The Threonine That Carries Fucose, but Not Fucose, Is Required for Cripto to Facilitate Nodal Signaling

Shaolin Shi,Changhui Ge,Yi Luo,Xinghua Hou,Robert S Haltiwanger,Pamela Stanley

doi:10.1074/jbc.m702593200

Abstract

Cripto is a membrane-bound co-receptor for Nodal, a member of the transforming growth factor-beta superfamily. Mouse embryos lacking either Cripto or Nodal have the same lethal phenotype at embryonic day 7.5. Previous studies suggest that O-fucosylation of the epidermal growth factor-like (EGF) repeat in Cripto is essential for the facilitation of Nodal signaling. Substitution of Ala for the Thr to which O-fucose is attached led to functional inactivation of both human and mouse Cripto. However, embryos null for protein O-fucosyltransferase 1, the enzyme that adds O-fucose to EGF repeats, do not exhibit a Cripto null phenotype and die at about embryonic day 9.5. This suggested that the loss of O-fucose from the EGF repeat may not have led to the inactivation of Cripto in previous studies. Here we investigate this hypothesis and show the following: 1) protein O-fucosyltransferase 1 is indeed the enzyme that adds O-fucose to Cripto; 2) Pofut1(-/-) embryonic stem cells behave the same as Pofut1(+/+) embryonic stem cells in a Nodal signaling assay; 3) Pofut1(-/-) and Pofut1(+/+) embryoid bodies are indistinguishable in their ability to differentiate into cardiomyocytes; and 4) none of 10 amino acid substitutions at Thr(72), including Ser which acquires O-fucose, rescues the activity of mouse Cripto in Nodal signaling assays. Therefore, the Thr to which O-fucose is linked in Cripto plays a key functional role, but O-fucose at Thr(72) is not required for Cripto to function in cell-based signaling assays or in vivo. By contrast, we show that O-fucose, and not the Thr to which it is attached, is required in the ligand-binding domain of Notch1 for Notch1 signaling.

Highlights

Mesoderm formation and the generation of left-right asymmetry [1]
O-Fucosylation is a comparatively rare form of glycosylation in which fucose is transferred to Thr or Ser in epidermal growth factor-like (EGF) repeats with the consensus sequence, C2-X4–5-(T/S)-C3, which occur in a variety of proteins, including urinary-type plasminogen activator [21], clotting factors VII and IX [22], Notch receptors [23] and their ligands [24], and Cripto [25, 26]
In the case of Cripto, Thr to Ala substitution in the EGF repeat of human or mouse Cripto leads to an inactive Cripto unable to facilitate Nodal signaling in cell-based assays [25, 26]

Summary

Introduction

Mesoderm formation and the generation of left-right asymmetry [1]. The major components of the Nodal-signaling pathway are the soluble ligand Nodal, activin membrane receptors (ActRIIB and ALK4) to which Nodal binds, Smad2 and Smad4 signal-transducing molecules, and the transcription factor Fast1 (FoxH1). In the case of Cripto, Thr to Ala substitution in the EGF repeat of human or mouse Cripto leads to an inactive Cripto unable to facilitate Nodal signaling in cell-based assays [25, 26].

Results

Conclusion