009687:Nuclear Proteins; D049452:Green Fluorescent Proteins; D004318:Doxycycline