TY - JOUR
T1 - The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling
AU - Shi, Shaolin
AU - Ge, Changhui
AU - Luo, Yi
AU - Hou, Xinghua
AU - Haltiwanger, Robert S.
AU - Stanley, Pamela
PY - 2007/7/13
Y1 - 2007/7/13
N2 - 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.
AB - 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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U2 - 10.1074/jbc.M702593200
DO - 10.1074/jbc.M702593200
M3 - Article
C2 - 17504756
AN - SCOPUS:34547111586
SN - 0021-9258
VL - 282
SP - 20133
EP - 20141
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 28
ER -