Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling

Mammalian Notch receptors contain 29–36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required fo...

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Bibliographic Details
Published in:The Journal of biological chemistry 2008-05, Vol.283 (20), p.13638-13651
Main Authors: Stahl, Mark, Uemura, Kazuhide, Ge, Changhui, Shi, Shaolin, Tashima, Yuko, Stanley, Pamela
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cell Membrane - metabolism
CHO Cells
Cricetinae
Cricetulus
Drosophila
Endoplasmic Reticulum - metabolism
Fucose - chemistry
Fucose - physiology
Fucosyltransferases - metabolism
Fucosyltransferases - physiology
Humans
Mice
Mice, Transgenic
Models, Biological
Receptors, Notch - metabolism
Signal Transduction
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Summary:Mammalian Notch receptors contain 29–36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1–/– ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1–/– cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated, inactive, endoplasmic reticulum glucosidase. Therefore, mammalian Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but, in contrast to Drosophila, Pofut1 is not required for stable cell surface expression of Notch. Importantly, we also show that, under certain circumstances, mammalian Notch receptors are capable of signaling in the absence of Pofut1 and O-fucose.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M802027200