Roles for cyclin D1 and O -fucose in Notch signaling

Abstract

Notch receptors are transmembrane glycoproteins that control growth and cell fate in the metazoa and are dysregulated in several diseases and neoplasms. The four Notch receptors in mammals contain 29--36 epidermal growth factor-like (EGF) repeats, many potentially modified by O-fucose glycans that control their ligand interactions. Protein O-fucosyltransferase-1 (Pofut1) adds fucose to specific Ser/Thr residues allowing further elongation by other glycotransferases to di-, tri-, or tetrasaccharides. Notch ligand binding activates target genes, many via specific binding sequences in their promoters. The function of these O-fucose modifications and the downstream signaling of Notch to the target gene cyclin D1 was the focus of this work.;RKE-1 cells overexpressing activated Notch1 exhibit increased cyclin D1 transcripts and become transformed by a previously unknown mechanism. Mutational analysis was used to identify the sole Notch1-responsive region of the cyclin D1 promoter. This region also responds to activated Notch4 and ligand-induced Notch signaling. The cyclin D1 gene was subsequently found to be a physiologic target of Notch signaling in Pofut1-null mouse embryos and in embryos with inactivated Notch1. In vitro, Notch1 induced transformation of RKE cells was found to be dependent on increased expression of cyclin D1 protein through the use of cyclin D1 antisense.;Pofut1 is essential for Notch signaling both in vitro and in vivo; mice lacking the Pofut1 gene die at mid-gestation. In Drosophila, Pofut1 is thought to be required for trafficking Notch to the cell surface. We found that Pofut1-null murine ES cells do not bind Notch ligands and do not transduce Notch signals in vitro. However, they are similar to wild type ES cells in cell-surface Notch expression and in ligand-independent Notch signaling. Expression of wild-type Pofut1 corrected these deficiencies more efficiently than a fucosyltransferase-mutant Pofut1. CHO cells with reduced Pofut1 levels are similarly deficient. Lec13 CHO mutant cells, which have reduced GDP-fucose, bind less ligand than wild-type cells and are also deficient in ligand-induced Notch signaling but can be corrected by fucose supplementation. Thus, mammalian Notch receptors require O-fucose in order to optimally bind Notch ligands and thereby signal.