Transcription factor Oct4 plays critical roles in maintaining pluripotency and controlling lineage commitment of embryonic stem cells （ESCs）. Our previous study indicates that Wwp2, a mouse HECT-type E3 ubiquitin li...Transcription factor Oct4 plays critical roles in maintaining pluripotency and controlling lineage commitment of embryonic stem cells （ESCs）. Our previous study indicates that Wwp2, a mouse HECT-type E3 ubiquitin ligase, ubiquitinates Oct4 and promotes its degradation in a heterologous system. However, roles of Wwp2 in regulating en- dogenous Oct4 protein levels as well as molecular characteristics of the function of Wwp2 have not been determined. Here, we report that Wwp2 plays an important role in Oct4 ubiquitination and degradation during differentiation of embryonal carcinoma cells （ECCs）, although it does not appear to affect Oct4 protein levels in the undifferentiated ECCs and ESCs. Importantly, inhibition of Wwp2 expression by specific RNA interference elevates the Oct4 protein level, leading to attenuation in retinoid acid-induced activation of differentiation-related marker genes. Mechanisti- cally, Wwp2 catalyzes Oct4 poly-ubiquitination via the lysine 63 linkage in a dosage-dependent manner. Interest- ingly, Wwp2 also regulates its own ligase activity in a similar manner. Moreover, auto-ubiquitination of Wwp2 occurs through an intra-molecular mechanism. Taken together, these results demonstrate a crucial role of Wwp2 in con- trolling endogenous Oct4 protein levels during differentiation processes of ECCs and suggest an interesting dosage- dependent mechanism for regulating the catalytic activity of the E3 ubiquitin ligase, Wwp2.展开更多
基金We thank Dr Richard Baer （Pathology, Columbia University, New York, USA） for generously providing various Ub mutant plasmids. This study was supported by Grants from the National Natural Science Foundation of China （30871257, 30730051） and the National High Technology Research, Development Program of China （2006CB943901 and 2007CB947904）, the Shanghai Sci- ence and Technology Developmental Foundation （08JC1413100） and the Shanghai Leading Academic Discipline Project （$30201）.
文摘Transcription factor Oct4 plays critical roles in maintaining pluripotency and controlling lineage commitment of embryonic stem cells （ESCs）. Our previous study indicates that Wwp2, a mouse HECT-type E3 ubiquitin ligase, ubiquitinates Oct4 and promotes its degradation in a heterologous system. However, roles of Wwp2 in regulating en- dogenous Oct4 protein levels as well as molecular characteristics of the function of Wwp2 have not been determined. Here, we report that Wwp2 plays an important role in Oct4 ubiquitination and degradation during differentiation of embryonal carcinoma cells （ECCs）, although it does not appear to affect Oct4 protein levels in the undifferentiated ECCs and ESCs. Importantly, inhibition of Wwp2 expression by specific RNA interference elevates the Oct4 protein level, leading to attenuation in retinoid acid-induced activation of differentiation-related marker genes. Mechanisti- cally, Wwp2 catalyzes Oct4 poly-ubiquitination via the lysine 63 linkage in a dosage-dependent manner. Interest- ingly, Wwp2 also regulates its own ligase activity in a similar manner. Moreover, auto-ubiquitination of Wwp2 occurs through an intra-molecular mechanism. Taken together, these results demonstrate a crucial role of Wwp2 in con- trolling endogenous Oct4 protein levels during differentiation processes of ECCs and suggest an interesting dosage- dependent mechanism for regulating the catalytic activity of the E3 ubiquitin ligase, Wwp2.
