Abstract:

Objective To investigate whether WW domain containing E3 ubiquitin protein ligase 1（WWP1） and WW domain containing E3 ubiquitin protein ligase 2（WWP2） modulate the protein levels of octamer-binding transcription factor 4（OCT4）,sex-determining region Y-box protein 2（SOX2） and NANOG. Methods Exogenous WWP1 or WWP2 was enforced to expression in HEK 293FT cells along with OCT4,SOX2 and NANOG respectively by plasmid transfection. The influence of WWP1 or WWP2 overexpression on the protein levels of OCT4,SOX2 and NANOG was determined by western blotting. GST pull-down and immunoprecipitation experiments were carried out to verified the protein interaction between WWP1 and SOX2. In vitro ubiquitination assay was conducted to demonstrate that WWP1 was a new E3 ubiquitin ligase of SOX2. Results In the co-expression systems of WWP1 and OCT4,WWP1 and SOX2,and WWP2 and OCT4,both WWP1 and WWP2 showed dose-dependent down-regulation of the corresponding co-expression protein OCT4 or SOX2,and the down-regulation of SOX2 protein was the most obvious in the co-expression system of WWP1 and SOX2. In the other co-expression systems,WWP1 or WWP2 had no significant down-regulation effect on the corresponding co-expressed proteins. The lysosome inhibitor chloroquine partially prevented the decrease in SOX2 protein levels. After 24 h treatment with cyclohexmide（CHX）,the SOX2 protein level in WWP1 and SOX2 co-expression system decreased faster than that of SOX2 overexpression alone（P<0.05）. WWP1 protein interacted with SOX2 protein directly and catalyzed ubiquitin modification on SOX2 in vitro. Conclusions WWP1 is a new specific E3 ubiquitin ligase of SOX2,and WWP1 may promote SOX2 protein turnover through ubiquitin-lysosome pathway,resulting in attenuation of protein stability and abundance of SOX2.

Key words: WW domain containing E3 ubiquitin protein ligase 1, Sex-determining region Y-box protein 2, Ubiquitination, Protein degradation