The Notch Intracellular Domain Is Ubiquitinated and Negatively Regulated by the Mammalian Sel-10 Homolog

Abstract

The Caenorhabditis elegans sel-10 protein is structurally similar to E3 ubiquitin ligases and is a negative regulator of Notch (lin-12) and presenilin signaling. In this report, we characterize the mammalian Sel-10 homolog (mSel-10) and analyze its effects on Notch signaling. We find that mSel-10 localizes to the cell nucleus, and that it physically interacts with the Notch 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 ubiquitination requires the presence of the most carboxyl-terminal region of the Notch IC, including the PEST domain. In the presence of the proteasome inhibitor MG132, the amount of Notch 1 IC and its level of ubiquitination are increased. Interestingly, this accumulation of Notch 1 IC in the presence of MG132 is accompanied by decreased activation of the HES 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 proteasome-mediated degradation for the activity and turnover of Notch ICs, and demonstrate that mSel-10 plays a key role in this process.

Up‐regulated deubiquitinase USP4 plays an oncogenic role in melanoma

“…For example, the inactivating somatic mutations of BAP1 , a nuclear ubiquitin carboxyl‐terminal hydrolase (UCH), were frequently detected in metastasizing uveal melanomas, and melanoma cell with BAP1 gene deficiency grew as multi‐cellular non‐adherent spheroids with rounded epithelioid morphology, paralleled with increased metastatic capacity 26, 27. In addition, the mutation of ubiquitin ligase FBXW7 resulted in hyper‐activation of Notch1 signalling and subsequent sustained proliferation of melanoma cells by up‐regulation of cell‐cycle mediators 28, 29, 30. What is more, the pro‐proliferative NF‐κB pathway was potentiated due to the facilitated degradation of IκB by E3 ubiquitin ligase β‐Trcp, which was up‐regulated by BRAF over‐activation in melanoma 12.…”

Fbw7 regulates the activity of endoreduplication mediators and the p53 pathway to prevent drug-induced polyploidy

“…This finding explains the frequently elevated expression of cyclin E in breast tumors lacking active Fbw7 (Ekholm-Reed et al, 2004). Other substrates include the oncoproteins c-Myc (Moberg et al, 2004;Welcker et al, 2004;Yada et al, 2004), c-Jun (Nateri et al, 2004;Wei et al, 2005) and Notch (Gupta-Rossi et al, 2001;Oberg et al, 2001;Wu et al, 2001), all of which act to increase cell volume, proliferation and de-differentiation. In addition, Aurora A kinase, an important regulator of mitosis that is frequently overexpressed in human cancer tissues, also serves as a substrate of Fbw7 (Mao et al, 2004;Fujii et al, 2006).…”

FBW7 is associated with prognosis, inhibits malignancies and enhances temozolomide sensitivity in glioblastoma cells

“…Notch signaling pathway has been widely recognized for its vital roles in cell differentiation, cancer stemness, as well as EMT. Among these the cleaved intracellular domain of Notch1, NICD, is the most important intermediate for Notch signaling transduction, which is another substrate of FBW7 13. From the western blot assay, the mechanism of inhibited migration and invasion upon FBW7 elevation in our study can be partially explained by the early downregulation of Notch1.…”

“…It is an important tumor suppressor with dominant‐negative mutations in a multitude of tumors, including gastric, colorectal, breast, pancreatic, liver, prostate and endometrial cancers, as well as leukemia and osteosarcoma 5, 7. To date, multiple substrates of FBW7 have been identified, which include a series of important oncoproteins like Cyclin E, c‐Myc, Jun, Mcl1 and Sox9 that are involved in the regulation of cell division, apoptosis, differentiation and some classical oncogenic signaling pathways, such as Notch and mTOR 8, 9, 10, 11, 12, 13, 14. Among them, the distinct regulation of several substrates needs to be examined in glioma cells.…”