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

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Cripto is a membrane-bound co-receptor for Nodal, a member of the transforming growth factor-β 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 Thr72, 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 Thr72 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.

Original languageEnglish (US)
Pages (from-to)20133-20141
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number28
DOIs
StatePublished - Jul 13 2007

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Fucose
Threonine
Epidermal Growth Factor
Assays
Embryonic Stem Cells
Stem cells
Substitution reactions
Embryonic Structures
Embryoid Bodies
Phenotype
Transforming Growth Factors
Enzymes
Amino Acid Substitution
Cardiac Myocytes
Proteins
Ligands
Membranes
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling. / Shi, Shaolin; Ge, Changhui; Luo, Yi; Hou, Xinghua; Haltiwanger, Robert S.; Stanley, Pamela.

In: Journal of Biological Chemistry, Vol. 282, No. 28, 13.07.2007, p. 20133-20141.

Research output: Contribution to journalArticle

Shi, Shaolin ; Ge, Changhui ; Luo, Yi ; Hou, Xinghua ; Haltiwanger, Robert S. ; Stanley, Pamela. / The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 28. pp. 20133-20141.
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abstract = "Cripto is a membrane-bound co-receptor for Nodal, a member of the transforming growth factor-β 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 Thr72, 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 Thr72 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.",
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