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

2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês

10.1074/jbc.m702593200

ISSN

1083-351X

Autores

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

Tópico(s)

Genomics and Chromatin Dynamics

Resumo

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. 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. Nodal, a member of the TGF-β 4The abbreviations used are: TGF-β, transforming growth factor-β; EGF, epidermal growth factor-like; CFC, cripto-FRL-cryptic; Pofut, protein O-fucosyltransferase; TSR, thrombospondin type 1 repeat; CHO, Chinese hamster ovary; FBS, fetal bovine serum; EB, embryoid body; RT, reverse transcriptase; E, embryonic day; uPA, urinary-type plasminogen activator; MAPK, mitogen-activated protein kinase. 4The abbreviations used are: TGF-β, transforming growth factor-β; EGF, epidermal growth factor-like; CFC, cripto-FRL-cryptic; Pofut, protein O-fucosyltransferase; TSR, thrombospondin type 1 repeat; CHO, Chinese hamster ovary; FBS, fetal bovine serum; EB, embryoid body; RT, reverse transcriptase; E, embryonic day; uPA, urinary-type plasminogen activator; MAPK, mitogen-activated protein kinase. superfamily, plays essential roles in the embryonic development of vertebrates, including mesoderm formation and the generation of left-right asymmetry (1Schier A.F. Annu. Rev. Cell Dev. Biol. 2003; 19: 589-621Crossref PubMed Scopus (519) Google Scholar). 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 addition, Cripto, a membrane glycoprotein with a glycosylphosphatidylinositol anchor, is an essential co-receptor for Nodal and is required for Nodal signaling. Cripto contains the following two functional domains that play distinct roles in Nodal signaling: a truncated epidermal growth factor-like (EGF) repeat and the CFC domain (2Shen M.M. Schier A.F. Trends Genet. 2000; 16: 303-309Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Cripto and Nodal null mouse embryos exhibit extremely similar phenotypes, both lacking embryonic mesoderm and definitive endoderm (2Shen M.M. Schier A.F. Trends Genet. 2000; 16: 303-309Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 3Ding J.X.Y. Yan Y.T. Chen A. Desai N. Wynshaw-Boris A. Shen M.M. Nature. 1998; 395: 702-707Crossref PubMed Scopus (396) Google Scholar, 4Brennanj L.C. Norris D.P. Rodriguez T.A. Beddington R.S. Robertson E.J. Nature. 2001; 411: 965-969Crossref PubMed Scopus (409) Google Scholar, 5Xu C. Liguori G. Persico M.G. Adamson E.D. Development (Camb.). 1999; 126: 483-494Crossref PubMed Google Scholar). Cripto is also a co-receptor for GDF1/Vg1, another member of the TGF-β superfamily (6Cheng S.K. Olale F. Bennett J.T. Brivanlou A.H. Schier A.F. Genes Dev. 2003; 17: 31-36Crossref PubMed Scopus (147) Google Scholar). In addition, mouse Gdf3 (growth-differentiation factor 3) is similar to Nodal in requiring Cripto for its signaling activity (7Chen C. Ware S.M. Sato A. Houston-Hawkins D.E. Habas R. Matzuk M.M. Shen M.M. Brown C.W. 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O-Fucosylation is a comparatively rare form of glycosylation in which fucose is transferred to Thr or Ser in EGF repeats with the consensus sequence, C2-X4–5-(T/S)-C3, which occur in a variety of proteins, including urinary-type plasminogen activator (uPA) (21Rabbani S.A. Mazar A.P. Bernier S.M. Haq M. Bolivar I. Henkin J. Goltzman D. J. Biol. Chem. 1992; 267: 14151-14156Abstract Full Text PDF PubMed Google Scholar), clotting factors VII and IX (22Harris R.J. Ling V.T. Spellman M.W. J. Biol. Chem. 1992; 267: 5102-5107Abstract Full Text PDF PubMed Google Scholar), Notch receptors (23Moloney D.J. Shair L.H. Lu F.M. Xia J. Locke R. Matta K.L. Haltiwanger R.S. J. Biol. Chem. 2000; 275: 9604-9611Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar) and their ligands (24Panin V.M. Shao L. Lei L. Moloney D.J. Irvine K.D. Haltiwanger R.S. J. Biol. Chem. 2002; 277: 29945-29952Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), and Cripto (25Schiffer S.G. Foley S. Kaffashan A. Hronowski X. Zichittella A.E. Yeo C.Y. Miatkowski K. Adkins H.B. Damon B. Whitman M. Salomon D. Sanicola M. Williams K.P. J. Biol. Chem. 2001; 276: 37769-37778Abstract Full Text Full Text PDF PubMed Google Scholar, 26Yan Y.T. Liu J.J. Luo Y.E.C. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Crossref PubMed Scopus (167) Google Scholar) (for an extensive list of predicted targets, see Ref. 27Rampal R. Luther K.B. Haltiwanger R.S. Curr. Mol. Med. 2007; (in press)PubMed Google Scholar). To date, O-fucose has been reported to be required for the function of uPA (21Rabbani S.A. Mazar A.P. Bernier S.M. Haq M. Bolivar I. Henkin J. Goltzman D. J. Biol. Chem. 1992; 267: 14151-14156Abstract Full Text PDF PubMed Google Scholar), Cripto (25Schiffer S.G. Foley S. Kaffashan A. Hronowski X. Zichittella A.E. Yeo C.Y. Miatkowski K. Adkins H.B. Damon B. Whitman M. Salomon D. Sanicola M. Williams K.P. J. Biol. Chem. 2001; 276: 37769-37778Abstract Full Text Full Text PDF PubMed Google Scholar, 26Yan Y.T. Liu J.J. Luo Y.E.C. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Crossref PubMed Scopus (167) Google Scholar), and Notch receptors (28Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar, 29Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (719) Google Scholar, 30Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (323) Google Scholar, 31Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 32Rampal R. Arboleda-Velasquez J.F. Nita-Lazar A. Kosik K.S. Haltiwanger R.S. J. Biol. Chem. 2005; 280: 32133-32140Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 33Xu A. Lei L. Irvine K.D. J. Biol. Chem. 2005; 280: 30158-30165Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). uPA lacking O-fucose loses its mitogenic but not its receptor binding activity, suggesting that O-fucose is required for uPA-induced signaling (21Rabbani S.A. Mazar A.P. Bernier S.M. Haq M. Bolivar I. Henkin J. Goltzman D. J. Biol. Chem. 1992; 267: 14151-14156Abstract Full Text PDF PubMed Google Scholar). 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 (25Schiffer S.G. Foley S. Kaffashan A. Hronowski X. Zichittella A.E. Yeo C.Y. Miatkowski K. Adkins H.B. Damon B. Whitman M. Salomon D. Sanicola M. Williams K.P. J. Biol. Chem. 2001; 276: 37769-37778Abstract Full Text Full Text PDF PubMed Google Scholar, 26Yan Y.T. Liu J.J. Luo Y.E.C. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Crossref PubMed Scopus (167) Google Scholar). The requirement of O-fucose for Notch receptors to function has been demonstrated in several contexts. Lec13, a CHO mutant cell line, which is defective in GDP-fucose synthesis and thereby has insufficient O-fucosylation on Notch receptors, has impaired Jagged1-induced Notch signaling (28Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar, 29Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (719) Google Scholar). In addition, inactivation of Pofut1 (protein O-fucosyltransferase 1), the glycosyltransferase that catalyzes the addition of fucose to EGF-containing proteins, results in severe Notch signaling defects in both Drosophila and mouse (30Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (323) Google Scholar, 31Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 34Sasamura T. Sasaki N. Miyashita F. Nakao S. Ishikawa H.O. Ito M. Kitagawa M. Harigaya K. Spana E. Bilder D. Perrimon N. Matsuno K. Development (Camb.). 2003; 130: 4785-4795Crossref PubMed Scopus (146) Google Scholar). Finally, mutation of O-fucose glycosylation sites (Thr to Ala mutations) within either EGF repeat 12 or 27 of mouse Notch1 causes a significant reduction in Notch signaling activity in cell-based assays (32Rampal R. Arboleda-Velasquez J.F. Nita-Lazar A. Kosik K.S. Haltiwanger R.S. J. Biol. Chem. 2005; 280: 32133-32140Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). O-Fucosylation also occurs in thrombospondin type 1 repeats (TSRs) (35Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. J. Biol. Chem. 2001; 276: 6485-6498Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 36Gonzalez de Peredo A. Klein D. Macek B. Hess D. Peter-Katalinic J. Hofsteenge J. Mol. Cell. Proteomics. 2002; 1: 11-18Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Pofut1 cannot add O-fucose to TSRs, suggesting the presence of another enzyme (37Luo Y. Nita-Lazar A. Haltiwanger R.S. J. Biol. Chem. 2006; 281: 9385-9392Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). A distantly related protein O-fucosyltransferase encoded by the Pofut2 gene in the metazoa (38Roos C. Matlila K.M. Renkonen R. J. Biol. Chem. 2002; 277: 3168-3175Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) has been shown recently to catalyze the transfer of fucose to Thr or Ser in TSR with the consensus CXX(S/T))CXXG (37Luo Y. Nita-Lazar A. Haltiwanger R.S. J. Biol. Chem. 2006; 281: 9385-9392Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 39Luo Y. Koles K. Vorndam W. Haltiwanger R.S. Panin V.M. J. Biol. Chem. 2006; 281: 9393-9399Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The Drosophila form of Pofut2 (OFUT2) is not capable of adding O-fucose to an EGF repeat from human factor VII in vitro (39Luo Y. Koles K. Vorndam W. Haltiwanger R.S. Panin V.M. J. Biol. Chem. 2006; 281: 9393-9399Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The O-linked fucose on TSRs can be elongated by a β1,3-linked glucose (36Gonzalez de Peredo A. Klein D. Macek B. Hess D. Peter-Katalinic J. Hofsteenge J. Mol. Cell. Proteomics. 2002; 1: 11-18Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 37Luo Y. Nita-Lazar A. Haltiwanger R.S. J. Biol. Chem. 2006; 281: 9385-9392Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 40Kozma K. Keusch J.J. Hegemann B. Luther K.B. Klein D. Hess D. Haltiwanger R.S. Hofsteenge J. J. Biol. Chem. 2006; 281: 36742-36751Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 41Sato T. Sato M. Kiyohara K. Sogabe M. Shikanai T. Kikuchi N. Togayachi A. Ishida H. Ito H. Kameyama A. Gotoh M. Narimatsu H. Glycobiology. 2006; 16: 1194-1206Crossref PubMed Scopus (60) Google Scholar). Because O-fucose appeared to be essential for Cripto/Nodal signaling, we had expected that targeted mutation of Pofut1 would give rise to a Cripto–/– phenotype. Cripto null embryos are described as "trunkless" and die at about E7.5 (2Shen M.M. Schier A.F. Trends Genet. 2000; 16: 303-309Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 3Ding J.X.Y. Yan Y.T. Chen A. Desai N. Wynshaw-Boris A. Shen M.M. Nature. 1998; 395: 702-707Crossref PubMed Scopus (396) Google Scholar, 4Brennanj L.C. Norris D.P. Rodriguez T.A. Beddington R.S. Robertson E.J. Nature. 2001; 411: 965-969Crossref PubMed Scopus (409) Google Scholar, 5Xu C. Liguori G. Persico M.G. Adamson E.D. Development (Camb.). 1999; 126: 483-494Crossref PubMed Google Scholar). However, Pofut1–/– embryos die later at about E9.5 with a phenotype consistent with a global deficiency in the canonical Notch signaling pathway (30Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (323) Google Scholar). Therefore, an important question is why the ablation of Pofut1 in mouse embryos did not inactivate Cripto and give rise to a Cripto null phenotype? It has been shown that there are no wild type Pofut1 gene transcripts in E6.5 Pofut1–/– embryos that could rescue a Cripto phenotype (43Shi S. Stahl M. Lu L. Stanley P. Mol. Cell. Biol. 2005; 25: 9503-9508Crossref PubMed Scopus (51) Google Scholar). Possible alternative explanations are that Pofut1 is not the enzyme that transfers fucose to Cripto, that another enzyme is also capable of adding fucose to Cripto, or that the Thr in the EGF domain to which fucose is attached is critical, rather than the fucose. Here we show the following: Cripto is O-fucosylated in Pofut1+/+ but not Pofut1–/– ES cell extracts; Nodal signaling based on activation of a FAST2 reporter construct occurs equivalently in Pofut1+/+ and Pofut1–/– ES cells; Pofut1–/– and Pofut1+/+ embryoid bodies differentiate into cardiomyocytes equivalently in culture, a process that requires functional Cripto/Nodal signaling (44Xu C. Liguori G. Adamson E.D. Persico M.G. Dev. Biol. 1998; 196: 237-247Crossref PubMed Scopus (100) Google Scholar, 45Parisi S. D'Andrea D. Lago C.T. Adamson E.D. Persico M.G. Minchiotti G. J. Cell Biol. 2003; 163: 303-314Crossref PubMed Scopus (137) Google Scholar); Ser instead of Thr at amino acid 72 in mouse Cripto acquires O-fucose but is not functional in Cripto/Nodal signaling; and eight other amino acids in place of Thr72 in mouse Cripto gave Cripto with greatly impaired activity in Nodal signaling assays. Therefore, Thr72 and not fucose is required for Cripto function. ES Cell Lines—ES cell lines were isolated from outgrowths of blastocysts obtained from mating Pofut1+/– heterozygotes and genotyped as described previously (43Shi S. Stahl M. Lu L. Stanley P. Mol. Cell. Biol. 2005; 25: 9503-9508Crossref PubMed Scopus (51) Google Scholar). Independent cell lines with the Pofut1+/+, Pofut1+/– or Pofut1–/– genotype were obtained. Expression Constructs—The constructs for Nodal signaling assays (all derived from mouse) were kindly provided by Dr. Michael Shen (Rutgers University) (26Yan Y.T. Liu J.J. Luo Y.E.C. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Crossref PubMed Scopus (167) Google Scholar). They were pcDNA3-FLAG-Cripto, pcDNA3-HA-Cripto, pcDNA3-FLAG-Cripto (tr1) carrying three point mutations in the EGF-like domain (L75A/S77A/F78A), pcDNA-FLAG-Cripto(T72A), pcDNA3-Nodal, pcDNA-Fast-2, and the A3-luc luciferase reporter, which contains three tandem repeats of a Nodal-responsive element from the Xenopus Mix.2 gene (26Yan Y.T. Liu J.J. Luo Y.E.C. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Crossref PubMed Scopus (167) Google Scholar). Renilla luciferase, pRL-TK (Promega), was used for normalization. pcDNA3 (Invitrogen) was used to maintain the same DNA concentration in transfection experiments. The mouse Notch1 fragment containing the N terminus and EGF repeats 1–18 with a Myc-His6 C-terminal tag was described previously (46Shao L. Moloney D.J. Haltiwanger R. J. Biol. Chem. 2003; 278: 7775-7782Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Preparation of Cell Extracts—Pofut1+/+ and Pofut1–/– ES cells grown in the absence of feeder cells were genotyped with the primers PS644 and PS645 as described previously (43Shi S. Stahl M. Lu L. Stanley P. Mol. Cell. Biol. 2005; 25: 9503-9508Crossref PubMed Scopus (51) Google Scholar). Cells were grown on 10-cm gelatin-coated dishes with ES cell culture medium. When the cells became confluent, they were scraped off the plate and washed three times with TBS (10 mm Tris-HCl, pH 7.5, 0.15 m NaCl). The cell pellet was placed on ice, and 2 ml of lysis buffer (TBS with 1% Nonidet P-40) and 1× protease inhibitor mixture (Roche Applied Science) was added. The cells were resuspended by pipetting and incubated on ice for 15 min with repeated vortexing. The cell lysate was spun at ∼12,000 × g for 10 min at 4 °C. The supernatant was snap-frozen using liquid nitrogen and stored at –80 °C. Protein O-Fucosyltransferase 1 Assays—Assays for Pofut1 activity were performed essentially as described previously (39Luo Y. Koles K. Vorndam W. Haltiwanger R.S. Panin V.M. J. Biol. Chem. 2006; 281: 9393-9399Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 47Wang Y. Shao L. Shi S. Harris R.J. Spellman M.W. Stanley P. Haltiwanger R.S. J. Biol. Chem. 2001; 276: 40338-40345Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Substrates for the assays were produced as follows. 1) ES cells cultured in a 10-cm gelatinized plate to 50% confluency were transfected with 24 μg of Notch1 EGF1–18 DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. After 24 h, the medium was collected and centrifuged, and the supernatant was incubated on a rotator with 50 μl of Ni2+-nitrilotriacetic acid-agarose (Qiagen) for 1 h at 4 °C. The agarose beads were washed five times in 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and complete protease inhibitor (Roche Applied Science) mixture. 2) Production of bacterially expressed EGF repeat 1 from human factor VII was described previously (39Luo Y. Koles K. Vorndam W. Haltiwanger R.S. Panin V.M. J. Biol. Chem. 2006; 281: 9393-9399Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 48Wang Y. Lee G.F. Kelley R.F. Spellman M.W. Glycobiology. 1996; 6: 837-842Crossref PubMed Scopus (57) Google Scholar, 49Rampal R. Li A.S. Moloney D.J. Georgiou S.A. Luther K.B. Nita-Lazar A. Haltiwanger R.S. J. Biol. Chem. 2005; 280: 42454-42563Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). 3) To produce the EGF repeat (amino acids 62–91) from mouse Cripto, the corresponding DNA sequences were amplified from pcDNA3-HA-Cripto and subcloned into the BamHI and XhoI sites of the pET20b+ plasmid, in-frame with the C-terminal His6 tag. The primers used were 5′-ACCGAAGGATCCTAAGTCGCTTAATAAAACTTGC-3′ and 5′-TGAAATCTCGAGGCGAACATCATGTTCACAGTTGCG-3′. The EGF repeat was expressed in bacteria and purified as described (49Rampal R. Li A.S. Moloney D.J. Georgiou S.A. Luther K.B. Nita-Lazar A. Haltiwanger R.S. J. Biol. Chem. 2005; 280: 42454-42563Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Bacterial expression and characterization of the third thrombospondin type 1 repeat from human thrombospondin-1 (TSR3-TSP1) were performed as described (37Luo Y. Nita-Lazar A. Haltiwanger R.S. J. Biol. Chem. 2006; 281: 9385-9392Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). All acceptors were assayed for their ability to be a substrate for the transfer of fucose from GDP-[3H]fucose by Pofut1 and Pofut2 present in extracts of ES cells as described (39Luo Y. Koles K. Vorndam W. Haltiwanger R.S. Panin V.M. J. Biol. Chem. 2006; 281: 9393-9399Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 47Wang Y. Shao L. Shi S. Harris R.J. Spellman M.W. Stanley P. Haltiwanger R.S. J. Biol. Chem. 2001; 276: 40338-40345Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Products of the assay were separated and counted or analyzed by SDS-PAGE. Site-directed Mutagenesis of Cripto and Notch1—pcDNA3-FLAG-Cripto was used as a template in the QuikChange site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions. All mutants except those provided by Michael Shen (Rutgers University) were generated in the Stony Brook University Molecular Cloning Service Core. All constructs were sequenced to confirm mutagenesis. A full-length construct of mouse Notch1, in which the C-terminal PEST domain is replaced by six tandem MYC epitopes in pCS2+, was a kind gift of Raphael Kopan (Washington University School of Medicine). The primers to generate N1/T12S and N1/EGF11–15/T12S were 5′-CCATGTCAGAATGATGCCTCGTGCCTGGACCAGATTG-3′ and 5′-CAATCTGGTCCAGGCACGAGGCATCATTCTGACATGG-3′; the primers to generate N1/T12A were 5′-CCATGTCAGAATGACGCCGCATGCCTGGACCAGATTG-3′ and 5′-CAATCTGGTCCAGGCACGTGGCGTCATTCTGACATGG-3′; the primers to generate the N1/T12A revertant were 5′-CCATGTCAGAATGATGCCACTTGCCTGGACCAGATTG-3′ and 5′-CAATCTGGTCCAGGCAAGTGGCATCATTCTGACATGG-3′; and the primers for N1/EGF11–15 and EGF11–15T12A were described previously (46Shao L. Moloney D.J. Haltiwanger R. J. Biol. Chem. 2003; 278: 7775-7782Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The QuikChange site-directed mutagenesis protocol from Stratagene was used for site-directed mutagenesis. All constructs were sequenced to confirm nucleotide changes. Analysis of O-Fucosylation—O-Fucosylation of wild type and mutant forms of mouse Cripto was evaluated as described (26Yan Y.T. Liu J.J. Luo Y.E.C. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Crossref PubMed Scopus (167) Google Scholar), and O-fucosylation of Notch fragments N1/EGF11–15 (with C-terminal Myc-His6 tags) and mutants T466A and T466S was determined essentially as described (46Shao L. Moloney D.J. Haltiwanger R. J. Biol. Chem. 2003; 278: 7775-7782Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In brief, expression constructs were transiently expressed in Pro–Lec1.3C Chinese hamster ovary (Lec1 CHO) cells (50Chen W. Stanley P. Glycobiology. 2003; 13: 43-50Crossref PubMed Scopus (94) Google Scholar) using FuGENE 6 (Roche Applied Science) or GenePorter (Genlantis). After 24 h the medium was replaced with fresh medium containing 20 μCi/ml [6-3H]fucose (American Radiochemical Corp., St. Louis). After 48 h, medium and lysates were collected, and Notch1 fragments were purified by rotating with Ni2+-nitrilotriacetic acid-agarose beads (Qiagen) overnight at 4 °C. After washing five times with 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS, fragments were eluted with 100 mm EDTA, pH 8.0. After electrophoresis on a 10% SDS gel, the dried gel was treated with EN3HANCER (PerkinElmer Life Sciences) and exposed for 1 week at –80 °C to a BioMax MS film (Eastman Kodak Co.). Nodal Signaling Assay with ES Cells and 293T Cells—A method to assay Nodal signaling was developed in ES cells based on Yan et al. (26Yan Y.T. Liu J.J. Luo Y.E.C. Haltiwanger R.S. Abate-Shen C. Shen M.M. Mol. Cell. Biol. 2002; 22: 4439-4449Crossref PubMed Scopus (167) Google Scholar). Briefly, ES cells (5 × 105) were plated in 24-well plates. Transfection was performed after 24 h (∼50% confluency) with 50 ng of Renilla luciferase DNA, 300 ng of A3-luc reporter, 300 ng of pcDNA3-Fast, 400 ng of pcDNA3-HA-Cripto, 400 ng of pcDNA3-Nodal, and various amounts of pcDNA3 to bring the DNA concentration to 1.45 μg. The DNA was mixed with 4 μl of Lipofectamine-2000 (Invitrogen) and added to each well with 1 ml of Opti-MEM medium (Invitrogen) containing an additional 3% fetal bovine serum (FBS; Gemini). After 24 h at 37 °C, the cells were lysed by overlaying 300 μl of lysis buffer from the Dual-Luciferase Reporter Assay System (E1910, Promega), and rocked at room temperature for 15 min. The cell lysate was transferred to an Eppendorf tube and frozen at –80 °C. After thawing and vortexing, luciferase activity assays were conducted according to the manufacturer's instruction using a luminometer. Renilla luciferase activity was used to normalize for differences in transfection efficiency. Each signaling assay was performed in duplicate and was repeated at least twice. The Nodal signaling assay in HEK-293T cells was the same except that the cell density at transfection was ∼80%. Differentiation of Embryoid Bodies into Cardiomyocytes—Independently derived Pofut1–/– and Pofut1+/+ ES cell lines were differentiated into embryoid bodies (EB) as described with minor modification (51Wobus A.M. Guan K. Yang H.T. Boheler K.R. Methods Mol. Biol. 2002; 185: 127-156PubMed Google Scholar). Briefly, undifferentiated ES cells passaged twice to remove feeder cells were cultured in hanging drops of 500 cells at 37 °C in 5% CO2. After 2 days the cells were

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