The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian sel-10 homolog

The Caenorhabditis elegans sel-10 protein is structurally similar to E3 ubi quitin ligases and is a negative regulator of Notch (lin-12) and presenilin signaling. In this report, we characterize the mammalian Sel-10 homolog (m Sel-10) and analyze its effects on Notch signaling. We find that mSel-10 lo calizes to the cell nucleus, and that it physically interacts with the Notc h 1 intracellular domain (IC) and reduces Notch 1 IC-mediated activation of the HES 1 promoter. Notch 1 IC is ubiquitinated by mSel-10, and ubiquitina tion requires the presence of the most carboxyl-terminal region of the Notc h IC, including the PEST domain. In the presence of the proteasome inhibito r MG132, the amount of Notch 1 IC and its level of ubiquitination are incre ased. Interestingly, this accumulation of Notch 1 IC in the presence of MG1 32 is accompanied by decreased activation of the RES 1 promoter, suggesting that ubiquitinated Notch 1 IC is a less potent transactivator. Finally, we show that mSel-10 itself is ubiquitinated and degraded by the proteasome. In conclusion, these data reveal the importance of ubiquitination and prote asome-mediated degradation for the activity and turnover of Notch ICs, and demonstrate that mSel-10 plays a key role in this process.