title: The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling.
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.
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, NY 10461, USA.
authors.forename: Shaolin
authors.initials: S
authors.lastname: Shi
authors.forename: Changhui
authors.initials: C
authors.lastname: Ge
authors.forename: Yi
authors.initials: Y
authors.lastname: Luo
authors.forename: Xinghua
authors.initials: X
authors.lastname: Hou
authors.forename: Robert S
authors.initials: RS
authors.lastname: Haltiwanger
authors.forename: Pamela
authors.initials: P
authors.lastname: Stanley
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D005643:Fucose / Q000235:genetics / Q000378:metabolism; D005647:Fucosyltransferases / Q000172:deficiency / Q000378:metabolism; D058851:GPI-Linked Proteins; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D055457:Nodal Protein; D046188:Protein Modification, Translational / Q000502:physiology*; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
chemical_list: D058851:GPI-Linked Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C526007:NODAL protein, human; C497761:NOTCH1 protein, human; D009363:Neoplasm Proteins; D055457:Nodal Protein; C526008:Nodal protein, mouse; C497762:Notch1 protein, mouse; D051881:Receptor, Notch1; C070636:TDGF1 protein, human; C489450:Tdgf1 protein, mouse; D016212:Transforming Growth Factor beta; D005643:Fucose; D013912:Threonine; D004815:Epidermal Growth Factor; D005647:Fucosyltransferases