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J. Biol. Chem., Vol. 282, Issue 28, 20133-20141, July 13, 2007

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

Shaolin Shi¹, Changhui Ge, Yi Luo², Xinghua Hou, Robert S. Haltiwanger, and Pamela Stanley³

From the Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461 and the Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, New York 11794-5215

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

Received for publication, March 26, 2007 , and in revised form, May 11, 2007.

^* This work was supported by NCI Grant CA95022 (to P. S.) and NIGMS Grant GM61126 (to R. S. H.) from the National Institutes of Health and in part by the Albert Einstein Cancer Center Grant PO1 13330. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Division of Nephrology, Dept. of Medicine, Mount Sinai School of Medicine, Box 1243, One Gustave L. Levy Place, New York, NY 10029.

² Present address: The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609.

³ To whom correspondence should be addressed: Dept. of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461. Tel.: 718-430-3346; Fax: 718-430-8574; E-mail: stanley{at}aecom.yu.edu.

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