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Originally published In Press as doi:10.1074/jbc.M702593200 on May 15, 2007

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 Shi{ddagger}1, Changhui Ge{ddagger}, Yi Luo§2, Xinghua Hou{ddagger}, Robert S. Haltiwanger§, and Pamela Stanley{ddagger}3

From the {ddagger}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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nodal, a member of the TGF-beta4 superfamily, plays essential roles in the embryonic development of vertebrates, including mesoderm formation and the generation of left-right asymmetry (1). 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 (2). Cripto and Nodal null mouse embryos exhibit extremely similar phenotypes, both lacking embryonic mesoderm and definitive endoderm (2–5). Cripto is also a co-receptor for GDF1/Vg1, another member of the TGF-beta superfamily (6). In addition, mouse Gdf3 (growth-differentiation factor 3) is similar to Nodal in requiring Cripto for its signaling activity (7). More recently, other factors were found to regulate Nodal signaling via interaction with Cripto, including activin (8), Lefty (9, 10), and Tomoregulin-1(TMEFF1) (11). In addition, Cripto has been shown to activate the Ras/Raf/MAPK pathway (12–18) and the phosphatidylinositol 3-kinase/AKT pathway (19) independently of Nodal signaling. In fact, these signaling events appear to be triggered by the direct interaction of Cripto with Glypican-1. Finally, Cripto has been implicated in the formation of multiple tumors, including breast, pancreatic, and colorectal cancer (reviewed in Ref. 20).

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) (21), clotting factors VII and IX (22), Notch receptors (23) and their ligands (24), and Cripto (25, 26) (for an extensive list of predicted targets, see Ref. 27). To date, O-fucose has been reported to be required for the function of uPA (21), Cripto (25, 26), and Notch receptors (28–33). uPA lacking O-fucose loses its mitogenic but not its receptor binding activity, suggesting that O-fucose is required for uPA-induced signaling (21). 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 (25, 26). 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 (28, 29). 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 (30, 31, 34). 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 (32).

O-Fucosylation also occurs in thrombospondin type 1 repeats (TSRs) (35, 36). Pofut1 cannot add O-fucose to TSRs, suggesting the presence of another enzyme (37). A distantly related protein O-fucosyltransferase encoded by the Pofut2 gene in the metazoa (38) has been shown recently to catalyze the transfer of fucose to Thr or Ser in TSR with the consensus CXX(S/T))CXXG (37, 39). The Drosophila form of Pofut2 (OFUT2) is not capable of adding O-fucose to an EGF repeat from human factor VII in vitro (39). The O-linked fucose on TSRs can be elongated by a beta1,3-linked glucose (36, 37, 40, 41).

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 (2–5). However, Pofut1–/– embryos die later at about E9.5 with a phenotype consistent with a global deficiency in the canonical Notch signaling pathway (30). 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 (43). 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 (44, 45); 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
ES Cell Lines—ES cell lines were isolated from outgrowths of blastocysts obtained from mating Pofut1+/– heterozygotes and genotyped as described previously (43). 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) (26). 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 (26). 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 (46).

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 (43). 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 1x 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 x 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 (39, 47). 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 (39, 48, 49). 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 (49). Bacterial expression and characterization of the third thrombospondin type 1 repeat from human thrombospondin-1 (TSR3-TSP1) were performed as described (37). 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 (39, 47). 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 (46). 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 (26), 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 (46). In brief, expression constructs were transiently expressed in Pro–Lec1.3C Chinese hamster ovary (Lec1 CHO) cells (50) 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. (26). Briefly, ES cells (5 x 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 (51). 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 cultured in suspension until EBs formed, and then replated onto gelatin-coated tissue culture plates. Differentiation medium was high glucose Dulbecco's modified Eagle's medium (Invitrogen) containing 20% heated-inactivated FBS (Gemini), 25 mM HEPES, 0.1 mM mercaptoethanol, nonessential amino acids, and 2 mM glutamine (Specialty Media), but lacking LIF. Rythmic beating of EBs was monitored daily by phase microscopy, and the numbers of beating EBs were counted on day 10.

Reverse Transcriptase-PCR (RT-PCR)—Total RNA from either undifferentiated ES cells or EBs was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. 1 µg of total RNA was reverse-transcribed to cDNA with Takara RNA PCR kit version 2.1 (Takara) using oligo(dT) in 20 µl, and 1 µl of the reverse transcription product was used for PCR. PCR was performed using the following primers and PCR conditions (51): for cardiac {alpha}-myosin heavy chain, 5'-GGAAGAGTGAGCGGCGCATCAAGG (reverse) and 5'-CTGCTGGAGAGGTTATTCCTGG (forward), 64 °C annealing temperature, 301-bp product; for myosin light chain isoform 2V (MLC-2V), 5'-TGTGGGTCACCTGAGGCTGTGGTTCAG (forward) and 5'-GAAGGCTGACTATGTCCGGGAGATGC (reverse), 60 °C annealing temperature, 189-bp product; for Nkx2.5, 5'-CGACGGAAGCCACGCGTGCT (forward) and 5'-CCGCTGTCGCTTGCACTTG (reverse), 60 °C annealing temperature, 181-bp product; for beta-tubulin control, 5'-GGAACATAGCCGTAAACTGC (forward) and 5'-TCACTGTGCCTGAACTTACC (reverse), 60 °C annealing temperature, 317-bp product.

Notch Signaling Assay—CHO cells (Pro–5; 1.5 x 105) were plated in a 6-well plate, and the next day were transiently transfected with 150 ng of wild type or mutant pCS2+Notch1, 200 ng of TP-1 luciferase Notch reporter construct, and 8 ng of CMV-Renilla luciferase reporter to normalize transfection efficiency using FuGENE 6 (Roche Applied Science) according to the manufacturer's specifications. After incubation for 16 h in a 37 °C incubator, FuGENE 6 reagent was removed, and fresh {alpha}-minimum Eagle's medium containing 10% FBS (Gemini) was added. L cells (1.5 x 106) or L cells selected by flow cytometry to express high levels of Delta1 (43) were added to duplicate wells. After 30 h of co-culture, cell lysates were prepared in the lysis buffer provided in the Promega dual luciferase kit. Fold activation after normalization was determined as described (28). To compare the effects of introduced wild type and mutant Notch1, Notch signaling because of endogenous Notch signaling that occurred in vector control cultures was subtracted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cripto Is the Substrate of Pofut1 but Not Pofut2—To examine whether Pofut1 is responsible for the addition of O-fucose to Cripto, we took advantage of ES cells from Pofut1–/– and Pofut1+/+ mice. To determine that Pofut1–/– cells lack Pofut1 activity, a mouse Notch1 N-terminal fragment containing EGF repeats 1–18 was transiently expressed in both Pofut1–/– and Pofut1+/+ ES cells, purified from media on nickel-nitrilotriacetic acid-agarose, and incubated with recombinant Pofut1 and GDP-[3H]fucose in vitro. After 12 h at 37 °C, the beads were washed extensively, boiled in SDS-PAGE loading buffer, and analyzed by Western blot analysis and fluorography. Significantly less EGF1–18 protein was recovered from Pofut1–/– compared with Pofut1+/+ cells, suggesting that the loss of Pofut1 affects secretion of this Notch1 fragment (Fig. 1A). The fluorograph of the same samples shows that Notch1 EGF1–18 from Pofut1–/– cells was a substrate for fucosylation with Pofut1 in vitro, indicating that O-fucose sites are unoccupied on this protein. In contrast, Notch1 EGF1–18 from Pofut1+/+ cells was not a substrate for in vitro fucosylation, even though it was present in excess, consistent with all O-fucose sites being already occupied. Therefore, loss of Pofut1 in ES cells significantly reduces or eliminates O-fucosylation of Notch1.


Figure 1
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  		FIGURE 1.
Pofut1 O-fucosylates EGF repeats in Notch1, factor VII, and Cripto. A, Pofut1+/+ and Pofut1–/– ES cells were transfected with mouse Notch1 EGF-(1–18); the secreted fragment was purified on nickel-nitrilotriacetic acid-agarose and incubated with GDP-[3H]fucose in the presence of recombinant Pofut1. The products were analyzed by Western analysis with anti-Myc antibody (Ab)(left panel) and fluorography (right panel). Only N1 EGF-(1–18) from Pofut1–/– ES cells was labeled by [3H]fucose, consistent with the presence of empty O-fucose sites. B, bacterially expressed factor VII-EGF, Cripto-EGF, or a thrombospondin repeat (TSR3-TSP1) were incubated with GDP-[3H]fucose in the presence of cell extract from Pofut1+/+ or Pofut1–/– ES cells.

 
As an alternative approach to analyzing the activity of Pofut1, cell extracts were prepared from Pofut1+/+ (8-8) and Pofut1–/– (5-6-1) ES cells, and incubated with GDP-[3H]fucose and a bacterially expressed (i.e. nonfucosylated) EGF repeat from human factor VII. As expected, extracts from Pofut1+/+ cells were able to fucosylate the factor VII EGF repeat, whereas those from the Pofut1–/– cells were not (Fig. 1B). The presence of Pofut2 in ES cell extracts was examined by incubating with a bacterially expressed (i.e. nonfucosylated) thrombospondin type 1 repeat from human thrombospondin-1 (TSR3-TSP1). TSR3-TSP1 can be modified by Pofut2, but not by Pofut1, in in vitro assays (37, 39). Transfer of [3H]fucose from GDP-[3H]fucose to TSR3-TSP1 was detected following incubation with either extract, demonstrating that Pofut2 activity is present in ES cells and unaffected by the elimination of Pofut1 (Fig. 1B). Significantly, when a bacterially expressed EGF repeat from mouse Cripto was incubated with the extracts, only the extract from the Pofut1+/+ ES cells was able to catalyze fucosylation. Taken together, these results demonstrate that Pofut1 is the protein O-fucosyltransferase that O-fucosylates Cripto.


Figure 2
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  		FIGURE 2.
Lack of O-fucose on Cripto does not affect its activity in Nodal signaling. A, Nodal signaling assays following transfection of different factors into Pofut1–/– and Pofut1+/+ ES cells performed in duplicate. The average and range of data are given. B, Pofut1+/+ and Pofut1–/– ES cells with endogenous Cripto gave ~25% increased signaling following co-transfection of Nodal, Fast2, A3-luc, and Renilla luciferase (n = 3; ± S.D.). C, Pofut1+/+ and Pofut1–/– ES cells with endogenous Nodal gave ~80% increased signaling following transfection of Cripto, Fast2, A3-luc, and Renilla luciferase (n = 3; ±S.D.).

 
Pofut1–/– ES Cells Behave Similarly to Pofut1+/+ ES Cells in Nodal Signaling Assays—The major components of Nodal signaling (Nodal, Cripto, Nodal receptors, Smad2, Smad3, Smad4, and Fast2) are expressed in ES cells (52). Nodal signaling by Pofut1+/+ (8-8) compared with Pofut1–/– (5-6-3) ES cells was assayed in three ways. First, a Nodal cDNA was transfected alone as a measure of endogenous Cripto activity; second, a Cripto cDNA was transfected alone as a measure of endogenous Nodal activity; and third, Nodal and Cripto cDNAs were transfected together. In all cases a Fast2-firefly luciferase reporter construct (A3-luc) and Renilla luciferase construct were co-transfected. The three different assays gave similar signaling results in both Pofut1+/+ (8-8) and Pofut1–/– (5-6-3) ES cells (Fig. 2A), suggesting that O-fucose on Cripto is not required for Nodal signaling. The results show that exogenous Nodal increased signaling by ~25% in both Pofut1+/+ and Pofut1–/– ES cells (Fig. 2B), indicating that endogenous Cripto does not need O-fucose to facilitate Nodal signaling. Exogenous Cripto reproducibly resulted in an ~80% increase in signaling in both wild type and mutant cells (Fig. 2C), demonstrating that there is no significant difference in Nodal signaling between ES cells that add O-fucose to Cripto and those that do not.


Figure 3
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  		FIGURE 3.
Cardiomyogenesis is similar in Pofut1+/+ and Pofut1–/– embryoid bodies. A, percentage of beating EB formed from Pofut1+/+, Pofut1+/–, and Pofut1–/– EB. B, RT-PCR for {alpha}-myosin heavy chain ({alpha}-MHC), NKX2.5, and MLC-2V and beta-tubulin. Wt, wild type, Pofut1+/+; Mut, Pofut1–/–.

 
O-Fucose Is Not Required for Cripto to Function in EB Differentiation into Cardiomyocytes—Cripto/Nodal signaling is required for cardiomyogenesis because ES cells lacking Cripto fail to form cardiomyocytes from EB (44, 45). Thus, if O-fucose is necessary for Cripto function, Pofut1–/– ES cells should have lost their potential to become cardiomyocytes. We compared the capacity of Pofut1+/+ and Pofut1–/– ES cells in cardiomyogenesis by using several independent ES lines, 8-8 (Pofut1+/+), 7-1-1 (Pofut1+/–) or 7-1-2 (Pofut1+/–), 8-4-1 (Pofut1–/–), 4-4 (Pofut1–/–), and 5-6-1 (Pofut1–/–). Each ES line had essentially the same potential to differentiate into cardiomyocytes as shown by the efficiency of formation of beating EBs (Fig. 3A). Under our experimental conditions, ~50% of the EBs derived from Pofut1+/+, Pofut1+/–, and Pofut1–/– ES cell lines were beating after 10 days of culture. To confirm that cardiomyocytes had been formed, RT-PCR for the expression of marker genes up-regulated in cardiomyogenesis was performed (Fig. 3B). It can be seen that whereas all ES cell lines do not express the myosin heavy chain, myosin light chain, or the transcription factor Nkx2.5, each of these genes is expressed by all EBs at day 10 of culture when EBs are beating. These data demonstrate that Cripto without O-fucose in three independent Pofut1–/– ES cell lines had the same ability to promote cardiomyogenesis as Cripto possessing O-fucose from Pofut1+/– and Pofut1+/+ ES cells.


Figure 4
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  		FIGURE 4.
Cripto T72S mutant is modified with O-fucose. Cripto-FLAG, Cripto-FLAG/T72A, and Cripto-FLAG/T72S expressed in 293T cells were radiolabeled with [3H]fucose as described under "Experimental Procedures." Following isolation with anti-FLAG beads, Western analysis revealed two major bands. N-Glycanase treatment eliminated the top bands. Fluorography showed that all three proteins were labeled with [3H]fucose, but only Cripto and Cripto/T72S remained labeled after N-glycanase treatment.

 
Cripto with a T72S Mutation Is O-Fucosylated but Poorly Facilitates Nodal Signaling—The results above strongly suggest that O-fucosylation is not essential for Cripto to activate Nodal signaling. Nonetheless, these findings are in conflict with previous mutagenesis studies showing that substitution of Thr72 with Ala abrogates Cripto function (25, 26). Therefore, we investigated an alternative possibility that the Thr at position 72 and not the attached O-fucose is essential for Cripto to function. To examine this question directly, a T72S mutant of Cripto was generated by site-directed mutagenesis. To determine whether T72S was O-fucosylated, wild type, T72A, and T72S Cripto expression constructs in the pcDNA3-FLAG vector were transfected into 293T cells and cultured in medium containing [3H]fucose. After immunoprecipitation with anti-FLAG antibody, N-glycanase digestion was used to remove [3H]fucose incorporated into complex N-glycans, and the sample was subjected to SDS-PAGE followed by Western analysis with anti-FLAG antibody or fluorography (Fig. 4). After N-glycanase digestion, wild type and T72S Cripto remained radiolabeled, indicating that both were O-fucosylated (Fig. 4). Comparison of protein expression levels detected by Western analysis suggests that the extent of O-fucosylation in wild type and T72S Cripto was similar. In contrast, Cripto T72A was no longer modified with [3H]fucose, consistent with the absence of O-fucose.

To determine whether O-fucosylated Cripto T72S retains activity, a Nodal signaling assay was performed in 293T cells expressing Cripto T72S and Cripto T72A. Although the expression level of both Cripto mutants was similar to wild type (Fig. 5A), Nodal signaling activity with Cripto T72S was reduced to a level similar to that of Cripto T72A (Fig. 5B). These results suggest that the methyl group of Thr72, but not O-fucose, is critical for Cripto to facilitate Nodal signaling.

Thr72 Cannot Be Replaced by a Variety of Different Amino Acids—Homology comparisons among EGF-CFC members show that Thr is present in the EGF repeats in all, suggesting it might be a critical residue for Cripto function. When Thr72 was changed by site-directed mutagenesis to any of eight additional residues (Asp, Glu, His, Lys, Asn, Gln, Val, and Tyr), all mutants lost most activity in the Nodal signaling assay, although they were well expressed as determined by Western analysis (Fig. 5A). Notably, none were as defective in signaling as a triple Cripto mutant (tr1) that carries three point mutations in the EGF repeat (L75A/S77A/F78A) (26). Point mutants almost as defective as Cripto(tr1) were Cripto with Asp, Glu, or Asn at position 72. The remaining Cripto mutants (Ala, His, Lys, Gln, Ser, Val, and Tyr at position 72) showed slightly more signaling activity. Similar results were obtained in Nodal signaling assays using a subset of these Cripto mutants expressed in Pofut1+/+ and Pofut1–/– ES cells (data not shown). The combined data clearly show that Thr72 is critical for the activity of Cripto.


Figure 5
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  		FIGURE 5.
Nodal signaling is optimal only in Cripto with Thr at position 72. Cripto mutants T72A, T72D, T72E, T72H, T72N, T72Q, T72S, T72V, T72Y, and Cripto(tr1) were compared with wild type Cripto for Nodal signaling. A, aliquot of each cell lysate was used for Western analysis with anti-FLAG antibody. B, Nodal signaling was determined following transfection of plasmids into 293T cells. Values are the average of duplicates, and bars are the range of values obtained. Similar results were obtained in other experiments.

 


Figure 6
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  		FIGURE 6.
Notch1 signaling requires O-fucose in EGF12. A, mouse Notch1 fragment EGF-(11–15) and the mutants EGF11–15/T466A and EGF11–15/T466S were prepared from Lec1 CHO cells grown in the presence of [3H]fucose, purified from medium and lysate using Ni3+ beads, and analyzed by Western analysis with anti-Myc antibody (Ab) and by fluorography. B, mouse Notch1 (N1) and mutant constructs N1/T466A and N1/T466S were transfected into Lec1 CHO cells along with the reporter TP-1-luciferase and CMV-Renilla luciferase. After 16 h, L cells or Delta1/L cells were overlaid. The dual luciferase assay was performed after 30 h. Data are shown as fold-activation of Notch signaling by Delta1/L versus L cells minus the background obtained following transfection of vector alone (average 13.7 ± 3; n = 10). Results are from two experiments performed in duplicate. Error bars represent S.E.

 
Notch1 Requires O-Fucose Not Thr in the Ligand-binding Domain for Notch Signaling—Another EGF repeat that has been reported to require O-fucose for function is EGF12 in Notch1. This O-fucosylation site is conserved in all Notch proteins (32, 53), although in some species (e.g. Drosophila), the fucose is attached to Ser and in others (e.g. mouse, human) to Thr. In Drosophila, a Ser to Val mutation at the O-fucosylation site of EGF12 of Notch results in Notch activation by Serrate even in the presence of Fringe, in the context of a developing wing disc (53). A Thr to Ala mutation (T466A) in EGF12 of mouse Notch1 has a profoundly inhibitory effect on Notch1 signaling induced by either Jagged1 or Delta-like1 in a co-culture reporter assay (32). To determine whether the presence of O-fucose or Thr466 is required in EGF12 of Notch1 for Notch signaling, Notch1 T466A and T466S mutants were compared for Notch1 signaling. [3H]Fucose was not incorporated into a mouse Notch1 EGF11–15 fragment with the T466A mutation (Fig. 6A) as shown previously (46). In contrast, [3H]fucose was incorporated into Notch1/T466S equivalently to Notch1T466 (Fig. 6A). Delta1-induced Notch signaling was greatly inhibited by the T466A mutation in a Notch signaling assay (Fig. 6B). As expected, signaling was recovered in the Notch1/A466T revertant. Importantly, when Ala at 466 was replaced with Ser that can accept O-fucose, Notch1 signaling was also recovered (Fig. 6B). Therefore, Notch1 signaling in this reporter assay requires O-fucose at amino acid 466 in EGF12, and the O-fucose can be attached to either Thr or Ser. Therefore, the lack of a functional requirement for O-fucose in the EGF repeat of Cripto may be specific to Cripto.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
O-Fucose Is Not Required for Cripto to Facilitate Nodal Signaling—Previously we showed that E6.5 embryos lacking Pofut1 do not exhibit Cripto null phenotypes (30). Rather, these embryos have defects typical of a global loss of Notch signaling through all four Notch receptors. In this study, we provide an explanation for these observations. Using several different experimental strategies, we show that O-fucose is dispensable for active Cripto in Nodal signaling. In summary we found the following. 1) Cripto is a substrate of Pofut1 and not Pofut2. 2) Cripto/Nodal signaling is not affected by the absence of Pofut1 from Pofut1–/– ES cells. 3) Pofut1–/– ES cells have the same potential as Pofut1+/+ ES cells for differentiation into beating EBs and cardiomyocytes. 4) The T72S Cripto mutant is O-fucosylated but has similarly poor activity to Cripto T72A in Nodal signaling assays. We further showed that Cripto with Asp, Glu, His, Lys, Asn, Gln, Val, or Tyr at position 72 has significantly impaired Cripto activity, indicating that Thr72 may be uniquely required for Cripto to facilitate Nodal signaling. The fact that Cripto T72S has similar activity to Cripto T72A suggests that the methyl group of Thr72 plays a critical role in the ability of Cripto to support Nodal signaling. This role is likely to be direct because Cripto T72A is stable at the cell surface and appears to be folded appropriately in that its binding to ActR1B at the cell surface is normal (54). Further evidence of appropriate folding is that both T72A and T72S Cripto are N-glycosylated at Asn63 (data not shown), which is close to Thr72 in a three-dimensional model (54). In fact, the O-fucosylation of T72S also indicates appropriate folding because Pofut1 will only transfer fucose to a folded EGF repeat (55).

Cripto T72A and Other Mutants Still Retain Some Activity—Although all of the Thr72 Cripto mutants had reduced activity in signaling assays, several retained the ability to support low levels of Nodal signaling (Fig. 6B). This observation is in agreement with previous results. The human T88A Cripto mutant exhibits a low level of activity in a Nodal signaling assay with F9 teratocarcinoma cells (25). In addition, purified T72A Cripto was able to restore the capacity of Cripto null ES cells to form beating EB, although a much higher concentration was needed compared with purified wild type Cripto (45). As a control, a mouse Cripto mutant bearing three point mutations (L75A/S77A/F78A) in the EGF repeat (26) were tested, and it exhibited the lowest activity compared with other mutants in cell-based signaling assays (Fig. 6B). In the ES cell-based signaling assays this mutant exhibited complete loss of activity (data not shown).

Thr72 Is Located in a Region That May Directly Interact with Nodal—Several residues that are crucial for the activity of mouse Cripto were identified previously based on their inability to rescue the Zebrafish MZoep mutant phenotype (54). These residues are exposed and on the same side of a three-dimensional model of Cripto. One of the mutations that is immediately adjacent to Thr72, G71N, resulted in a complete loss of activity of Cripto. Another critical residue is Phe78, mutation of which to Ala inactivates Cripto completely. This is also spatially close to Gly71 and Thr72. These residues form a surface in the three-dimensional model, and it may function as an interface that directly interacts with or binds to Nodal because Cripto binds to Nodal via its EGF repeat (15, 26, 56). The fact that the T72A mutation eliminates the binding of Cripto to Nodal (26) supports this hypothesis. In addition, Thr in the EGF domain is absolutely conserved across all EGF-CFC family members (26). Interestingly, there is no Ser at this position in any of the EGF-CFC family members, consistent with the idea that O-fucose may not be essential for their functions. Perhaps O-fucose on Cripto functions in a nonessential way to facilitate trafficking, stability, and Nodal or receptor binding that may be revealed by a subtle phenotype in vivo. Alternatively, O-fucose may be an addition that occurs because Thr72 is in the correct consensus sequence but has no functional consequence.

Is O-Fucose Required for Cripto to Facilitate GDF1/Vg1, Lefty, or Tomoregulin Signaling or Signal Blocking?—Cripto is also a co-receptor for another TGF-beta member, GDF1/Vg1 (6), and Cripto also interacts with GDF1/Vg1 through its EGF-like domain. Based on the fact that Pofut1–/– embryos successfully pass the stage in which GDF1/Vg1 is required for viability (57), O-fucose may not be required for the interaction of the EGF domain of Cripto with GDF1/Vg1.

Other molecules that regulate Nodal signaling via interacting with Cripto include Lefty and Tomoregulin. Lefty blocks Nodal signaling by binding and antagonizing Cripto (6, 9), and Tomoregulin also blocks Nodal signaling via direct binding to the CFC domain of Cripto (11). O-Fucose may not be involved in the interaction of Cripto with these molecules because Pofut1–/– embryos were normal through the stage at which these molecules block Nodal to ensure embryonic development (58).

Cripto has been shown to activate both Ras/Raf/MAPK and phosphatidylinositol 3-kinase/AKT pathways independently of Nodal signaling (59, 60) via direct interaction with Glypican-1 (61). The interacting region of Cripto with Glypican-1 most likely localizes to the EGF repeat because a refolded peptide containing only the EGF repeat of Cripto is able to activate the two pathways (59). It would be interesting to know if this interaction requires the O-fucose and/or Thr72.

The protein that was first reported to require O-fucose for activity was uPA (21). The removal of O-fucose from Thr18 in the EGF (GFD) domain of uPA by treatment of trifluoroacetic acid led to the loss of its mitogenic activity. In addition, uPA purified from Escherichia coli also fails to elicit a mitogenic response in cells although it binds to the receptor with equal affinity to wild type uPA. The authors speculated that O-fucosylation might serve as a trigger to elicit a mitogenic response. To further test this hypothesis, it would be helpful to prepare uPA from Pofut1–/– ES cells and test it for mitogenic activity.

O-Fucose Is Required for Notch1 Signaling in a Co-culture Assay—O-Fucose on Notch EGF12 is highly conserved in the ligand-binding domain of many species (33, 62). In Drosophila, a Ser to Val mutation at the O-fucosylation site of Notch EGF12 results in Notch activation even in the presence of Fringe, suggesting that O-fucose at this site is inhibitory in the developing wing disk (53). The absence of the O-fucose at this site facilitates ligand binding (53). In vitro binding of a bacterially expressed (and therefore unfucosylated) fragment of mouse Notch1 (EGF repeats 11–13) fragment to Delta1 also suggests that the O-fucose on EGF repeat 12 is not essential for ligand binding, although functional binding was not examined (63). Interestingly, a Thr to Ala mutation within the O-fucosylation site of mouse Notch1 had a profound effect on Notch activation by either Jagged1 or Delta-like 1 in cell-based assays (32). The differences in these results are most likely because of differences in the systems being studied. We show in Fig. 6 that O-fucose is required in EGF12 for mouse Notch1 to signal in a co-culture assay. Thus, Ser can replace Thr at amino acid 466 and give an active Notch1, but Ala, which cannot be modified by O-fucose, causes Notch1 to become inactive in this assay.

Split, a mutation of Ile578–Thr in EGF repeat 14 of Drosophila Notch, creates a new O-fucosylation site. The consequence of this mutation is the activation of Notch receptor in R8 cells to respond to Delta ligand in adjacent cells. Wild type Notch receptor in R8 cells is not able to respond to Delta on adjacent cells. This activation of Notch is considered to be associated with the O-fucose (42). However, based on our results, it is possible that the methyl group of the Thr may contribute to the phenotypes observed.

Advantages of Pofut1–/– ES Cells in the Functional Studies of O-Fucosylation—The Pofut1–/– ES cells used here in Nodal signaling and elsewhere in Notch signaling assays (43) can be used for the production of O-fucose-free proteins that normally carry fucose on their EGF repeats, allowing the role of O-fucose on these proteins to be evaluated. These cells are particularly advantageous when compared with other approaches to eliminate O-fucose from proteins, such as chemical treatments, site-directed mutagenesis, and bacterial expression, because these methods could potentially give rise to artifacts.


   FOOTNOTES
 
* 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. Back

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. Back

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

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.

4 The abbreviations used are: TGF-beta, transforming growth factor-beta; 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. Back


   ACKNOWLEDGMENTS
 
We thank Michael Shen and Raphael Kopan for providing plasmids and the Molecular Cloning Service at Stony Brook University for assistance in generating mutants.



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