Highly Conserved O-Fucose Sites Have Distinct Effects on Notch1 Function

Raajit Rampal,Joseph F Arboleda-Velasquez,Alexandra Nita-Lazar,Kenneth S Kosik,Robert S Haltiwanger

doi:10.1074/jbc.m506104200

Raajit Rampal, Joseph F Arboleda-Velasquez + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m506104200

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Abstract

The extracellular domain of mouse Notch1 contains 36 tandem epidermal growth factor-like (EGF) repeats, many of which are modified with O-fucose. Previous work from several laboratories has indicated that O-fucosylation plays an important role in ligand mediated Notch activation. Nonetheless, it is not clear whether all, or a subset, of the EGF repeats need to be O-fucosylated. Three O-fucose sites are invariantly conserved in all Notch homologues with 36 EGF repeats (within EGF repeats 12, 26, and 27). To investigate which O-fucose sites on Notch1 are important for ligand-mediated signaling, we mutated the three invariant O-fucose sites in mouse Notch1, along with several less highly conserved sites, and evaluated their ability to transduce Jagged1- and Delta1-mediated signaling in a cell-based assay. Our analysis revealed that mutation of any of the three invariant O-fucose sites resulted in significant changes in both Delta1 and Jagged1 mediated signaling, but mutations in less highly conserved sites had no detectable effect. Interestingly, mutation of each invariant site gave a distinct effect on Notch function. Mutation of the O-fucose site in EGF repeat 12 resulted in loss of Delta1 and Jagged1 signaling, while mutation of the O-fucose site in EGF repeat 26 resulted in hyperactivation of both Delta1 and Jagged1 signaling. Mutation of the O-fucose site in EGF repeat 27 resulted in faulty trafficking of the Notch receptor to the cell surface and a decreased S1 processing of the receptor. These results indicate that the most highly conserved O-fucose sites in Notch1 are important for both processing and ligand-mediated signaling in the context of a cell-based signaling assay.

Highlights

For modification by O-fucose [6, 7]
This mutant demonstrated increases in both Delta and Serrate binding to Notch in in vitro binding assays. This effect was specific to the epidermal growth factor-like (EGF) repeat 12 mutant, as mutation of other O-fucose sites did not result in changes in Notch activity
COS-7 cells were transiently transfected with a plasmid encoding mouse Notch1 along with a luciferase reporter and a ␤-galactosidase reporter

Summary

Introduction

For modification by O-fucose [6, 7]. Some of the O-fucose moieties on EGF repeats of Notch can be further elongated by the action of Fringe, a fucose-specific ␤1,3-N-acetylglucosaminyltransferase [8, 9]. This effect was specific to the EGF repeat 12 mutant, as mutation of other O-fucose sites did not result in changes in Notch activity. We have sought to determine the role these three O-fucose sites in mouse Notch1 by analyzing the effects of mutation on ligand-mediated signaling, S1 processing, and cellsurface expression.

Results

Conclusion